Temporal analysis of PI-3kinase and MAPK signal transduction during skeletal muscle overload

Skeletal muscles can adapt to increased mechanical forces (or loading) by increasing the size and strength of the muscle. Knowledge of the molecular mechanisms by which muscle responds to increased loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. The objective of this research was to examine the temporal associations between the activation of specific signaling pathway intermediates and their potential upstream regulator(s) in response to increased muscle loading. Previous work has demonstrated that focal adhesion kinase (FAK) activity is increased in overloaded hypertrophying skeletal muscle. Thus FAK is a candidate for transducing the loading stimulus in skeletal muscle, potentially by activating phosphatidylinositol 3-kinase (PI3K) and members of the mitogen-activated protein kinase (MAPK) family. However, it was unknown if muscle overload would result in activation of PI3K or the MAPKs. Thus, this work seeks to characterized the temporal response of (1) MAPK phosphorylation (including Erk 2, p38 MAPK and JNK), (2) PI3K activity, and (3) FAK tyrosine phosphorylation in response to 24 hours of compensatory overload in the rat soleus and plantaris muscles. In both muscles, overload resulted in transient Increases in the phosphorylation state of Erk2 and JNK, which peaked within the first hour of overload and returned to baseline thereafter. In contrast, p38 MAPK phosphorylation remained elevated throughout the entire 24-hour overload period. Moreover, overload increased PI3K activity only, in the plantaris and only at 12 hours. Moreover, 24 hours of overload induced a significant increase in total protein content in the plantaris but not the soleus. Thus an increase in total muscle protein content within the 24-hour loading period was observed only in muscle exhibiting increased PI3K activity. Surprisingly, FAK tyrosine phosphorylation was not increased during the overload period in either muscle, indicating that PI3K activation and increased MAPK phosphorylation were independent of increased FAK tyrosine phosphorylation. In summary, increased PI3K activity and sustained elevation of p38 MAPK phosphorylation were associated with muscle overload, identifying these pathways as potential mediators of the early hypertrophic response to skeletal muscle overload. This suggests that stimuli or mechanisms that activate these pathways may reduce/minimize muscle wasting and frailty. ^

Using incomplete citation data for MEDLINE results ranking.

Information overload is a significant problem for modern medicine. Searching MEDLINE for common topics often retrieves more relevant documents than users can review. Therefore, we must identify documents that are not only relevant, but also important. Our system ranks articles using citation counts and the PageRank algorithm, incorporating data from the Science Citation Index. However, citation data is usually incomplete. Therefore, we explore the relationship between the quantity of citation information available to the system and the quality of the result ranking. Specifically, we test the ability of citation count and PageRank to identify "important articles" as defined by experts from large result sets with decreasing citation information. We found that PageRank performs better than simple citation counts, but both algorithms are surprisingly robust to information loss. We conclude that even an incomplete citation database is likely to be effective for importance ranking.

Using incomplete citation data for MEDLINE results ranking.

Information overload is a significant problem for modern medicine. Searching MEDLINE for common topics often retrieves more relevant documents than users can review. Therefore, we must identify documents that are not only relevant, but also important. Our system ranks articles using citation counts and the PageRank algorithm, incorporating data from the Science Citation Index. However, citation data is usually incomplete. Therefore, we explore the relationship between the quantity of citation information available to the system and the quality of the result ranking. Specifically, we test the ability of citation count and PageRank to identify "important articles" as defined by experts from large result sets with decreasing citation information. We found that PageRank performs better than simple citation counts, but both algorithms are surprisingly robust to information loss. We conclude that even an incomplete citation database is likely to be effective for importance ranking.

Dynamic Remodeling of the Stressed Heart: Role of Protein Degradation Pathways

The heart is a remarkable organ. In order to maintain its function, it remodels in response to a variety of environmental stresses, including pressure overload, volume overload, mechanical or pharmacological unloading and hormonal or metabolic disturbances. All these responses are linked to the inherent capacity of the heart to rebuild itself. Particularly, cardiac pressure overload activates signaling pathways of both protein synthesis and degradation. While much is known about regulators of protein synthesis, little is known about regulators of protein degradation in hypertrophy. The ubiquitin-proteasome system (UPS) selectively degrades unused and abnormal intracellular proteins. I speculated that the UPS may play an important role in both qualitative and quantitative changes in the composition of heart muscle during hypertrophic remodeling. My study hypothesized that cardiac remodeling in response to hypertrophic stimuli is a dynamic process that requires activation of highly regulated mechanisms of protein degradation as much as it requires protein synthesis. My first aim was to adopt a model of left ventricular hypertrophy and determine its gene expression and structural changes. Male Sprague-Dawley rats were submitted to ascending aortic banding and sacrificed at 7 and 14 days after surgery. Sham operated animals served as controls. Effective aortic banding was confirmed by hemodynamic assessment by Doppler flow measurements in vivo. Banded rats showed a four-fold increase in peak stenotic jet velocities. Histomorphometric analysis revealed a significant increase in myocyte size as well as fibrosis in the banded animals. Transcript analysis showed that banded animals had reverted to the fetal gene program. My second aim was to assess if the UPS is increased and transcriptionally regulated in hypertrophic left ventricular remodeling. Protein extracts from the left ventricles of the banded and control animals were used to perform an in vitro peptidase assay to assess the overall catalytic activity of the UPS. The results showed no difference between hypertrophied and control animals. Transcript analysis revealed decreases in transcript levels of candidate UPS genes in the hypertrophied hearts at 7 days post-banding but not at 14 days. However, protein expression analysis showed no difference at either time point compared to controls. These findings indicate that elements of the UPS are downregulated in the early phase of hypertrophic remodeling and normalizes in a later phase. The results provide evidence in support of a dynamic transcriptional regulation of a major pathway of intracellular protein degradation in the heart. The discrepancy between transcript levels on the one hand and protein levels on the other hand supports post-transcriptional regulation of the UPS pathway in the hypertrophied heart. The exact mechanisms and the functional consequences remain to be elucidated.

