Comparative analysis of late functional outcome following preoperative radiation therapy or chemoradiotherapy and surgery or surgery alone in rectal cancer.

PURPOSE: This study evaluates the anorectal and genitourinary function of patients treated by preoperative short-term radiotherapy (RT) or chemoradiotherapy (CRT) followed by surgery and surgery alone for rectal cancer. METHODS: For this study, a total of 613 patients, who were identified from a prospective rectal cancer database, underwent anterior resection of the rectum between October 2001 and December 2007. Standardized questionnaires were used to determine fecal incontinence, urinary, and sexual function. Relevant clinical variables were evaluated using univariate and multivariate analyses. Independent predictors of functional outcome were identified by a binary logistic regression analysis. RESULTS: The data of 263 (43 %) patients were available for analysis. On multivariate analysis, neoadjuvant RT (P < 0.01) and low anterior resection (LAR) (P = 0.049) were associated with fecal incontinence. In univariate analysis, fecal incontinence was linked to preoperative neoadjuvant treatment (RT and/or CRT vs. LAR) (P < 0.01). The hazard ratio for developing fecal incontinence was 3.3 (1.6-6.8) for patients who received RT. One hundred twenty-five patients (51.2 %) experienced urinary incontinence following surgery, the majority of whom were female (P < 0.01). On univariate analysis, male sexual function was associated with age (P < 0.01), ASA class (P = 0.01) and LAR (P = 0.01). CONCLUSION: Multimodal therapy of low rectal cancer increases the incidence of fecal incontinence and negatively affects sexual function. The potential benefits of RT or CRT need to be balanced against the risk of increased bowel dysfunction when determining the appropriate treatment for individual patients with rectal cancer.

Development of an indole-based chemically cleavable linker concept for immobilizing bait compounds for protein pull-down experiments.

A novel linker chemistry based on a malondialdehyde-indole condensation reaction has been developed for the affinity-independent elution of targeted protein pull-downs. Previously developed in our lab for the tagging of tryptophan residues on proteins or peptides, the concept was extended for the design of a chemically cleavable linker system. Target molecules for interaction studies are immobilized on a solid support including the linker scaffold, and a typical pull-down experiment is carried out. After purification, the linker is cleaved by incubation with 50 mM pyrrolidine. A specific tyrosine kinase inhibitor, bosutinib, was coupled to agarose and acrylamide beads, respectively, via the new linker system, and a protein pull-down experiment of putative interaction partners from a K562 whole cell lysate was performed. The system was found to be compatible with targeted protein pull-downs; during the cleavage step, no protein hydrolysis or any degradation of amino acid side-chains was apparent. From the pull-down experiment, key targets of bosutinib such as the tyrosine kinase, Btk, were identified by liquid chromatography-tandem mass spectrometry.

miRanalyzer: a microRNA detection and analysis tool for next-generation sequencing experiments.

Next-generation sequencing allows now the sequencing of small RNA molecules and the estimation of their expression levels. Consequently, there will be a high demand of bioinformatics tools to cope with the several gigabytes of sequence data generated in each single deep-sequencing experiment. Given this scene, we developed miRanalyzer, a web server tool for the analysis of deep-sequencing experiments for small RNAs. The web server tool requires a simple input file containing a list of unique reads and its copy numbers (expression levels). Using these data, miRanalyzer (i) detects all known microRNA sequences annotated in miRBase, (ii) finds all perfect matches against other libraries of transcribed sequences and (iii) predicts new microRNAs. The prediction of new microRNAs is an especially important point as there are many species with very few known microRNAs. Therefore, we implemented a highly accurate machine learning algorithm for the prediction of new microRNAs that reaches AUC values of 97.9% and recall values of up to 75% on unseen data. The web tool summarizes all the described steps in a single output page, which provides a comprehensive overview of the analysis, adding links to more detailed output pages for each analysis module. miRanalyzer is available at http://web.bioinformatics.cicbiogune.es/microRNA/.

TargetSpy: a supervised machine learning approach for microRNA target prediction.

BACKGROUND: Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. RESULTS: We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences.In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. CONCLUSION: Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila, suggesting that it may be applicable to a broad range of species. Moreover, we have demonstrated that the application of machine learning techniques in combination with upcoming deep sequencing data results in a powerful microRNA target site prediction tool http://www.targetspy.org.

Probing the phosphoproteome of HeLa cells using nanocast metal oxide microspheres for phosphopeptide enrichment.

Metal oxide affinity chromatography (MOAC) has become a prominent method to enrich phosphopeptides prior to their analysis by liquid chromatography-mass spectrometry. To overcome limitations in material design, we have previously reported the use of nanocasting as a means to generate metal oxide spheres with tailored properties. Here, we report on the application of two oxides, tin dioxide (stannia) and titanium dioxide (titania), for the analysis of the HeLa phosphoproteome. In combination with nanoflow LC-MS/MS analysis on a linear ion trap-Fourier transform ion cyclotron resonance instrument, we identified 619 phosphopeptides using the new stannia material, and 896 phosphopeptides using titania prepared in house. We also compared the newly developed materials to commercial titania material using an established enrichment protocol. Both titania materials yielded a comparable total number of phosphopeptides, but the overlap of the two data sets was less than one-third. Although fewer peptides were identified using stannia, the complementarity of SnO(2)-based MOAC could be shown as more than 140 phosphopeptides were exclusively identified by this material.

