Metabolite Profiling of Alzheimer's Disease Cerebrospinal Fluid

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive functions. Today the diagnosis of AD relies on clinical evaluations and is only late in the disease. Biomarkers for early detection of the underlying neuropathological changes are still lacking and the biochemical pathways leading to the disease are still not completely understood. The aim of this study was to identify the metabolic changes resulting from the disease phenotype by a thorough and systematic metabolite profiling approach. For this purpose CSF samples from 79 AD patients and 51 healthy controls were analyzed by gas and liquid chromatography-tandem mass spectrometry (GC-MS and LC-MS/MS) in conjunction with univariate and multivariate statistical analyses. In total 343 different analytes have been identified. Significant changes in the metabolite profile of AD patients compared to healthy controls have been identified. Increased cortisol levels seemed to be related to the progression of AD and have been detected in more severe forms of AD. Increased cysteine associated with decreased uridine was the best paired combination to identify light AD (MMSE>22) with specificity and sensitivity above 75%. In this group of patients, sensitivity and specificity above 80% were obtained for several combinations of three to five metabolites, including cortisol and various amino acids, in addition to cysteine and uridine

Dynamic Modelling under Uncertainty: The Case of Trypanosoma brucei Energy Metabolism

Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis). Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies

Anti-Tumor Activity and Immunotherapeutic Potential of a Bisphosphonate Prodrug

Bisphosphonates have benefits in breast cancer and multiple myeloma patients and have been used with adoptive immunotherapy with γδ T cells expressing Vγ2?Vδ2 TCRs. Although treatment with γδ T cells is safe, it has shown limited efficacy. Present bisphosphonates stimulate γδ T cells but were designed to inhibit bone resorption rather than treating cancer and have limited oral absorption, tumor cell entry, and cause bone side effects. The development of phosphate and phosphonate nucleotide prodrugs has led to important drugs for hepatitis C and HIV. Using a similar approach, we synthesized bisphosphonate prodrugs and found that they efficiently limit tumor cell growth. Pivoxil bisphosphonate esters enter cells where esterases convert them to their active acids. The bisphosphonate esters stimulated γδ T cells to secrete TNF-α in response to a variety of tumor cells more efficiently than their corresponding acids. The most active compound, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1- bisphosphonate (7), specifically expanded γδ T cells and stimulated them to secrete interferon-γ and kill tumor cells. In preclinical studies, combination therapy with compound 7 and γδ T cells prolonged survival of mice inoculated with either human bladder cancer or fibrosarcoma cells. Therefore, bisphosphonate prodrugs could enhance the effectiveness of adoptive cancer immunotherapy with γδ T cells

Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

Background: MicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis. Results: The expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context. Conclusion: MiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in CRC provide a basis for understanding the functional role of miRNAs in cancer. © 2009 Arndt et al; licensee BioMed Central Ltd

Evidence for the Complexity of MicroRNA-Mediated Regulation in Ovarian Cancer: A Systems Approach

MicroRNAs (miRNAs) are short (∼22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. Previous studies have shown that miRNAs inhibit the translation and facilitate the degradation of their targeted messenger RNAs (mRNAs) making them attractive candidates for use in cancer therapy. However, the potential clinical utility of miRNAs in cancer therapy rests heavily upon our ability to understand and accurately predict the consequences of fluctuations in levels of miRNAs within the context of complex tumor cells. To evaluate the predictive power of current models, levels of miRNAs and their targeted mRNAs were measured in laser captured micro-dissected (LCM) ovarian cancer epithelial cells (CEPI) and compared with levels present in ovarian surface epithelial cells (OSE). We found that the predicted inverse correlation between changes in levels of miRNAs and levels of their mRNA targets held for only ∼11% of predicted target mRNAs. We demonstrate that this low inverse correlation between changes in levels of miRNAs and their target mRNAs in vivo is not merely an artifact of inaccurate miRNA target predictions but the likely consequence of indirect cellular processes that modulate the regulatory effects of miRNAs in vivo. Our findings underscore the complexities of miRNA-mediated regulation in vivo and the necessity of understanding the basis of these complexities in cancer cells before the therapeutic potential of miRNAs can be fully realized

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies

Micro-RNAs (miRs) are important regulators of mRNA and protein expression; the ability of miR expression profilings to distinguish different cancer types and classify their sub-types has been well-described. They also represent a novel biological entity with potential value as tumour biomarkers, which can improve diagnosis, prognosis, and monitoring of treatment response for human cancers. This endeavour has been greatly facilitated by the stability of miRs in formalin-fixed paraffin-embedded (FFPE) tissues, and their detection in circulation. This review will summarize some of the key dysregulated miRs described to date in human epithelial malignancies, and their potential value as molecular bio-markers in FFPE tissues and blood samples. There remain many challenges in this domain, however, with the evolution of different platforms, the complexities of normalizing miR profiling data, and the importance of evaluating sufficiently-powered training and validation cohorts. Nonetheless, well-conducted miR profiling studies should contribute important insights into the molecular aberrations driving human cancer development and progression