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Molecular interactions are key drivers of biological function. Providing interaction resources to the research community is important since they allow functional interpretation and network-based analysis of molecular data. ConsensusPathDB (http://consensuspathdb.org) is a meta-database combining interactions of diverse types from 31 public resources for humans, 16 for mice and 14 for yeasts. Using ConsensusPathDB, researchers commonly evaluate lists of genes, proteins and metabolites against sets of molecular interactions defined by pathways, Gene Ontology and network neighborhoods and retrieve complex molecular neighborhoods formed by heterogeneous interaction types. Furthermore, the integrated protein–protein interaction network is used as a basis for propagation methods. Here, we present the 2022 update of ConsensusPathDB, highlighting content growth, additional functionality and improved database stability. For example, the number of human molecular interactions increased to 859 848 connecting 200 499 unique physical entities such as genes/proteins, metabolites and drugs. Furthermore, we integrated regulatory datasets in the form of transcription factor–, microRNA– and enhancer–gene target interactions, thus providing novel functionality in the context of overrepresentation and enrichment analyses. We specifically emphasize the use of the integrated protein–protein interaction network as a scaffold for network inferences, present topological characteristics of the network and discuss strengths and shortcomings of such approaches.

Many methods for haplotyping have materialized, but their application on a significant scale has been rare to date. Here we summarize analyses that were carried out in 1092 genomes from the 1000 Genomes Consortium and validated in an unprecedented number of 184 PGP genomes that have been experimentally haplotype-resolved by application of the Long-Fragment Read (LFR) technology. These analyses provided first insights into the diplotypic nature of human genomes and its potential functional implications. Thus, protein-changing variants were not randomly distributed between the two homologues of 18,121 autosomal protein-coding genes but occurred significantly more frequently in cis than in trans configurations in virtually each of the 1276 phased genomes. This resulted in global cis/trans ratios of ~60:40, establishing "cis abundance" as a universal characteristic of diploid human genomes. This phenomenon was based on two different classes of genes, a larger one exhibiting cis configurations of protein-changing variants in excess, so-called "cis-abundant" genes, and a smaller one of "trans-abundant" genes. These two gene classes, which together constitute a common diplotypic exome, were further functionally distinguished by means of gene ontology (GO) and pathway enrichment analysis. Moreover, they were distinguishable in terms of their effects on the human interactome, where they constitute distinct cis and trans modules, as shown with network propagation on a large integrated protein-protein interaction network. These analyses, recently performed with updated database and analysis tools, further consolidated the characterization of cis- and trans-abundant genes while expanding previous results. In this chapter, we present the key results along with the materials and methods to motivate readers to investigate these findings independently and gain further insights into the diplotypic nature of genes and genomes.

Background: We recently showed that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) has a solid antitumoral effect on the Jurkat cell line due to the fact of its antioxidative, caspase-3 and apoptosis activities, but no negative effect on human fibroblasts was obtained. The question arises how the single compounds, aKG and 5-HMF, affect peroxynitrite (ONOO−) and nitration of tyrosine residues, Jurkat cell proliferation and caspase-activated apoptosis. Methods: The ONOO− luminol-induced chemiluminescence reaction was used to measure the ONOO− scavenging function of aKG or 5-HMF, and their protection against nitration of tyrosine residues on bovine serum albumin was estimated with the ELISA technique. The Jurkat cell line was cultivated in the absence or presence of aKG or 5-HMF solutions between 0 and 3.5 µM aKG or 0 and 4 µM 5-HMF. Jurkat cells were tested for cell proliferation, mitochondrial activity and caspase-activated apoptosis. Results: aKG showed a concentration-dependent reduction in ONOO−, resulting in a 90% elimination of ONOO− using 200 mM aKG. In addition, 20 and 200 mM 5-HMF were able to reduce ONOO− only by 20%, while lower concentrations of 5-HMF remained stable in the presence of ONOO−. Nitration of tyrosine residues was inhibited 4 fold more effectively with 5-HMF compared to aKG measuring the IC50%. Both substances, aKG and 5-HMF, were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time. The aKG effectively reduced Jurkat cell growth down to 50% after 48 and 72 h of incubation using the highest concentration of 3.5 µM, and 1, 1.6, 2, 3 and 4 µM 5-HMF inhibited any cell growth within (i) 24 h; 1.6, 2, 3 and 4 µM 5-HMF within 48 h (ii); 2, 3 and 4 µM 5-HMF within 72 h (iii). Furthermore, 4 µM was able to eliminate the starting cell number of 20,000 cells after 48 and 72 h down to 11,233 cells. The mitochondrial activity measurements supported the data on aKG or 5-HMF regarding cell growth in Jurkat cells, in both a dose- and incubation-time-dependent manner: the highest concentration of 3.5 µM aKG reduced the mitochondrial activity over 24 h (67.7%), 48 h (57.9%) and 72 h (46.8%) of incubation with Jurkat cells compared to the control incubation without aKG (100%). 5-HMF was more effective compared to aKG; the mitochondrial activity in the presence of 4 µM 5-HMF decreased after 24 h down to 68.4%, after 48 h to 42.9% and after 72 h to 32.0%. Moreover, 1.7 and 3.4 µM aKG had no effect on caspase-3-activated apoptosis (0.58% and 0.56%) in the Jurkat cell line. However, 2 and 4 µM 5-HMF increased the caspase-3-activated apoptosis up to 22.1% and 42.5% compared to the control (2.9%). A combined solution of 1.7 µM aKG + 0.7 µM 5-HMF showed a higher caspase-3-activated apoptosis (15.7%) compared to 1.7 µM aKG or 2 µM 5-HMF alone. In addition, 3.5 µM µg/mL aKG + 1.7 µM 5-HMF induced caspase-activated apoptosis up to 55.6% compared to 4.5% or 35.6% caspase-3 activity using 3.5 µM aKG or 4 µM 5-HMF. Conclusion: Both substances showed high antioxidative potential in eliminating either peroxynitrite or nitration of tyrosine residues, which results in a better inhibition of cell growth and mitochondrial activity of 5-HMF compared to aKG. However, caspase-3-activated apoptosis measurements revealed that the combination of both substances synergistically is the most effective compared to single compounds.

Background: Epirubicin (EPI) is an important anticancer drug that is well-known for its cardiotoxic side effect. Studying epigenetic modification such as DNA methylation can help to understand the EPI-related toxic mechanisms in cardiac tissue. In this study, we analyzed the DNA methylation profile in a relevant human cell model and inspected the expression of differentially methylated genes at the transcriptome level to understand how changes in DNA methylation could affect gene expression in relation to EPI-induced cardiotoxicity. Methods: Human cardiac microtissues were exposed to either therapeutic or toxic (IC20) EPI doses during 2 weeks. The DNA and RNA were collected from microtissues in triplicates at 2, 8, 24, 72, 168, 240, and 336 hours of exposure. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) analysis was used to detect DNA methylation levels in EPI-treated and control samples. The MeDIP-seq data were analyzed and processed using the QSEA package with a recently published workflow. RNA sequencing (RNA-seq) was used to measure global gene expression in the same samples. Results: After processing the MeDIP-seq data, we detected 35, 37, 15 candidate genes which show strong methylated alterations between all EPI-treated, EPI therapeutic and EPI toxic dose-treated samples compared to control, respectively. For several genes, gene expressions changed compatibly reflecting the DNA methylation regulation. Conclusions: The observed DNA methylation modifications provide further insights into the EPI-induced cardiotoxicity. Multiple differentially methylated genes under EPI treatment, such as SMARCA4, PKN1, RGS12, DPP9, NCOR2, SDHA, POLR2A, and AGPAT3, have been implicated in different cardiac dysfunction mechanisms. Together with other differentially methylated genes, these genes can be candidates for further investigations of EPI-related toxic mechanisms. Data Repository: The data has been generated by the HeCaToS project (http://www.ebi.ac.uk/biostudies) under accession numbers S-HECA433 and S-HECA434 for the MeDIP-seq data and S-HECA11 for the RNA-seq data. The R code is available on Github (https://github.com/NhanNguyen000/MeDIP).