Cardiomyocyte PDGFR-beta signaling is an essential component of the mouse cardiac response to load-induced stress.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

Design and implementation of a multicriteria medical decision support system for diagnosis and treatment

Health care providers face the problem of trying to make decisions with inadequate information and also with an overload of (often contradictory) information. Physicians often choose treatment long before they know which disease is present. Indeed, uncertainty is intrinsic to the practice of medicine. Decision analysis can help physicians structure and work through a medical decision problem, and can provide reassurance that decisions are rational and consistent with the beliefs and preferences of other physicians and patients. ^ The primary purpose of this research project is to develop the theory, methods, techniques and tools necessary for designing and implementing a system to support solving medical decision problems. A case study involving “abdominal pain” serves as a prototype for implementing the system. The research, however, focuses on a generic class of problems and aims at covering theoretical as well as practical aspects of the system developed. ^ The main contributions of this research are: (1) bridging the gap between the statistical approach and the knowledge-based (expert) approach to medical decision making; (2) linking a collection of methods, techniques and tools together to allow for the design of a medical decision support system, based on a framework that involves the Analytic Network Process (ANP), the generalization of the Analytic Hierarchy Process (AHP) to dependence and feedback, for problems involving diagnosis and treatment; (3) enhancing the representation and manipulation of uncertainty in the ANP framework by incorporating group consensus weights; and (4) developing a computer program to assist in the implementation of the system. ^

Design and implementation of a clinical dashboard

In a large health care system, the importance of accurate information as feedback mechanisms about its performance is necessary on many levels from the senior level management to service level managers for valid decision-making purposes. The implementation of dashboards is one way to remedy the problem of data overload by providing up-to-date, accurate, and concise information. As this health care system seeks to have an organized, systematic review mechanism in place, dashboards are being created in a variety of the hospital service departments to monitor performance indicators. The Infection Control Administration of this health care system is one that does not currently utilize a dashboard but seeks to implement one. ^ The purpose of this project is to research and design a clinical dashboard for the Infection Control Administration. The intent is that the implementation and usefulness of the clinical dashboard translates into improvement in the measurement of health care quality.^

Anticancer activity of OSW-1: Induction of apoptotic pathway in leukemia and autophagic death in pancreatic cancer through a calcium mediated mechanism

OSW-1 is a natural compound found in the bulbs of Orninithogalum saudersiae, a member of the lily family. This compound exhibits potent antitumor activity in vitro with the IC50 values in the low nanomolar concentration range and demonstrating its ability to kill drug resistant cancer cells. In an effort to discover the unknown mechanism of action of this novel compound as a potential anticancer agent, the main objective of this research project was to test the cytotoxicity of OSW-1 against various cancer lines, and to elucidate the biochemical and molecular mechanism(s) responsible for the anticancer activity of OSW-1. My initial investigation revealed that OSW-1 is effective in killing various cancer cells including pancreatic cancer cells and primary leukemia cells resistant to standard chemotherapeutic agents, and that non-malignant cells were less sensitive to this compound. Further studies revealed that in leukemia cells, OSW-1 causes a significant increase in cytosolic calcium and activates rapid calcium-dependent apoptosis by the intrinsic pathway. Additionally, OSW-1 treatment leads to the degradation of the ER chaperone GRP78/BiP implicated in the survival of cancer cells. Meanwhile, it shows a reduced sensitivity in respiration-deficient sub-clones of leukemia cells which had higher basal levels of Ca2+. Mechanistically, it was further demonstrated that cytosolic Ca2+ elevations were observed together with Na+ decreases in the cytosol, suggesting OSW-1 caused the calcium overload through inhibition of the Na+/Ca 2+exchanger (NCX). Although similar calcium disturbances were observed in pancreatic cancer cells, mechanistic studies revealed that autophagy served as an initial pro-survival mechanism subsequent to OSW-1 treatment but extended autophagy caused inevitable cell death. Furthermore, combination of OSW-1 with autophagy inhibitors significantly enhances the cytotoxicity against pancreatic cancer cells. Taken together, this study revealed the novel mechanism of OSW-1 which is through inhibition of the Na+/Ca2+ exchanger and provides a basis for using this compound in combination with other agents for the treatment of pancreatic cancer which is resistant to available anticancer drugs. ^