R-etodolac is a more potent Wnt signaling inhibitor than enantiomer,S-etodolac

Etodolac is an FDA-approved nonsteroidal anti-inflammatory drug (NSAID) used to treat a variety of inflammatory diseases. The drug is administered as a racemate (50/50 mixture of R- and S- enantiomers), however, studies have shown that the two enantiomers have distinct biologic and pharmacokinetic differences. Wnt signaling, which plays key roles in cell proliferation, polarity, and differentiation, has been shown to be inhibited by R-etodolac; however, comparative analyses of R- and S-etodolac in this function have not been conducted. We used in silico molecular docking and TOPflash functional biologic assays to compare R- and S-enantiomers effect on Wnt signaling inhibition. Further, we used a cultivated limbal stem epithelial cell (cLSCs) model to investigate enantiospecific changes in the colony-forming efficiency (CFE) of cLSCs. The data shows that R-etodolac is a more potent inhibitor of Wnt signaling. In addition, consistently, while both enantiomers demonstrate a dose-dependent decrease in CFE of cLSCs, R-etodolac is a more potent inhibitor.     

Community engagement: leadership tool for catastrophic health events

Disasters and epidemics are immense and shocking disturbances that require the judgments and efforts of large numbers of people, not simply those who serve in an official capacity. This article reviews the Working Group on Community Engagement in Health Emergency Planning's recommendations to government decision makers on why and how to catalyze the civic infrastructure for an extreme health event. Community engagement--defined here as structured dialogue, joint problem solving, and collaborative action among formal authorities, citizens at-large, and local opinion leaders around a pressing public matter--can augment officials' abilities to govern in a crisis, improve application of communally held resources in a disaster or epidemic, and mitigate community wide losses. The case of limited medical options in an influenza pandemic serves to demonstrate the civic infrastructure's preparedness, response, and recovery capabilities and to illustrate how community engagement can improve pandemic contingency planning.     

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function ( n = 11–14, P < 0.05) and decreased right ventricular wall stress ( n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 ± 1.1% versus 27.8 ± 1.2%; n = 6–8, P < 0.001) and greater capillary density (338.5 ± 14.7 versus 259.8 ± 9.2 vessels per mm; n = 6–8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 ± 0.03% versus 0.42 ± 0.03%; n = 6, P = 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit⁺ and CD34⁺ stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood ( n = 7, P < 0.05) and 48 hrs in the infarcted hearts ( n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts ( n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit⁺ and CD34⁺ stem cells via the enhanced expression of chemoattractant SDF-1.     

The Prostate Cancer-Associated Human Retrovirus XMRV Lacks Direct Transforming Activity but Can Induce Low Rates of Transformation in Cultured Cells

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5α (Ref1), showing that XMRV is resistant to TRIM5α restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.The association of human prostate cancer with mutations that impair the function of the antiviral defense protein RNase L suggested a role for virus in prostate cancer. Indeed, analysis of cDNA from prostate tumors by use of a DNA microarray (Virochip) containing conserved DNA sequences from all known virus families indicated the presence of a novel gammaretrovirus in 40% of prostate cancer patients having homozygous R462Q mutations in RNase L (35). Cloning and sequencing of the virus revealed a close similarity to mouse xenotropic retroviruses; thus, the new virus was named XMRV (xenotropic murine leukemia virus-related virus) (35). Importantly, XMRV has been found integrated into human genomic DNA from tumor-bearing prostatic tissue samples of 11 patients, showing that XMRV can indeed infect humans and is not a laboratory contaminant (7, 13). Although an initial study found XMRV only in tumor stromal cells (35), recent studies have found XMRV in the prostate carcinoma cell line 22Rv1 (14) and in malignant epithelial cells in prostate tumors (34).XMRV lacks a host cell-derived oncogene, but examples of oncogenic activity in Env proteins from other retroviruses (1, 6, 16, 24) raise the possibility that the Env protein of XMRV might also be oncogenic. Such activity could be a result of interaction of the XMRV Env protein with the virus entry receptor Xpr1 (7, 14), which shows similarity to a yeast protein involved in G protein-coupled signal transduction (2), or interaction with other cellular proteins that do not function as virus entry receptors, as is the case for jaagsiekte sheep retrovirus (JSRV) Env (interacting protein unknown) (16) and the Env protein of spleen focus-forming virus, which interacts with and activates the erythropoietin receptor and the receptor tyrosine kinase Stk (24). Detection of XMRV oncogenic activity would strengthen the argument for a role for XMRV in prostate cancer.In addition, while XMRV shows the highest sequence similarity to the mouse xenotropic retroviruses, it is also similar to the mink cell focus-forming (MCF) retroviruses of mice, which are highly leukemogenic due to their ability to multiply reinfect cells, leading to more-frequent activation of cellular oncogenes (36). MCF viruses were first defined by their ability to induce foci of altered cells in mink cell layers (11). Initially, it was unclear whether these foci were the result of cell transformation or cytopathic effects of the virus (11), but it is clear now that these foci result from cytopathic effects related to the ability of MCF viruses to multiply reinfect cells in what can be a receptor-independent manner, leading to cell apoptosis (23, 36, 37). It was thus important to determine if XMRV has similar properties and might be able to more frequently activate cellular oncogenes.Here, we have found that while XMRV lacks direct transforming activity in the fibroblast and epithelial cell lines tested and does not induce cytopathic effects typical of multiple reinfection by MCF viruses, it is able to induce rare transformed foci in a rat fibroblast cell line. Interestingly, in one case, transformation led to the production of a highly active oncogenic retrovirus.     

