Excision of Titin's Cardiac Pevk Spring Element Abolishes PKCα-Induced Increases in Myocardial Stiffness

Protein Kinase C-alpha (PKCalpha) was recently reported to increase myocardial stiffness, an effect that was proposed to be due to phosphorylation of two highly conserved sites (S11878 and S12022) within the proline-gluatamic acid-valine-lysine (PEVK) rich spring element of titin. To test this proposal we investigated the effect of PKCalpha on phosphorylation and passive stiffness in a mouse model lacking the titin exons that contain these two phosphorylation sites, the PEVK knockout (KO). We used skinned, gelsolin-extracted, left ventricular, myocardium from wildtype and PEVK KO mice. Consistent with previous work we found that PKCalpha increased passive stiffness in the WT myocardium by 27 +/-6%. Importantly, this effect was completely abolished in KO myocardium. In addition, increases in the elastic and viscous moduli at a wide range of frequencies (properties important in diastolic filling) following PKCalpha incubation (27 +/-3% and 20 +/-4%, respectively) were also ablated in the KO. Back phosphorylation assays showed that titin phosphorylation following incubation with PKCalpha was significantly reduced by 36+/-12% in skinned PEVK KO myocardial tissues. The remaining phosphorylation in the KO suggests that PKCalpha sites exist in the titin molecule outside the PEVK region; these sites are not involved in increasing passive stiffness. Our results firmly support that the PEVK region of cardiac titin is phosphorylated by PKCalpha and that this increases passive tension. Thus, the PEVK spring element is the critical site of PKCalpha's involvement in passive myocardial stiffness

The Foreign Direct Investment Location Decision: A Contingency Model of the Foreign Direct Investment Location Decision-Making Process