Cancer survival prediction is typically done with uninterpretable machine learning techniques, e.g., gradient tree boosting. Therefore, additional steps are needed to infer biological plausibility of the predictions. Here, we describe a protocol that combines pan-cancer survival prediction with XGBoost tree- ensemble learning and subsequent propagation of the learned feature weights on protein interaction networks. This protocol is based on TCGA transcriptome data of 8,024 patients from 25 cancer types but can easily be adapted to cancer patient data from other sources. For complete details on the use and execution of this protocol, please refer to Thedinga and Herwig (2022).

To nominate novel disease genes for obesity and type 2 diabetes (T2D), we recently generated two mouse backcross populations of the T2D-susceptible New Zealand Obese (NZO/HI) mouse strain and two genetically different, lean and T2D-resistant strains, 129P2/OlaHsd and C3HeB/FeJ. Comparative linkage analysis of our two female backcross populations identified seven novel body fat-associated quantitative trait loci (QTL). Only the locus Nbw14 (NZO body weight on chromosome 14) showed linkage to obesity-related traits in both backcross populations, indicating that the causal gene variant is likely specific for the NZO strain as NZO allele carriers in both crosses displayed elevated body weight and fat mass. To identify candidate genes for Nbw14, we used a combined approach of gene expression and haplotype analysis to filter for NZO-specific gene variants in gonadal white adipose tissue, defined as the main QTL-target tissue. Only two genes, Arl11 and Sgcg, fulfilled our candidate criteria. In addition, expression QTL analysis revealed cis-signals for both genes within the Nbw14 locus. Moreover, retroviral overexpression of Sgcg in 3T3-L1 adipocytes resulted in increased insulin-stimulated glucose uptake. In humans, mRNA levels of SGCG correlated with body mass index and body fat mass exclusively in diabetic subjects, suggesting that SGCG may present a novel marker for metabolically unhealthy obesity. In conclusion, our comparative-cross analysis could substantially improve the mapping resolution of the obesity locus Nbw14. Future studies will throw light on the mechanism by which Sgcg may protect from the development of obesity.

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL), patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.

Background There is a huge body of scientific literature describing the relation between tumor types and anti-cancer drugs. The vast amount of scientific literature makes it impossible for researchers and physicians to extract all relevant information manually. Methods In order to cope with the large amount of literature we applied an automated text mining approach to assess the relations between 30 most frequent cancer types and 270 anti-cancer drugs. We applied two different approaches, a classical text mining based on named entity recognition and an AI-based approach employing word embeddings. The consistency of literature mining results was validated with 3 independent methods: first, using data from FDA approvals, second, using experimentally measured IC-50 cell line data and third, using clinical patient survival data. Results We demonstrated that the automated text mining was able to successfully assess the relation between cancer types and anti-cancer drugs. All validation methods showed a good correspondence between the results from literature mining and independent confirmatory approaches. The relation between most frequent cancer types and drugs employed for their treatment were visualized in a large heatmap. All results are accessible in an interactive web-based knowledge base using the following link: https://knowledgebase.microdiscovery.de/heatmap. Conclusions Our approach is able to assess the relations between compounds and cancer types in an automated manner. Both, cancer types and compounds could be grouped into different clusters. Researchers can use the interactive knowledge base to inspect the presented results and follow their own research questions, for example the identification of novel indication areas for known drugs.

Current molecular tumor diagnostics encompass panel sequencing to detect mutations, copy number alterations, and rearrangements. However, tumor suppressor genes can also be inactivated by methylation within their promoter region. These epigenetic alterations are so far rarely assessed in the clinical setting. Therefore, we established the AllCap protocol facilitating the combined detection of mutations and DNA methylation at the coding and promoter regions of 342 DNA repair genes in one experiment. We demonstrate the use of the protocol by applying it to ovarian cancer cell lines with different responsiveness to poly(ADP-ribose) polymerase inhibition. BRCA1, ATM, ATR, and EP300 mutations and methylation of the BRCA1 promoter were detected as potential predictors for therapy response. The required amount of input DNA was optimized, and the application to formalin-fixed, paraffin-embedded tissue samples was verified to improve the clinical applicability. Thus, by adding DNA methylation values to panel resequencings, the AllCap assay will add another important level of information to clinical tests and will improve stratification of patients for systemic therapies.

To fully understand human genetic variation and its functional consequences, the specific distribution of variants between the two chromosomal homologues of genes must be known. The ‘phase’ of variants can significantly impact gene function and phenotype. To assess patterns of phase at large scale, we have analyzed 18 121 autosomal genes in 1092 statistically phased genomes from the 1000 Genomes Project and 184 experimentally phased genomes from the Personal Genome Project. Here we show that genes with cis-configurations of coding variants are more frequent than genes with trans-configurations in a genome, with global cis/trans ratios of ∼60:40. Significant cis-abundance was observed in virtually all genomes in all populations. Moreover, we identified a large group of genes exhibiting cis-configurations of protein-changing variants in excess, so-called ‘cis-abundant genes’, and a smaller group of ‘trans-abundant genes’. These two gene categories were functionally distinguishable, and exhibited strikingly different distributional patterns of protein-changing variants. Underlying these phenomena was a shared set of phase-sensitive genes of importance for adaptation and evolution. This work establishes common patterns of phase as key characteristics of diploid human exomes and provides evidence for their functional significance, highlighting the importance of phase for the interpretation of protein-coding genetic variation and gene function.

Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data., Using a network propagation approach with integrated multi-omic data, Selevsek et al. develop a reproducible workflow for identifying drug toxicity effects in cellular systems. This is demonstrated with the analysis of anthracycline cardiotoxicity in cardiac microtissues under the effect of multiple drugs.

To identify novel disease genes for type 2 diabetes (T2D) we generated two backcross populations of obese and diabetes-susceptible New Zealand Obese (NZO/HI) mice with the two lean mouse strains 129P2/OlaHsd and C3HeB/FeJ. Subsequent whole-genome linkage scans revealed 30 novel quantitative trait loci (QTL) for T2D-associated traits. The strongest association with blood glucose [12 cM, logarithm of the odds (LOD) 13.3] and plasma insulin (17 cM, LOD 4.8) was detected on proximal chromosome 7 (designated Nbg7p, NZO blood glucose on proximal chromosome 7) exclusively in the NZOxC3H crossbreeding, suggesting that the causal gene is contributed by the C3H genome. Introgression of the critical C3H fragment into the genetic NZO background by generating recombinant congenic strains and metabolic phenotyping validated the phenotype. For the detection of candidate genes in the critical region (30–46 Mb), we used a combined approach of haplotype and gene expression analysis to search for C3H-specific gene variants in the pancreatic islets, which appeared to be the most likely target tissue for the QTL. Two genes, Atp4a and Pop4, fulfilled the criteria from our candidate gene approaches. The knockdown of both genes in MIN6 cells led to decreased glucose-stimulated insulin secretion, indicating a regulatory role of both genes in insulin secretion, thereby possibly contributing to the phenotype linked to Nbg7p. In conclusion, our combined- and comparative-cross analysis approach has successfully led to the identification of two novel diabetes susceptibility candidate genes, and thus has been proven to be a valuable tool for the discovery of novel disease genes.