Serum alters the uptake and biologic activity of CpG oligodeoxynucleotides in B cell chronic lymphocytic leukemia

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) have a number of effects on B cells, including upregulation of immunogenic molecules, and, therefore, appear attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). Previous in vitro studies investigating the effect of CpG ODN on B-CLL cells used serum-low conditions and did not account for the longer-half life of CpG ODN in vitro. The present study was designed to explore how the presence of serum and exposure time affect CpG ODN-mediated changes on B-CLL cells. The optimal concentration for CpG ODN-mediated effects in the presence of 100% serum or plasma was higher (10-20 microg/ml) than for serum-low conditions. Maximal CpG ODN-mediated effects required the presence of ODN for no longer than 3 hours. The inhibition of CpG ODN-mediated effects by serum correlated with lower uptake of ODN into B-CLL cells in the presence of serum. A threshold effect on biologic response was observed, with a given amount of ODN internalized, resulting in phenotypic changes. In conclusion, systemic short-term application of CpG ODN appears to be sufficient to induce phenotypic changes, but higher doses of CpG ODN than previously thought may be necessary because of inhibition of their uptake by serum.     

Biological significance of nitric oxide-mediated protein modifications

Nitric oxide (NO), despite an apparently simple diatomic structure, has a wide variety of functions in both physiology and pathology and within every major organ system. It has become an increasingly important scientific challenge to decipher how this wide range of activity is achieved. To this end a number of investigators have begun to explore how NO-mediated posttranslational modifications of proteins may represent mechanisms of cellular signaling. These modifications include: 1). binding to metal centers; 2). nitrosylation of thiol and amine groups; 3). nitration of tyrosine, tryptophan, amine, carboxylic acid, and phenylalanine groups; and 4). oxidation of thiols (both cysteine and methionine residues) and tyrosine. However, two particular modifications have recently received much attention, nitrosylation of thiols to produce S-nitrosothiol and nitration of tyrosine residues to produce nitrotyrosine. It is the purpose of this review to examine the possibility that these modifications may play a role in NO-mediated signaling.     

Use of Expert Panels to Define the Reference Standard in Diagnostic Research: A Systematic Review of Published Methods and Reporting

Background

In diagnostic studies, a single and error-free test that can be used as the reference (gold) standard often does not exist. One solution is the use of panel diagnosis, i.e., a group of experts who assess the results from multiple tests to reach a final diagnosis in each patient. Although panel diagnosis, also known as consensus or expert diagnosis, is frequently used as the reference standard, guidance on preferred methodology is lacking. The aim of this study is to provide an overview of methods used in panel diagnoses and to provide initial guidance on the use and reporting of panel diagnosis as reference standard.

Methods and Findings

PubMed was systematically searched for diagnostic studies applying a panel diagnosis as reference standard published up to May 31, 2012. We included diagnostic studies in which the final diagnosis was made by two or more persons based on results from multiple tests. General study characteristics and details of panel methodology were extracted. Eighty-one studies were included, of which most reported on psychiatry (37%) and cardiovascular (21%) diseases. Data extraction was hampered by incomplete reporting; one or more pieces of critical information about panel reference standard methodology was missing in 83% of studies. In most studies (75%), the panel consisted of three or fewer members. Panel members were blinded to the results of the index test results in 31% of studies. Reproducibility of the decision process was assessed in 17 (21%) studies. Reported details on panel constitution, information for diagnosis and methods of decision making varied considerably between studies.

Conclusions

Methods of panel diagnosis varied substantially across studies and many aspects of the procedure were either unclear or not reported. On the basis of our review, we identified areas for improvement and developed a checklist and flow chart for initial guidance for researchers conducting and reporting of studies involving panel diagnosis. Please see later in the article for the Editors'' Summary     

The Genetic Origin of Daunians and the Pan-Mediterranean Southern Italian Iron Age Context

The geographical location and shape of Apulia, a narrow land stretching out in the sea at the South of Italy, made this region a Mediterranean crossroads connecting Western Europe and the Balkans. Such movements culminated at the beginning of the Iron Age with the Iapygian civilization which consisted of three cultures: Peucetians, Messapians, and Daunians. Among them, the Daunians left a peculiar cultural heritage, with one-of-a-kind stelae and pottery, but, despite the extensive archaeological literature, their origin has been lost to time. In order to shed light on this and to provide a genetic picture of Iron Age Southern Italy, we collected and sequenced human remains from three archaeological sites geographically located in Northern Apulia (the area historically inhabited by Daunians) and radiocarbon dated between 1157 and 275 calBCE. We find that Iron Age Apulian samples are still distant from the genetic variability of modern-day Apulians, they show a degree of genetic heterogeneity comparable with the cosmopolitan Republican and Imperial Roman civilization, even though a few kilometers and centuries separate them, and they are well inserted into the Iron Age Pan-Mediterranean genetic landscape. Our study provides for the first time a window on the genetic make-up of pre-Roman Apulia, whose increasing connectivity within the Mediterranean landscape, would have contributed to laying the foundation for modern genetic variability. In this light, the genetic profile of Daunians may be compatible with an at least partial autochthonous origin, with plausible contributions from the Balkan peninsula.     

Glioma-associated endothelial cells show evidence of replicative senescence

The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-beta-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.