Analysis of quaternary ammonium and phosphonium ionic liquids by reversed-phase high-performance liquid chromatography with charged aerosol detection and unified calibration.

Several hydrophobic ionic liquids (ILs) based on long-chain aliphatic ammonium- and phosphonium cations and selected aromatic anions were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) employing trifluoroacetic acid as ion-pairing additive to the acetonitrile-containing mobile phase and adopting a step-gradient elution mode. The coupling of charged aerosol detection (CAD) for the non-chromophoric aliphatic cations with diode array detection (DAD) for the aromatic anions allowed their simultaneous analysis in a set of new ILs derived from either tricaprylmethylammonium chloride (Aliquat 336) and trihexyltetradecylphosphonium chloride as precursors. Aliquat 336 is a mix of ammonium cations with distinct aliphatic chain lengths. In the course of the studies it turned out that CAD generates an identical detection response for all the distinct aliphatic cations. Due to lack of single component standards of the individual Aliquat 336 cation species, a unified calibration function was established for the quantitative analysis of the quaternary ammonium cations of the ILs. The developed method was validated according to ICH guidelines, which confirmed the validity of the unified calibration. The application of the method revealed molar ratios of cation to anion close to 1 indicating a quantitative exchange of the chloride ions of the precursors by the various aromatic anions in the course of the synthesis of new ILs. Anomalies of CAD observed for the detection of some aromatic anions (thiosalicylate and benzoate) are discussed.

Tools for analyzing the phosphoproteome and other phosphorylated biomolecules: a review.

Enrichment, separation and mass spectrometric analysis of biomolecules carrying a phosphate group plays an important role in current analytical chemistry. Application areas range from the preparative enrichment of phospholipids for biotechnological purposes and the separation and purification of plasmid DNA or mRNA to the specific preconcentration of phosphoproteins and -peptides to facilitate their later identification and characterization by mass spectrometry. Most of the recent improvements in this field were triggered by the need for phosphopeptide enrichment technology for the analysis of cellular protein phosphorylation events with the help of liquid chromatography-mass spectrometry. The high sensitivity of mass spectrometry and the possibility to combine this technique with different separation modes in liquid chromatography have made it the method of choice for proteome analysis. However, in the case of phosphoprotein analysis, the low abundance of the resulting phosphopeptides and their low quality fragment spectra interfere with the identification of phosphorylation events. Recent developments in phosphopeptide enrichment and fragmentation technologies successfully helped to overcome these limitations. In this review, we will focus on sample preparation techniques in the field of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules.

Optimizing the performance of tin dioxide microspheres for phosphopeptide enrichment.

Phosphopeptide enrichment based on metal oxide affinity chromatography is one of the most powerful tools for studying protein phosphorylation on a large scale. To complement existing metal oxide sorbents, we have recently introduced tin dioxide as a promising alternative. The preparation of SnO(2) microspheres by the nanocasting technique, using silica of different morphology as a template, offers a strategy to prepare materials that vary in their particle size and their porosity. Here, we demonstrate how such stannia materials can be successfully generated and their properties fine-tuned in order to obtain an optimized phosphopeptide enrichment material. We combined data from liquid chromatography-mass spectrometry experiments and physicochemical characterization, including nitrogen physisorption and energy-dispersive X-ray spectroscopy (EDX), to explain the influence of the various experimental parameters.

Abnormal antinuclear antibody titers are less common than generally assumed in established cases of systemic lupus erythematosus.

OBJECTIVE: To evaluate antinuclear antibody (ANA) tests in established cases of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) by indirect immunofluorescence microscopy (F-ANA) and enzyme-immunoassays detecting antinucleosomal antibodies (ANSA-EIA). METHODS: Sera from 50 patients with SLE and 65 patients with RA were analyzed regarding abnormal concentrations of F-ANA (serum dilution>or=1:200=95th percentile among 300 healthy blood donors). The sera were also analyzed with 2 commercial ANSA-EIA kits. RESULTS: An abnormal F-ANA titer occurred in 76% of the SLE sera compared to 23% in RA, and was not related to present use of antirheumatic drugs. At dilution 1:50, 84% of the SLE sera were F-ANA-positive compared to 20% of healthy women. Forty percent and 56%, respectively, of the SLE sera tested positive in the 2 ANSA-EIA kits. By the most sensitive assay, 96% of the ANSA-positive SLE sera produced a homogenous (chromosomal) F-ANA staining pattern compared to 18% of the ANSA-negative SLE sera. Ten of the 15 F-ANA-positive RA sera (63%) generated homogenous F-ANA staining and 13 (20%) tested positive in the most sensitive ANSA-EIA, but with no correlation to the F-ANA staining pattern. CONCLUSION: The sensitivity of F-ANA at an abnormal titer was surprisingly low (76%) in established cases of SLE. ANSA occurred in 56% of the SLE sera, but also in a fair number (20%) of RA sera. Practically all ANSA-positive SLE sera were identified by chromosomal F-ANA staining. We conclude that the antigen-specific antinucleosomal EIA does not have high enough diagnostic specificity to justify use of this analysis for routine diagnostic purposes.