Though clinical trials for medical applications of dimethyl sulfoxide (DMSO) reported toxicity in the 1960s, later, the FDA classified DMSO in the safest solvent category. DMSO became widely used in many biomedical fields and biological effects were overlooked. Meanwhile, biomedical science has evolved towards sensitive high-throughput techniques and new research areas, including epigenomics and microRNAs. Considering its wide use, especially for cryopreservation and in vitro assays, we evaluated biological effect of DMSO using these technological innovations. We exposed 3D cardiac and hepatic microtissues to medium with or without 0.1% DMSO and analyzed the transcriptome, proteome and DNA methylation profiles. In both tissue types, transcriptome analysis detected >2000 differentially expressed genes affecting similar biological processes, thereby indicating consistent cross-organ actions of DMSO. Furthermore, microRNA analysis revealed large-scale deregulations of cardiac microRNAs and smaller, though still massive, effects in hepatic microtissues. Genome-wide methylation patterns also revealed tissue-specificity. While hepatic microtissues demonstrated non-significant changes, findings from cardiac microtissues suggested disruption of DNA methylation mechanisms leading to genome-wide changes. The extreme changes in microRNAs and alterations in the epigenetic landscape indicate that DMSO is not inert. Its use should be reconsidered, especially for cryopreservation of embryos and oocytes, since it may impact embryonic development.

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence.

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

Deletions at the C-terminus of the proto-oncogene protein c-Src kinase are found in the viral oncogene protein v-Src as well as in some advanced human colon cancers. They are associated with increased kinase activity and cellular invasiveness. Here, we analyzed the mRNA expression signature of a constitutively active C-terminal mutant of c-Src, c-Src(mt), in comparison with its wild-type protein, c-Src(wt), in the human non-transformed breast epithelial cell line MCF-10A. We demonstrated previously that the mutant altered migratory and metastatic properties. Genome-wide transcriptome analysis revealed that c-Src(mt) de-regulated the expression levels of approximately 430 mRNAs whose gene products are mainly involved in the cellular processes of migration and adhesion, apoptosis and protein synthesis. 82.9% of these genes have previously been linked to cellular migration, while the others play roles in RNA transport and splicing processes, for instance. Consistent with the transcriptome data, cells expressing c-Src(mt), but not those expressing c-Src(wt), showed the capacity to metastasize into the lungs of mice in vivo. The mRNA expression profile of c-Src(mt)-expressing cells shows significant overlap with that of various primary human tumor samples, possibly reflecting elevated Src activity in some cancerous cells. Expression of c-Src(mt) led to elevated migratory potential. We used this model system to analyze the transcriptional changes associated with an invasive cellular phenotype. These genes and pathways de-regulated by c-Src(mt) may provide suitable biomarkers or targets of therapeutic approaches for metastatic cells. DATABASE: This project was submitted to the National Center for Biotechnology Information BioProject under ID PRJNA288540. The Illumina RNA-Seq reads are available in the National Center for Biotechnology Information Sequence Read Archive under study ID SRP060008 with accession numbers SRS977414 for MCF-10A cells, SRS977717 for mock cells, SRS978053 for c-Src(wt) cells and SRS978046 for c-Src(mt) cells.

We present NetCore, a novel network propagation approach based on node coreness, for phenotype–genotype associations and module identification. NetCore addresses the node degree bias in PPI networks by using node coreness in the random walk with restart procedure, and achieves improved re-ranking of genes after propagation. Furthermore, NetCore implements a semi-supervised approach to identify phenotype-associated network modules, which anchors the identification of novel candidate genes at known genes associated with the phenotype. We evaluated NetCore on gene sets from 11 different GWAS traits and showed improved performance compared to the standard degree-based network propagation using cross-validation. Furthermore, we applied NetCore to identify disease genes and modules for Schizophrenia GWAS data and pan-cancer mutation data. We compared the novel approach to existing network propagation approaches and showed the benefits of using NetCore in comparison to those. We provide an easy-to-use implementation, together with a high confidence PPI network extracted from ConsensusPathDB, which can be applied to various types of genomics data in order to obtain a re-ranking of genes and functionally relevant network modules.

[Background] Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide and is primarily treated with radiation, surgery, and platinum-based drugs like cisplatin and carboplatin. The major challenge in the treatment of NSCLC patients is intrinsic or acquired resistance to chemotherapy. Molecular markers predicting the outcome of the patients are urgently needed., [Methods] Here, we employed patient-derived xenografts (PDXs) to detect predictive methylation biomarkers for platin-based therapies. We used MeDIP-Seq to generate genome-wide DNA methylation profiles of 22 PDXs, their parental primary NSCLC, and their corresponding normal tissues and complemented the data with gene expression analyses of the same tissues. Candidate biomarkers were validated with quantitative methylation-specific PCRs (qMSP) in an independent cohort., [Results] Comprehensive analyses revealed that differential methylation patterns are highly similar, enriched in PDXs and lung tumor-specific when comparing differences in methylation between PDXs versus primary NSCLC. We identified a set of 40 candidate regions with methylation correlated to carboplatin response and corresponding inverse gene expression pattern even before therapy. This analysis led to the identification of a promoter CpG island methylation of LDL receptor-related protein 12 (LRP12) associated with increased resistance to carboplatin. Validation in an independent patient cohort (n = 35) confirmed that LRP12 methylation status is predictive for therapeutic response of NSCLC patients to platin therapy with a sensitivity of 80% and a specificity of 84% (p, [Conclusions] Using an epigenome-wide sequencing approach, we find differential methylation patterns from primary lung cancer and PDX-derived cancers to be very similar, albeit with a lower degree of differential methylation in primary tumors. We identify LRP12 DNA methylation as a powerful predictive marker for carboplatin resistance. These findings outline a platform for the identification of epigenetic therapy resistance biomarkers based on PDX NSCLC models., This work was supported by the Volkswagenstiftung (Lichtenberg program to MRS), the German Federal Ministry of Education and Research (0316190A (EPITREAT), the Center for Molecular Medicine Cologne (CMMC), the state of North Rhine-Westphalia (EFRE-0800397), and the German Research Foundation (DFG KFO286).