Despite considerable prior research into foreign direct investment (FDI) location decisions, our understanding of the processes underlying such decisions is still limited. Findings from work based in the economics and behavioral theories of the multinational enterprise (MNE) both acknowledge that FDI is not a point-of-time decision but a gradual process that yields important changes over its duration. However, these competing traditions both fall short when attempting to portray the actual process by which FDI location decisions are made by managers in MNEs. This gap has been recently attributed to two interrelated limitations. Firstly, level of analysis concerns have artificially separated managerial decision-making processes from the organizational and environmental structures within which they are made. Secondly, because of the complexity inherent in the FDI location decision environment, the study of these decisions has not taken contextual factors into consideration. This study addresses three important questions in order to build our understanding of the FDI location decision-making processes: (1) What are the decision-making processes that lead to FDI location choice? (2) What is the impact of contextual variables on FDI location decision-making processes at different levels of analysis, and are there any patterns of variation in decision processes under different decision conditions? (3) What factors drive final FDI location choice, and can a useful framework or theory be developed that links FDI location decision-making processes and context to drivers of FDI location choice? In order to address level of analysis concerns, the study places the manager at the center of the FDI location decision in modeling and in research, a strategy recommended by an emerging stream of behavioral-focused international business research (Aharoni, 2010; Buckley et al., 2007; Devinney, 2011). By examining FDI location decisions from the perspective of the managers who implement them, it is possible to clarify the nature of processes that lead to FDI location choice, and identify the impact of different elements of decision maker, firm and environmental context on such processes. The conceptual framework builds on Aharoni’s (1966) pivotal research while incorporating findings from broader behavioral managerial decision models and international business research. The framework is based on the assumption that FDI location decision-making processes and final choice are contingent upon interactions between the environmental, firm and decision maker context under which the decision is made. The research was undertaken in three phases. Phase 1 included a literature review that covered research on the MNE, internationalization, and decision making. The findings of the review identified key aspects of FDI location decision context and led to the development of an initial contingency framework of strategic decision making. Phase 2 consisted of an exploratory case study of twenty four FDI location decisions. The initial contingency framework developed during the literature review was used during this stage to identify the relationship between decision-making processes and contextual variables at the case decisions. By drawing on results from the exploratory research, an initial conceptual model and a set of propositions were developed. In Phase 3, twenty case studies were theoretically sampled from a pool of MNEs of varying size and parent-country nationality within the knowledge-based industries. The data collection and analysis followed a process, event-driven approach to case study research involving the mapping of key sequences of events as well as within- and cross-case analysis. The results identify the key elements of the decision process that explain FDI location behavior and develop a framework that links them together and makes them sensible. The four key elements of the FDI location decision that comprise the framework include: (i) the process, (ii) the context, (iii) patterns, and (iv) location. Research findings show the FDI location decision process as comprising of five broad stages, the content of each driven by a dynamic and evolving interpretation of maximum subjective expected utility. Utility preferences are identified as the consequence of shifting and opaque goals, founded upon imperfect information, operating in an environment marked by uncertainty. Five variations in the overall orientation of utility at case decisions, classified in the study as ‘decision rules,’ proved to be more useful predictors of decision-making behavior than traditional notions of bounded rationality seeking rent extraction and profitability. Decision processes were found to vary in five prototypical patterns, according to clusters of contextual variables that together moderated the level of decision-maker autonomy, hierarchical centralization, rule formalization, commitment to strategy, and politicization of the decision. Patterns are described as FDI location decision-making models, and proposed as an initial step towards the development of a taxonomy of FDI location decision-making processes. Because of the dynamic and staged nature of the process, findings showed that factors that were important at one stage of the decision were not as important at the next. As such, the task of identifying universal drivers of FDI location was deemed an unfeasible one. In place of universal drivers, the initiating force of the investment, the purpose of investment and information sources and networks are identified as the key context-specific determinants of location in FDI decisions. Bounded by uncertainty, chance, the dynamics of the process and decision-maker effects, each of these aspects of the decision served to limit the possible consideration set for investment, and formed the value basis and measures from which to select the most attractive location choice. Despite the contextual differences in these drivers, however, the study revealed a strong pattern that showed that the importance of specific location considerations differed in much the same way across case decisions. During the first stage of case decisions primarily strategic aspects of locations were considered; during the second, considerations relating to the system; operational concerns in the third; implementation concerns in the fourth; and added value factors in the final choice. How each of these concerns was interpreted to reach final location choice differed according to the drivers mentioned previously, although the patterns were the same. This study develops a contingency framework for examining the FDI location decision-making processes of MNEs under different operating conditions. By identifying the four key components of the FDI location decision, their interrelationships and many sources of variance, this thesis shows that despite its complexity, the FDI location decision is amenable to useful conceptual structuring. From an academic standpoint, the framework answers Aharoni’s most recent call to action in ‘Behavioral Elements in Foreign Direct Investment’ (2010) by developing a replicable structure within which to think about incorporating managerial decision models and context into the theory of the MNE. These findings enhance understandings of decision making at MNEs, reconcile a number of inconsistencies between opposing perspectives of MNE theory, and thereby update extant theory so that it has greater relevance in today’s diverse international business environment. From a managerial standpoint, the thesis helps managers to recognize the opportunities and limitations posed by different aspects of decision context so that they are able to tailor their FDI location decision strategies to best suit their needs. Finally, from the perspective of policy markers, research findings provide great support for the use of investment attraction schemes through the use of targeted location marketing and investment incentives.

