Analyzing M-CSF dependent monocyte/macrophage differentiation: Expression modes and meta-modes derived from an independent component analysis

<p>Abstract</p> <p>Background</p> <p>The analysis of high-throughput gene expression data sets derived from microarray experiments still is a field of extensive investigation. Although new approaches and algorithms are published continuously, mostly conventional methods like hierarchical clustering algorithms or variance analysis tools are used. Here we take a closer look at independent component analysis (ICA) which is already discussed widely as a new analysis approach. However, deep exploration of its applicability and relevance to concrete biological problems is still missing. In this study, we investigate the relevance of ICA in gaining new insights into well characterized regulatory mechanisms of M-CSF dependent macrophage differentiation.</p> <p>Results</p> <p>Statistically independent gene expression modes (GEM) were extracted from observed gene expression signatures (GES) through ICA of different microarray experiments. From each GEM we deduced a group of genes, henceforth called <it>sub-mode</it>. These <it>sub-modes </it>were further analyzed with different database query and literature mining tools and then combined to form so called <it>meta-modes</it>. With them we performed a knowledge-based pathway analysis and reconstructed a well known signal cascade.</p> <p>Conclusion</p> <p>We show that ICA is an appropriate tool to uncover underlying biological mechanisms from microarray data. Most of the well known pathways of M-CSF dependent monocyte to macrophage differentiation can be identified by this unsupervised microarray data analysis. Moreover, recent research results like the involvement of proliferation associated cellular mechanisms during macrophage differentiation can be corroborated.</p

Reliability of different body temperature measurement sites during aortic surgery

Objective: We retrospectively performed a comparative analysis of temperature measurement sites during surgical repair of the thoracic aorta. Methods: Between January 2004 and May 2006, 22 patients (mean age: 63±12 years) underwent operations on the thoracic aorta with arterial cannulation of the aortic arch concavity and selective antegrade cerebral perfusion (ACP) during deep hypothermic circulatory arrest (HCA). Indications for surgical intervention were acute type A dissection in 14 (64%) patients, degenerative aneurysm in 6 (27%), aortic infiltration of thymic carcinoma in 1 (4.5%) and intra-aortic stent refixation in 1 (4.5%). Rectal, tympanic and bladder temperatures were evaluated to identify the best reference to arterial blood temperature during HCA and ACP. Results: There were no operative deaths and the 30-day mortality rate was 13% (three patients). Permanent neurological deficits were not observed and transient changes occurred in two patients (9%). During re-warming, there was strong correlation between tympanic and arterial blood temperatures (r= 0.9541, p<0.001), in contrast to the rectal and bladder temperature (r=0.7654, p= n.s; r=0.7939, p= n.s., respectively). Conclusion: We conclude that tympanic temperature measurements correlate with arterial blood temperature monitoring during aortic surgery with HCA and ACP and, therefore, should replace bladder and rectal measurements

Conjugation with Tris Decreases the Risk of Ketoprofen-Induced Mucosal Damage and Reduces Inflammation-Associated Methane Production in a Rat Model of Colitis

We have designed a new compound from the non-steroidal anti-inflammatory drug (NSAID) ketoprofen (Ket) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) precursors, with the aim to reduce the gastrointestinal (GI) side effects of NSAID therapies. We investigated mucosal reactions in a standard rat model of colitis together with methane generation as a possible indicator of pro-inflammatory activation under this condition (approval number: V./148/2013). Whole-body methane production (photoacoustic spectroscopy) and serosal microcirculation (intravital videomicroscopy) were measured, and mucosal damage was assessed (conventional histology; in vivo laser-scanning endomicroscopy). Inflammatory markers were measured from tissue and blood samples. Colitis induced an inflammatory response, morphological colonic damage and increased methane output. Ket treatment lowered inflammatory activation and colonic mucosal injury, but macroscopic gastric bleeding and increased methane output were present. Ket-Tris reduced inflammatory activation, methane emission and colonic mucosal damage, without inducing gastric injury. Conjugation with Tris reduces the GI side effects of Ket and still decreases the inflammatory response in experimental colitis. Methane output correlates with the mucosal inflammatory response and non-invasively demonstrates the effects of anti-inflammatory treatments

Preparative Separation and Identification of the Flavonoid Phlorhizin from the Crude Extract of Lithocarpus Polystachyus Rehd

The flavonoid phlorhizin is abundant in the leaves of Sweet Tea(ST, Lithocarpus Polystachyus Rehd). Phlorhizinwas preparatively separated and purified from a crude ST extract containing 40% total flavonoids by static adsorption and dynamic desorption on ADS-7 macroporous resin and neutral alumina column chromatography. Only water and ethanol were used as solvents and eluants throughout the whole separation and purification process. Using a phlorhizin standard as the reference compound, the target compound separated from the crude ST extracts was analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and electrosprayionization mass spectrometry (EIS-MS) and identified as 99.87% pure (by HPLC-UV) phlorhizin. The results showed that 10g of the targetcompound could be obtained from 40g of the crude extracts in a single operation, indicating a 40% recovery. Therefore, this represents an efficientand environmentally-friendly technology for separating and purifying phlorhizinfrom ST leaves

Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification

Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification out-performs relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs

Genetic Determinants of Circulating Sphingolipid Concentrations in European Populations

Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic beta-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08 x 10(-66). The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1-3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10(-4) or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be tested for a role in the development of common cardiovascular, metabolic, neurological, and psychiatric diseases