For more than a decade pharmaceutical R&D has been hampered by considerable attrition rates during clinical trials. The main reasons for drug failure is related to the lack of efficacy, limitations with respect to ADME (absorption, distribution, metabolism and excretion) properties, and—in approximately 30% of the cases—unforeseen toxicity (Kola and Landis 2004). The majority of adverse drug reactions observed in the clinical phase refer to organ injuries, e.g. of the cardiovascular system, the liver, the central nervous system and skeletal muscle (Cook et al. 2014). This clearly demonstrates the limited predictive accuracy of current preclinical models such as the rodent bioassay in evaluating repeated dose toxicity for predicting human toxic risks. It has been argued that overall, only 43% of toxic effects in humans may be correctly predicted by applying rodent-based safety evaluation protocols due to the fact that these assays tend to generate relatively large numbers of false negative as well as false positive read outs (Hartung 2009). ISSN:0340-5761 ISSN:1432-0738

Objective: We evaluated the effectiveness of pelvic vein embolization with aethoxysclerol in aero-block technique for the treatment of impotence due to venous leakage in men using sildenafil for intercourse. The aim of the procedure was to reduce the use of sildenafil. Methods: A total of 96 patients with veno-occlusive dysfunction, severe enough for the need of PDE5 inhibitors for vaginal penetration, underwent pelvic venoablation with aethoxysclerol. The mean patient age was 53.5 years. Venous leaks were identified by Color Doppler Ultrasound after intracavernous alprostadil injection. Under local anesthesia a 20-gauge needle was inserted into the deep dorsal penile vein. The pelvic venogram was obtained through deep dorsal venography. Aethoxysclerol 3% as sclerosing agent was injected after air-block under Valsalva manoeuver. Success was defined as the ability to achieve vaginal insertion without the aid of any drugs, vasoactive injections, penile prosthesis, or vacuum device. Additionally, a pre- and post- therapy IIEF score and a digital overnight spontaneous erections protocol (OSEP) with the NEVA™-system was performed. Results: At 3 month follow-up 77 out of 96 patients (80.21%) reported to have erections sufficient for vaginal insertion without the use of any drug or additional device. Four (4.17%) patients did not report any improvement. Follow up with color Doppler ultrasound revealed a new or persistent venous leakage in 8 (8.33%) of the patients. No serious complications occurred. Conclusions: Our new pelvic venoablation technique using aethoxysclerol in air-block technique was effective, minimally invasive, and cost-effective. All patients were able to perform sexual intercourse without the previously used dosage of PDE5 inhibitor. This new method may help in patients with contra-indications against PDE5 inhibitors, in patients who cannot afford the frequent usage of expensive oral medication or those who do not fully respond to PDE5-inhibitors.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab., The heterogeneity of colorectal cancer has important clinical and therapeutic implications. Here the authors analysed the responses of a large biobank of organoids and xenografts derived from colorectal patients to a panel of clinically relevant therapeutic agents to identify genes signatures associated with drug response.

We evaluated a dual energy, split bolus computerized tomography protocol that provides virtual noncontrast, parenchymal and urographic phases in a single scan. We assessed the sensitivity of the virtual noncontrast phase using this protocol to detect urinary stones compared to the gold standard of the true noncontrast phase.We prospectively enrolled in the study 81 patients who underwent unenhanced single energy computerized tomography at 120 kV/200 mA as well as contrast enhanced dual energy computerized tomography on a Somatom® Definition Flash-CT (tube A 80 kV/233 mA and tube B SN 140 kV/180 mA with 1/0.8 mm slice thickness). For the split bolus protocol 400 mg/ml Iomeron® were injected at 2 time points, that is 15 ml 10 minutes before the scan and 80 ml 65 seconds before the scan. In a consensus reading 2 readers evaluated the presence and diameter of stones on the true and virtual noncontrast phases.Of the 350 stones noted on the true noncontrast phase we found 289 on the virtual noncontrast phase as well as 13 false-positive and 66 false-negative stones. Sensitivity was 98.4%, 89.8% and 82.6% per patient, segment and stone, respectively. The diameter measured on the virtual noncontrast phase corresponded to a mean ± SD 92.5% ± 31.6% of the diameter on the true noncontrast phase. The mean effective dose was 4.8 ± 1.8 and 10.5 ± 3.7 mSv for the true and virtual noncontrast phases, respectively.The proposed protocol allows for combining 3 phases in a single scan while still enabling the detection of urinary stones at high sensitivity. This technique halves the radiation dose and provides the surgeon with better anatomical information on the calyceal system. Therefore, it is a valuable diagnostic tool for kidney stone treatment planning and followup.

Current testing models for predicting drug-induced liver injury are inadequate, as they basically under-report human health risks. We present here an approach towards developing pathways based on hepatotoxicity-associated gene groups derived from two types of publicly accessible hepatotoxicity databases, in order to develop drug-induced liver injury biomarker profiles. One human liver ‘omics-based and four text-mining-based databases were explored for hepatotoxicity-associated gene lists. Over-representation analysis of these gene lists with a hepatotoxicant-exposed primary human hepatocytes data set showed that human liver ‘omics gene lists performed better than text-mining gene lists and the results of the latter differed strongly between databases. However, both types of databases contained gene lists demonstrating biomarker potential. Visualizing those in pathway format may aid in interpreting the biomolecular background. We conclude that exploiting existing and openly accessible databases in a dedicated manner seems promising in providing venues for translational research in toxicology and biomarker development.

Purpose: The Post-Ureteroscopic Lesion Scale (PULS) offers a simple grading system for the description of ureteral lesions after ureteroscopy. In this article, we present the results of a video-based multicenter evaluation of the inter-rater reliability of clinically important PULS grades 0-3. Methods: Video sequences at the end of ureteroscopy (final passage) were recorded for 100 consecutive patients at a single institution and assessed by experienced urologists (n = 20) and senior residents (n = 17) at 19 international centers. The cohort included only patients with lesions grades 0-3 (with grades 2 and 3 subsumed as 2 + since distinction is defined by an extravasation of contrast medium in fluoroscopy). The gradings were evaluated for inter-rater reliability and in terms of simplicity, validity, comprehensibility, reproducibility, and usefulness. Results: Overall, inter-rater reliability was high (Kendall's W = 0.69, p < 0.001) and was comparable between specialists (Kendall's W = 0.69, p < 0.001) and residents (Kendall's W = 0.71, p < 0.001). The matched ratings showed grade 0 in 43.0 % of patients and grades 1 or 2 + in 44.0 and 13.0 % of patients, respectively. Results of the questionnaires indicated a high degree of acceptance, with an overall rating of 1.76 (1.64-1.93 for different items, scale 1-6). Conclusions: Inter-rater reliability of the endoscopically assessable PULS was high among urologists with different levels of experience in different countries worldwide. The validated PULS system may be used for standardized reporting of ureteral lesions/injuries after ureteroscopy. In addition, PULS will enable more selective standardization of indications for postoperative DJ stenting based on the randomized controlled trials.

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein-protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.

When evaluating compound similarity, addressing multiple sources of information to reach conclusions about common pharmaceutical and/or toxicological mechanisms of action is a crucial strategy. In this chapter, we describe a systems biology approach that incorporates analyses of hepatotoxicant data for 33 compounds from three different sources: a chemical structure similarity analysis based on the 3D Tanimoto coefficient, a chemical structure-based protein target prediction analysis, and a cross-study/cross-platform meta-analysis of in vitro and in vivo human and rat transcriptomics data derived from public resources (i.e., the diXa data warehouse). Hierarchical clustering of the outcome scores of the separate analyses did not result in a satisfactory grouping of compounds considering their known toxic mechanism as described in literature. However, a combined analysis of multiple data types may hypothetically compensate for missing or unreliable information in any of the single data types. We therefore performed an integrated clustering analysis of all three data sets using the R-based tool iClusterPlus. This indeed improved the grouping results. The compound clusters that were formed by means of iClusterPlus represent groups that show similar gene expression while simultaneously integrating a similarity in structure and protein targets, which corresponds much better with the known mechanism of action of these toxicants. Using an integrative systems biology approach may thus overcome the limitations of the separate analyses when grouping liver toxicants sharing a similar mechanism of toxicity.