Assessment of nanoindentation in stiffness measurement of soft biomaterials: kidney, liver, spleen and uterus

Nanoindentation technology with high spatial resolution and force sensitivity is widely used to measure the mechanical properties of hard biomaterials and tissues. However, its reliability to analyze soft biomaterials and organs has not been tested. Here, we evaluated the utility of nanoindentation to measure the passive mechanical properties of soft biological specimen. Kidney, liver, spleen and uterus samples were harvested from C57BL/6 N mice. We assessed test–retest repeatability in biological specimen and hydrogel controls using Bland–Altman diagrams, intraclass correlation coefficients (ICCs) and the within-subject coefficients of variation (COVs). The results were calculated using Hertzian, JKR and Oliver & Pharr models. Similar to hydrogels, Bland–Altman plots of all biological specimen showed good reliability in stiffness test and retest examinations. In gels, ICCs were larger than 0.8 and COVs were smaller than 15% in all three models. In kidney, liver, spleen and uterus, ICCs were consistently larger than 0.8 only in the Hertzian model but not in the JKR and Oliver & Pharr models. Similarly, COVs were consistently smaller than 15% in kidney, liver, spleen and uterus only in the Hertzian model but not in the other models. We conclude that nanoindentation technology is feasible in detecting the stiffness of kidney, liver, spleen and uterus. The Hertzian model is the preferred method to provide reliable results on ex vivo organ stiffness of the biological specimen under study

68Ga-labelled exendin-3, a new agent for the detection of insulinomas with PET

Contains fulltext : 89596.pdf (publisher's version ) (Closed access)PURPOSE: Insulinomas are neuroendocrine tumours derived from pancreatic beta-cells. The glucagon-like peptide 1 receptor (GLP-1R) is expressed with a high incidence (>90%) and high density in insulinomas. Glucagon-like peptide 1 (GLP-1), the natural ligand of GLP-1R, is rapidly degraded in vivo. A more stable agonist of GLP-1R is exendin-3. We investigated imaging of insulinomas with DOTA-conjugated exendin-3 labelled with (68)Ga. METHODS: Targeting of insulinomas with [Lys(40)(DOTA)]exendin-3 labelled with either (111)In or (68)Ga was investigated in vitro using insulinoma tumour cells (INS-1). [Lys(40)((111)In-DTPA)]Exendin-3 was used as a reference in this study. In vivo targeting was investigated in BALB/c nude mice with subcutaneous INS-1 tumours. PET imaging was performed using a preclinical PET/CT scanner. RESULTS: In vitro exendin-3 specifically bound and was internalized by GLP-1R-positive cells. In BALB/c nude mice with subcutaneous INS-1 tumours a high uptake of [Lys(40)((111)In-DTPA)]exendin-3 in the tumour was observed (33.5 +/- 11.6%ID/g at 4 h after injection). Uptake was specific, as determined by coinjection of an excess of unlabelled [Lys(40)]exendin-3 (1.8 +/- 0.1%ID/g). The pancreas also exhibited high and specific uptake (11.3 +/- 1.0%ID/g). High uptake was also found in the kidneys (144 +/- 24%ID/g) and this uptake was not receptor-mediated. In this murine tumour model optimal targeting of the GLP-1R expressing tumour was obtained at exendin doses < or =0.1 microg. Remarkably, tumour uptake of (68)Ga-labelled [Lys(40)(DOTA)]exendin-3 was lower (8.9 +/- 3.1%ID/g) than tumour uptake of (111)In-labelled [Lys(40)(DTPA)]exendin-3 (25.4 +/- 7.2%ID/g). The subcutaneous tumours were clearly visualized by small-animal PET imaging after injection of 3 MBq of [Lys(40)((68)Ga-DOTA)]exendin-3. CONCLUSION: [Lys(40)((68)Ga-DOTA)]Exendin-3 specifically accumulates in insulinomas, although the uptake is lower than that of [Lys(40)((111)In-DTPA)]exendin-3. Therefore, [Lys(40)((68)Ga-DOTA)]exendin-3 is a promising tracer to visualize insulinomas with PET.01 juli 201

Temperature controlled martensitic phase transformations in a model NiAl alloy

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Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