Motivation: Extensive drug treatment gene expression data have been generated in order to identify biomarkers that are predictive for toxicity or to classify compounds. However, such patterns are often highly variable across compounds and lack robustness. We and others have previously shown that supervised expression patterns based on pathway concepts rather than unsupervised patterns are more robust and can be used to assess toxicity for entire classes of drugs more reliably. Results: We have developed a database, ToxDB, for the analysis of the functional consequences of drug treatment at the pathway level. We have collected 2694 pathway concepts and computed numerical response scores of these pathways for 437 drugs and chemicals and 7464 different experimental conditions. ToxDB provides functionalities for exploring these pathway responses by offering tools for visualization and differential analysis allowing for comparisons of different treatment parameters and for linking this data with toxicity annotation and chemical information. Database URL: http://toxdb.molgen.mpg.de

Hepatocyte-like cells derived from the differentiation of human embryonic stem cells (hES-Hep) have potential to provide a human relevant in vitro test system in which to evaluate the carcinogenic hazard of chemicals. In this study, we have investigated this potential using a panel of 15 chemicals classified as noncarcinogens, genotoxic carcinogens, and nongenotoxic carcinogens and measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA model that identified 592 genes highly discriminative for the panel of chemicals. Supervised classification with these genes achieved a cross-validation accuracy of > 95%. Moreover, the expression of the response genes in hES-Hep was strongly correlated with that in human primary hepatocytes cultured in vitro. In order to infer mechanistic information on the consequences of chemical exposure in hES-Hep, we developed a computational method that measures the responses of biochemical pathways to the panel of treatments and showed that these responses were discriminative for the three toxicity classes and linked to carcinogenesis through p53, mitogen-activated protein kinases, and apoptosis pathway modules. It could further be shown that the discrimination of toxicity classes was improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, for the first time, the potential of human embryonic stem cell--derived hepatic cells as an in vitro model for hazard assessment of chemical carcinogenesis, although it should be noted that more compounds are needed to test the robustness of the assay.

OBJECTIVE Prostate cancer is still the most frequent noncutaneous male malignancy and is the second most common cause of cancer death. Genetic factors have been extensively studied in different countries. In addition, numerous genome–wide association studies have been performed in developed countries. Genetic tests will be applied in the near future for diagnosis, therapeutic, and prognostic significance. Therefore, we reviewed the association of several important pathways and genes with critical functions in prostate cancer development or progression. MATERIALS AND METHODS We performed a PubMed® search using several key words such as prostate cancer, names of important genes with critical function, and polymorphisms. Then, we reviewed retrieved articles as well as relevant articles from 1997 to 2009. RESULTS There are conflicting results of studies on some gene polymorphisms in association with prostate cancer. Most of the inconsistent results have been reported in studies investigating the vitamin D receptor gene polymorphism in association with prostate cancer. Genes related to angiogenesis and cell adhesion genes are more promising. Following results of future studies, the use of antibodies blocking over-expressed genes or proteins may be supported in patients with prostate cancer. CONCLUSIONS The difference between the results of studies on gene polymorphisms in prostate cancer may be explained partly by ethnic differences, limited sample size, and other risk or protective factors modifying these effects. Genome-wide studies are currently performed in developed countries and extensive use of this type of analysis may merit consideration in other countries. Furthermore, future studies are needed to further investigate environmental and diet factors interactions with genetic factors.

OBJECTIVE To analyse the morphological appearance of horseshoe kidneys (HKs) and crossed fused ectopia (CFE) and to assess the frequency and clinical significance of associated anomalies and diseases. PATIENTS AND METHODS The findings and images of 209 patients with fused kidneys (FKs) were reviewed; in all, 244 scans from computed tomography (CT), 233 ultrasonograms and 89 micturition cysto-urethrograms, urograms, magnetic resonance images and angiograms were taken. RESULTS HKs (found in one of 474 abdominal CT scans) and CFEs (found in one of 3078 CT scans) showed a high variability of vasculature that could not be classified. However, some generalized conclusions were possible about the renal vasculature (430 arteries in 103 kidneys). Variants of the most cephalad artery of both sides were rare. The second artery on the right had a pre-caval course. The origins of vessels located further caudal were more ventral. CFEs were anatomically different from HKs with respect to lower position, greater axial rotation, smaller pelvic width, more caudal origin, and fewer vessels, but not in accompanying anomalies. Severe anomalies or malformations were found in 23% of patients, with half of them in the urogenital system. Malformations were found considerably more often in children than in adults. There was no increased incidence of diseases such as stones or inflammation of the renal pelvis. CONCLUSION Concomitant anomalies and diseases were equally frequent for HK and CFE, but less frequent than generally assumed. Individual cases of complex anatomical situations require special examination strategies, and CT appears to be the most reliable imaging method.

ConsensusPathDB is a database system for the integration of human functional interactions. Current knowledge of these interactions is dispersed in more than 200 databases, each having a specific focus and data format. ConsensusPathDB currently integrates the content of 12 different interaction databases with heterogeneous foci comprising a total of 26,133 distinct physical entities and 74,289 distinct functional interactions (protein-protein interactions, biochemical reactions, gene regulatory interactions), and covering 1738 pathways. We describe the database schema and the methods used for data integration. Furthermore, we describe the functionality of the ConsensusPathDB web interface, where users can search and visualize interaction networks, upload, modify and expand networks in BioPAX, SBML or PSI-MI format, or carry out over-representation analysis with uploaded identifier lists with respect to substructures derived from the integrated interaction network. The ConsensusPathDB database is available at: http://cpdb.molgen.mpg.de.

OBJECTIVE To investigate whether a mechanism of action of α-blockers on lower urinary tract symptoms (LUTS) involves improved perfusion of the LUT. PATIENTS, SUBJECTS AND METHODS The accuracy of perfusion measurements using transrectal colour Doppler ultrasound (TRCDUS) and colour pixel density (CPD) was initially confirmed in a porcine model. Following this confirmation, measurements were taken from four healthy male volunteers and 19 patients with LUTS. The urinary bladder was filled slowly (50 mL/min) with 0.2 m KCl, which resembles the osmolarity of concentrated urine, and evaluated by cystometry. In parallel, TRCDUS and measurement of the CPD of the LUT were performed. The patients with LUTS were then treated with daily α-blocker (0.4 mg tamsulosin) for 5 weeks and urodynamic variables as well as perfusion were evaluated again. RESULTS In the healthy men, perfusion of the LUT increased considerably (157%) during filling of the bladder to a mean (sd) maximum cystometric capacity (Cmax) of 481 (28.9) mL. All the patients with LUTS had a reduced mean Cmax during filling with KCl at 322.4 (58.5) mL. The mean CPD in the urinary bladder and the prostate were only increased by 58.4% during filling with KCl. After α-blocker therapy the mean Cmax during filling with KCl rose to 382.5 (42.9) mL; furthermore, perfusion of the LUT measured by CPD was significantly increased (132.8%). CONCLUSIONS The present data strongly suggest that LUTS are associated with chronic ischaemia of the prostate and urinary bladder. α-blockers increase perfusion in the LUT and Cmax. These results might explain the therapeutic effects of α-blockers on LUTS.

The present study investigated mRNA expression profiles of bovine oocytes and blastocysts by using a cross-species hybridization approach employing an array consisting of 15,529 human cDNAs as probe, thus enabling the identification of conserved genes during human and bovine preimplantation development. Our analysis revealed 419 genes that were expressed in both oocytes and blastocysts. The expression of 1,324 genes was detected exclusively in the blastocyst, in contrast to 164 in the oocyte including a significant number of novel genes. Genes indicative for transcriptional and translational control ( ELAVL4, TACC3) were overexpressed in the oocyte, whereas cellular trafficking ( SLC2A14, SLC1A3), proteasome ( PSMA1, PSMB3), cell cycle ( BUB3, CCNE1, GSPT1), and protein modification and turnover ( TNK1, UBE3A) genes were found to be overexpressed in blastocysts. Transcripts implicated in chromatin remodeling were found in both oocytes ( NASP, SMARCA2) and blastocysts ( H2AFY, HDAC7A). The trophectodermal markers PSG2 and KRT18 were enriched 5- and 50-fold in the blastocyst. Pathway analysis revealed differential expression of genes involved in 107 distinct signaling and metabolic pathways. For example, phosphatidylinositol signaling and gluconeogenesis were prominent pathways identified in the blastocyst. Expression patterns in bovine and human blastocysts were to a large extent identical. This analysis compared the transcriptomes of bovine oocytes and blastocysts and provides a solid foundation for future studies on the first major differentiation events in blastocysts and identification of a set of markers indicative for regular mammalian development.

Purpose: To evaluate the role of laparoscopic ureteropyelostomy and subtotal ureterectomy for management of an ectopic ureter to the prostatic urethra. Case Report: A 54-year-old man presented with complaints of paravertebral pain and recurrent prostatitis with elevation of the serum prostate specific antigen concentration. Diagnostic evaluation revealed right renal duplication with an ectopic ureter to the prostatic urethra. Using a four-port transperitoneal laparoscopic technique, a laparoscopic ureteropyelostomy and subtotal ureterectomy were performed. Diagnostic follow-up documented successful treatment with the minimally invasive technique. Conclusions: Laparoscopic ureteropyelostomy and subtotal ureterectomy provide effective treatment for this rare condition. The minimally invasive technique may also have clinical applications in the pediatric population.

Background Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts. Methodology/Principal Findings We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation. Conclusions/Significance Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis., Author Summary Leishmania parasites belong to different species, each one characterized by specific vectors and reservoirs, and causes cutaneous or visceral disease(s) of variable clinical presentation and severity. In its mammalian host, the parasite is an obligate intracellular pathogen infecting the monocyte/macrophage lineage. Leishmania have developed ambiguous relationships with macrophages. Indeed, these cells are the shelter of invading parasites, where they will grow and eventually will reside in a silent state for life. But macrophages are also the cells that participate, through the induction of several pro-inflammatory mediators and antigen presentation, to shape the host immune response and ultimately kill the invader. To subvert these anti-parasite responses, Leishmania manipulate the host machinery for their own differentiation and survival. We aimed to evaluate the impact of L. major (the causative agent of zoonotic cutaneous leishmaniasis) infection on deregulation of non-coding miRNAs, a class of important regulators of gene expression. Our results revealed the implication of several miRNAs on macrophage fate upon parasite infection through regulation of different pathways, including cell death. Our findings provided a new insight for understanding mechanisms governing this miRNA deregulation by parasite infection and will help to provide clues for the development of control strategies for this disease.

The development of novel high-throughput technologies has opened up the opportunity to deeply characterize patient tissues at various molecular levels and has given rise to a paradigm shift in medicine towards personalized therapies. Computational analysis plays a pivotal role in integrating the various genome data and understanding the cellular response to a drug. Based on that data, molecular models can be constructed that incorporate the known downstream effects of drug-targeted receptor molecules and that predict optimal therapy decisions. In this article, we describe the different steps in the conceptual framework of computational modeling. We review resources that hold information on molecular pathways that build the basis for constructing the model interaction maps, highlight network analysis concepts that have been helpful in identifying predictive disease patterns, and introduce the basic concepts of kinetic modeling. Finally, we illustrate this framework with selected studies related to the modeling of important target pathways affected by drugs.El desarrollo de nuevas tecnologías de alto rendimiento ha abierto la posibilidad de caracterizar en profundidad tejidos de los pacientes en varios niveles moleculares y ha dado lugar a un cambio de paradigma en la medicina hacia terapias personalizadas. El análisis computacional juega un papel central para integrar diversos datos del genoma y comprender la respuesta celular a un fármaco. En base a estos datos se pueden construir modelos moleculares que incorporen los efectos posteriores conocidos de los receptores moleculares blanco de los fármacos y así predecir las decisiones terapéuticas óptimas. En este artículo se describen las diferentes etapas del marco conceptual del diseño computacional. Se revisan las fuentes que contienen información sobre las vías moleculares que construyen las bases para diseñar los mapas de interacción del modelo, se destacan los conceptos de análisis de redes que han sido útiles en la identificación de patrones patológicos predictivos y se introducen los conceptos básicos de diseño cinético. Por último, se ilustra este marco con estudios seleccionados que se relacionan con el diseño de importantes vías blanco que son afectadas por los fármacos.Le développement des nouvelles technologies à haut débit a permis de décrire en détail les tissus des patients à différents niveaux moléculaires et a provoqué un changement de praradigme en médecine, vers les traitements personnalisés. L'analyse informatique joue un rôle central dans l'intégration des différentes données du génome et dans la compréhension de la réponse cellulaire à un médicament. Selon ces données, il est possible de construire des modèles moléculaires qui comprennent les effets connus des molécules en aval du récepteur cible par le médicament ; cela permet un choix optimal lors des décisions thérapeutiques. Nous décrivons dans cet article les différentes étapes du cadre conceptuel de la modélisation informatique. Nous passons en revue les sources des informations sur les voies moléculaires, bases de construction des cartes d'interaction des modèles ; nous soulignons les concepts d'analyse de réseaux utiles pour identifier quelles configurations des maladies ont une valeur prédictive ; nous expliquons les idées de base de la modélisation cinétique. Enfin, nous illustrons ces concepts à l'aide d'études de la modélisation des cibles importantes influencées par les médicaments.

The importance of oxidative stress in the pathogenesis of neuroimmunological and neurodegenerative diseases, such as multiple sclerosis (MS), has been discussed for a long time. However, markers for oxidative stress in cerebrospinal fluid are hardly detected. The aim of the present study is to assess whether carbonyl proteins as end products of metabolic processes may serve as a marker for oxidative stress in the cerebrospinal fluid (CSF) of patients with neuroimmunological and neurodegenerative diseases. Levels of carbonyl proteins in the CSF were assessed in 15 patients suffering from MS, four patients with neurodegenerative diseases, including one patient with dementia complicated by carcinomatous meningitis due to breast cancer, and four control subjects with no established neurological disease. Levels of carbonyl proteins were measured with a commercially available KIT. A significant difference (P = 0.025) was shown for mean values of various subgroups with highest levels for patients with neurodegenerative diseases (756.1 pmol/mg), followed by the MS (630.8 pmol/mg) and the control group (356.5 pmol/mg). Post-hoc pair wise comparisons showed significant differences between the MS group and healthy controls (P = 0.016) as well as for patients with neurodegenerative diseases and healthy controls (P = 0.02). This pilot trial showed that carbonyl proteins might serve as measure for oxidative stress in the CSF of relapsing as well as progressive MS patients and in patients with neurodegenerative diseases. Larger trials have to show whether they may serve as biomarkers and be helpful in monitoring patients with MS or neurodegenerative diseases.

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies., Wellcome Trust (London, England) (Core Award 090532/Z/09/Z), Wellcome Trust (London, England) (Senior Investigator Award 095552/Z/11/Z ), Wellcome Trust (London, England) (WT095908), Wellcome Trust (London, England) (WT109497), Wellcome Trust (London, England) (WT098051), Wellcome Trust (London, England) (WT086084/Z/08/Z), Wellcome Trust (London, England) (WT100956/Z/13/Z ), Wellcome Trust (London, England) (WT097307), Wellcome Trust (London, England) (WT0855322/Z/08/Z ), Wellcome Trust (London, England) (WT090770/Z/09/Z ), Wellcome Trust (London, England) (Major Overseas program in Vietnam grant 089276/Z.09/Z), Medical Research Council (Great Britain) (grant G0801823), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I02593X/1), Biotechnology and Biological Sciences Research Council (Great Britain) (grant BB/I021213/1), Zhongguo ke xue ji shu qing bao yan jiu suo. Office of 863 Programme of China (2012AA02A201), National Basic Research Program of China (2011CB809201), National Basic Research Program of China (2011CB809202), National Basic Research Program of China (2011CB809203), National Natural Science Foundation of China (31161130357), Shenzhen Municipal Government of China (grant ZYC201105170397A), Canadian Institutes of Health Research (grant 136855), Quebec Ministry of Economic Development, Innovation, and Exports (PSR-SIIRI-195), Germany. Bundesministerium für Bildung und Forschung (0315428A), Germany. Bundesministerium für Bildung und Forschung (01GS08201), Germany. Bundesministerium für Bildung und Forschung (BMBF-EPITREAT grant 0316190A), Deutsche Forschungsgemeinschaft (Emmy Noether Grant KO4037/1-1), Beatriu de Pinos Program (2006 BP-A 10144), Beatriu de Pinos Program (2009 BP-B 00274), Spanish National Institute for Health (grant PRB2 IPT13/0001-ISCIII-SGEFI/FEDER), Japan Society for the Promotion of Science (fellowship number PE13075), Marie Curie Actions Career Integration (grant 303772), Fonds National Suisse del la Recherche, SNSF, Scientifique (31003A_130342), National Center for Biotechnology Information (U.S.) (U54HG3067), National Center for Biotechnology Information (U.S.) (U54HG3273), National Center for Biotechnology Information (U.S.) (U01HG5211), National Center for Biotechnology Information (U.S.) (U54HG3079), National Center for Biotechnology Information (U.S.) (R01HG2898), National Center for Biotechnology Information (U.S.) (R01HG2385), National Center for Biotechnology Information (U.S.) (RC2HG5552), National Center for Biotechnology Information (U.S.) (U01HG6513), National Center for Biotechnology Information (U.S.) (U01HG5214), National Center for Biotechnology Information (U.S.) (U01HG5715), National Center for Biotechnology Information (U.S.) (U01HG5718), National Center for Biotechnology Information (U.S.) (U01HG5728), National Center for Biotechnology Information (U.S.) (U41HG7635), National Center for Biotechnology Information (U.S.) (U41HG7497), National Center for Biotechnology Information (U.S.) (R01HG4960), National Center for Biotechnology Information (U.S.) (R01HG5701), National Center for Biotechnology Information (U.S.) (R01HG5214), National Center for Biotechnology Information (U.S.) (R01HG6855), National Center for Biotechnology Information (U.S.) (R01HG7068), National Center for Biotechnology Information (U.S.) (R01HG7644), National Center for Biotechnology Information (U.S.) (DP2OD6514), National Center for Biotechnology Information (U.S.) (DP5OD9154), National Center for Biotechnology Information (U.S.) (R01CA166661), National Center for Biotechnology Information (U.S.) (R01CA172652), National Center for Biotechnology Information (U.S.) (P01GM99568), National Center for Biotechnology Information (U.S.) (R01GM59290), National Center for Biotechnology Information (U.S.) (R01GM104390), National Center for Biotechnology Information (U.S.) (T32GM7790), National Center for Biotechnology Information (U.S.) (R01HL87699), National Center for Biotechnology Information (U.S.) (R01HL104608), National Center for Biotechnology Information (U.S.) (T32HL94284), National Center for Biotechnology Information (U.S.) (HHSN268201100040C), National Center for Biotechnology Information (U.S.) (HHSN272201000025C), Lundbeck Foundation (grant R170-2014-1039, Simons Foundation (SFARI award SF51), National Science Foundation (U.S.) (Research Fellowship DGE-1147470)

Peyronie’s disease is a connective tissue disorder in the soft tissue of the penis. The underlying cause of Peyronie’s disease is not well understood but is thought to be caused by trauma or injury to the penis during sexual intercourse. The purpose of the interdisciplinary cooperation between urological surgery and physics is the development of a physical simulation tool in order to give prognosis of possible tunica albuginea fibre rupture at a certain degree of deviation of the penis. For our group the first challenge was to translate the human organ of the penis into a physical model. Starting and marginal parameters had to be defined, whereby some of them had to be based on assumption, as physical data of the human living tissue have rarely been measured up to now. The algorithm and its dependencies had to be developed. This paper is a first step of a three-dimensional mathematical-physical simulation with the assumption of a 100% filled rigid penis. The calculation gives proof of the hypothesis that the fibre-load-angle of the penis is less than 12 degrees. Thus physical simulation is able to provide the surgeon with a simple instrument to calculate and forecast the risk of the individual patient.

The importance of hormones in endogenous aging has been displayed by recent studies performed on animal models and humans. To decipher the molecular mechanisms involved in aging we maintained human sebocytes at defined hormone-substituted conditions that corresponded to average serum levels of females from 20 (f20) to 60 (f60) years of age. The corresponding hormone receptor expression was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunocytochemistry. Cells at f60 produced significantly lower lipids than at f20. Increased mRNA and protein levels of c-Myc and increased protein levels of FN1, which have been associated with aging, were detected in SZ95 sebocytes at f60 compared to those detected at f20 after 5 days of treatment. Expression profiling employing a cDNA microarray composed of 15 529 cDNAs identified 899 genes with altered expression levels at f20 vs. f60. Confirmation of gene regulation was performed by real-time RT-PCR. The functional annotation of these genes according to the Gene Ontology identified pathways related to mitochondrial function, oxidative stress, ubiquitin-mediated proteolysis, cell cycle, immune responses, steroid biosynthesis and phospholipid degradation - all hallmarks of aging. Twenty-five genes in common with those identified in aging kidneys and several genes involved in neurodegenerative diseases were also detected. This is the first report describing the transcriptome of human sebocytes and its modification by a cocktail of hormones administered in age-specific levels and provides an in vitro model system, which approximates some of the hormone-dependent changes in gene transcription that occur during aging in humans.

A 32-year-old man recovered completely from hypokalemic hypertension that had been caused by primary reninism after the ablation of an ectopic left testis, epididymis and ductus deferens. For several years, severe hypertension has been resistant to treatment, even the concurrent administration of up to seven antihypertensive agents. In this case, cryptorchidism was associated with an indirect inguinal hernia and an open peritoneo-vaginal process on both sides, aplasia of the posterior wall of the inguinal canal on the right side, an umbilical hernia, and a retroperitoneal tendrillar hemangioma.

Acne vulgaris is a skin disorder of the sebaceous follicles, involving hyperkeratinization and perifollicular inflammation. Matrix metalloproteinases (MMP) have a predominant role in inflammatory matrix remodeling and hyperproliferative skin disorders. We investigated the expression of MMP and tissue inhibitors of MMP (TIMP) in facial sebum specimens from acne patients, before and after treatment with isotretinoin. Gelatin zymography and Western-blot analysis revealed that sebum contains proMMP-9, which was decreased following per os or topical treatment with isotretinoin and in parallel to the clinical improvement of acne. Sebum also contains MMP-1, MMP-13, TIMP-1, and TIMP-2, as assessed by ELISA and western blot, but only MMP-13 was decreased following treatment with isotretinoin. The origin of MMP and TIMP in sebum is attributed to keratinocytes and sebocytes, since we found that HaCaT keratinocytes in culture secrete proMMP-2, proMMP-9, MMP-1, MMP-13, TIMP-1, and TIMP-2. SZ95 sebocytes in culture secreted proMMP-2 and proMMP-9, which was also confirmed by microarray analysis. Isotretinoin inhibited the arachidonic acid-induced secretion and mRNA expression of proMMP-2 and -9 in both cell types and of MMP-13 in HaCaT keratinocytes. These data indicate that MMP and TIMP of epithelial origin may be involved in acne pathogenesis, and that isotretinoin-induced reduction in MMP-9 and -13 may contribute to the therapeutic effects of the agent in acne.

Previous studies have demonstrated the efficacy of local application of estrogen in treating postmenopausal women with recurrent urinary tract infections (RUTI) and urinary incontinence. Younger women under oral contraceptives (OC) can suffer from similar symptoms. The aim of this pilot study was to evaluate the effectiveness of local estrogens on RUTI and the impact of local hormonal supplementation on bladder neck vascularization.30 women (mean age 22.7 years) with a longstanding history of RUTI were included. Pre-treatment investigation included complete clinical history, urinalysis, urine culture and cystoscopy. All subjects completed a questionnaire about onset and duration of disease and quality of life before and after treatment. Local (vaginal) estrogen therapy consisted of 1mg estriol (E3) 7 times a week for two weeks and twice a week for two additional weeks. Sonographic examination of bladder vascularization was performed before and after treatment using transperineal color Doppler ultrasound (6 MHz, Acuson Sequoia 512, Mountain View, CA, USA) with a filled bladder. After angle correction, peak systolic blood flow velocity (PSBFV) and end diastolic blood flow velocity (EDBFV) were measured in 2 bladder arteries; and the Resistive Index (RI) was calculated. Flow velocity in each vessel was measured at least four times and the mean value determined.All patients completed the therapy course without severe side effects. Patients had a mean history of RUTI over 2.3 years; the mean period under OC was 3.2 years. In the follow-up period of 11 months after treatment, 24/30 patients reported no symptoms of cystitis and used no additional medication. Normal bladder epithelium in control cystoscopy after E3 therapy was seen in all patients with trigonal metaplasia and vulnerable, highly vascularized urothelium at the initial investigation. RI decreased from 0.945 to 0.705 after treatment (p0.001), concomitantly the mean EDBFV increased highly significantly from 0.82 cm/sec to 4.45 cm/sec after estrogen treatment (p0.001).In a majority of young patients under OC and a longstanding history of RUTI, a considerable infection-free period was achieved after local application of estrogen. Decreased RI and increased EDBFV indicate vasodilatation and less peripheral vascular resistance. Responsiveness to local E3 may correspond to improved cystoscopic findings as a consequence of increased bladder perfusion.

Patients: In order to determine if there are areas of major and minor perfusion in a single testicle and if the quality of sperm is correlated with quantity of perfusion we collected testicle tissue for TESE in accordance to the local testicle tissue perfusion. Methods: A patient undergoing TESE underwent testicular perfusion mapping using contrast enhanced ultrasound. The exposed tissue was scanned with a Laser Doppler scanner and perfusion rates were determined measuring tissue perfusion units (TPUs). Tissue was biopsied and sperm were selected and prepared for assisted reproduction. Results: The total amount of isolated sperm correlated highly with the intensity of tissue perfusion showing high number of sperm in areas with high TPUs. Conclusions: This is the first demonstration that sperm quality and quantity is depending on tissue perfusion within the testicle. To further improve infertility treatment we propose that random biopsies could be replaced by perfusion-dependent collection of testicular tissue.

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.

The 2R hypothesis predicting two genome duplications at the origin of vertebrates is highly controversial. Studies published so far include limited sequence data from organisms close to the hypothesized genome duplications. Through the comparison of a gene catalog from amphioxus, the closest living invertebrate relative of vertebrates, to 3453 single-copy genes orthologous between Caenorhabditis elegans (C), Drosophila melanogaster (D), and Saccharomyces cerevisiae (Y), and to Ciona intestinalis ESTs, mouse, and human genes, we show with a large number of genes that the gene duplication activity is significantly higher after the separation of amphioxus and the vertebrate lineages, which we estimate at 650 million years (Myr). The majority of human orthologs of 195 CDY groups that could be dated by the molecular clock appear to be duplicated between 300 and 680 Myr with a mean at 488 million years ago (Mya). We detected 485 duplicated chromosomal segments in the human genome containing CDY orthologs, 331 of which are found duplicated in the mouse genome and within regions syntenic between human and mouse, indicating that these were generated earlier than the human–mouse split. Model based calculations of the codon substitution rate of the human genes included in these segments agree with the molecular clock duplication time-scale prediction. Our results favor at least one large duplication event at the origin of vertebrates, followed by smaller scale duplication closer to the bird–mammalian split.

We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.

PURPOSE: The assessment of plasma cytokine levels adds a useful tool to the diagnostic measures in severe inflammatory diseases. Proinflammatory cytokine levels in abdominal fluid after abdominal surgery have been shown to far exceed plasma cytokine levels. Thus, we investigated the local release of interleukin 1β, interleukin 6, and tumor necrosis factor-α in patients after colorectal surgery during the early postoperative period to evaluate whether it may serve as an indicator of evolving peritonitis. METHOD: In a prospective, observational pilot study, the first 12 consecutive patients who did not develop any postoperative complications (Group I), and the first 12 patients with secondary peritonitis caused by an anastomotic leakage (Group II), were included in the study. Interleukin 6, interleukin 1β, and tumor necrosis factor-α levels were determined in the abdominal exudate and compared between the groups within the first four days after colorectal surgery. RESULTS: Abdominal fluid interleukin 6 levels in Group II patients were higher (162,500 ± 105,800 pg/ml) as early as the first postoperative day compared with Group I (27,940 ± 13,860 pg/ml; P < 0.0001); this lasted for the whole observation period. The same applies to tumor necrosis factor-α levels (461.4 ± 167.8 pg/ml vs. 175.8 ± 178.6 pg/ml on day 1; P = 0.0007). The difference in interleukin 1β cytokine levels became statistically significant on the third postoperative day. Moreover, abdominal fluid cytokine levels rose in Group II, whereas they remained virtually unchanged or even tended to decrease over time in Group I. CONCLUSION: We suggest that the estimation of the peritoneal cytokine levels might be an additional diagnostic tool that can support the early recognition of peritonitic complications in colorectal surgery.