Contains fulltext : 88022.pdf (publisher's version ) (Closed access)PURPOSE: In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin. METHODS: Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of (111)In-albumin, (111)In-minigastrin, (111)In-exendin and (111)In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of (111)In-minigastrin, (111)In-exendin and (111)In-octreotide was determined. RESULTS: FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of (111)In-albumin, (111)In-exendin and (111)In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide #6), was selected for in vivo testing. In rats, 5 mg of peptide #6 very efficiently inhibited the renal uptake of (111)In-minigastrin, by 88%. Uptake of (111)In-exendin and (111)In-octreotide was reduced by 26 and 33%, respectively. CONCLUSIONS: The albumin-derived peptide #6 efficiently inhibited the renal reabsorption of (111)In-minigastrin, (111)In-exendin and (111)In-octreotide and is a promising candidate for kidney protection in PRRT.1 februari 201

Renal uptake of different radiolabelled peptides is mediated by megalin: SPECT and biodistribution studies in megalin-deficient mice

Contains fulltext : 98302.pdf (publisher's version ) (Closed access)PURPOSE: Radiolabelled peptides used for peptide receptor radionuclide therapy are excreted mainly via the kidneys and are partly reabsorbed and retained in the proximal tubular cells. The resulting high renal radiation dose can cause nephrotoxicity, limiting the maximum activity dose and the effectiveness of peptide receptor radionuclide therapy. The mechanisms of kidney reabsorption of these peptides are incompletely understood, but the scavenger receptor megalin has been shown to play a role in the reabsorption of (111)In-octreotide. In this study, the role of megalin in the renal reabsorption of various relevant radiolabelled peptides was investigated. METHODS: Groups of kidney-specific megalin-deficient mice and wild-type mice were injected with (111)In-labelled somatostatin, exendin, neurotensin or minigastrin analogues. Single photon emission computed tomographic (SPECT) images of the kidneys were acquired and analysed quantitatively, or the animals were killed 3 h after injection and the activity concentration in the kidneys was measured. RESULTS: Megalin-deficient mice showed significantly lower uptake of all studied radiolabelled peptides in the kidneys, ranging from 22% ((111)In-octreotide) to 65% ((111)In-exendin) of uptake in wild-type kidneys. Quantitative analysis of renal uptake by SPECT and ex vivo measurements showed a very good correlation. CONCLUSION: Megalin is involved in the renal reabsorption of radiolabelled octreotide, octreotate, exendin, neurotensin and minigastrin. This knowledge may help in the design of strategies to reduce this reabsorption and the resulting nephrotoxicity in peptide receptor radionuclide therapy, enabling more effective therapy. Small-animal SPECT is an accurate tool, allowing in vivo quantification of renal uptake and serial measurements in individual mice

Epithelial coxsackievirus adenovirus receptor promotes house dust mite-induced lung inflammation

Airway inflammation and remodelling are important pathophysiologic features in asthma and other respiratory conditions. An intact epithelial cell layer is crucial to maintain lung homoeostasis, and this depends on intercellular adhesion, whilst damaged respiratory epithelium is the primary instigator of airway inflammation. The Coxsackievirus Adenovirus Receptor (CAR) is highly expressed in the epithelium where it modulates cell-cell adhesion stability and facilitates immune cell transepithelial migration. However, the contribution of CAR to lung inflammation remains unclear. Here we investigate the mechanistic contribution of CAR in mediating responses to the common aeroallergen, House Dust Mite (HDM). We demonstrate that administration of HDM in mice lacking CAR in the respiratory epithelium leads to loss of peri-bronchial inflammatory cell infiltration, fewer goblet-cells and decreased pro-inflammatory cytokine release. In vitro analysis in human lung epithelial cells confirms that loss of CAR leads to reduced HDM-dependent inflammatory cytokine release and neutrophil migration. Epithelial CAR depletion also promoted smooth muscle cell proliferation mediated by GSK3β and TGF-β, basal matrix production and airway hyperresponsiveness. Our data demonstrate that CAR coordinates lung inflammation through a dual function in leucocyte recruitment and tissue remodelling and may represent an important target for future therapeutic development in inflammatory lung diseases

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases