Int J Mol Sci

Phage-displayed peptide selections generate complex repertoires of several hundred thousand peptides as revealed by next-generation sequencing (NGS). In repeated peptide selections, however, even in identical experimental in vitro conditions, only a very small number of common peptides are found. The repertoire complexities are evidence of the difficulty of distinguishing between effective selections of specific peptide binders to exposed targets and the potential high background noise. Such investigation is even more relevant when considering the plethora of in vivo expressed targets on cells, in organs or in the entire organism to define targeting peptide agents. In the present study, we compare the published NGS data of three peptide repertoires that were obtained by phage display under identical experimental in vitro conditions. By applying the recently developed tool PepSimili we evaluate the calculated similarities of the individual peptides from each of these three repertoires and perform their mappings on the human proteome. The peptide-to-peptide mappings reveal high similarities among the three repertoires, confirming the desired reproducibility of phage-displayed peptide selections

ANASTASIA: An Automated Metagenomic Analysis Pipeline for Novel Enzyme Discovery Exploiting Next Generation Sequencing Data

Metagenomic analysis of environmental samples provides deep insight into the enzymatic mixture of the corresponding niches, capable of revealing peptide sequences with novel functional properties exploiting the high performance of next-generation sequencing (NGS) technologies. At the same time due to their ever increasing complexity, there is a compelling need for ever larger computational configurations to ensure proper bioinformatic analysis, and fine annotation. With the aiming to address the challenges of such an endeavor, we have developed a novel web-based application named ANASTASIA (automated nucleotide aminoacid sequences translational plAtform for systemic interpretation and analysis). ANASTASIA provides a rich environment of bioinformatic tools, either publicly available or novel, proprietary algorithms, integrated within numerous automated algorithmic workflows, and which enables versatile data processing tasks for (meta)genomic sequence datasets. ANASTASIA was initially developed in the framework of the European FP7 project HotZyme, whose aim was to perform exhaustive analysis of metagenomes derived from thermal springs around the globe and to discover new enzymes of industrial interest. ANASTASIA has evolved to become a stable and extensible environment for diversified, metagenomic, functional analyses for a range of applications overarching industrial biotechnology to biomedicine, within the frames of the ELIXIR-GR project. As a showcase, we report the successful in silico mining of a novel thermostable esterase termed “EstDZ4” from a metagenomic sample collected from a hot spring located in Krisuvik, Iceland

Sensitivity of first trimester ultrasound in the detection of congenital anomalies in twin pregnancies: population study and systematic review.

INTRODUCTION: The first aim of this study was to ascertain the diagnostic performance of first trimester ultrasound in detecting congenital anomalies in twins. The secondary aim was to explore the strength of association between different pregnancy characteristics and early detection of structural anomalies in a large unselected population of twin pregnancies. A systematic review of the published literature was also carried out MATERIAL AND METHODS: Retrospective analysis of prospectively collected data from consecutive twin pregnancies booked for antenatal care from 1996 till 2014. Predictive accuracy of those covariates independently associated with the occurrence of fetal anomalies were assessed with logistic regression analysis and ROC curves RESULTS: 1064 twin pregnancies (820 dichorionic and 264 monochorionic) were included in the analysis. 42 pregnancies had one or more fetuses with structural abnormalities. Detection of structural abnormalities using ultrasound was possible in the first trimester in 27.3% (95% CI: 15.0-42.8) of twin pregnancies. Mono-chorionicity (OR 2.3, 95% CI: 1.1-4.7) and discordance in crown-rump length and nuchal translucency were associated with an increased risk of fetal anomalies. However, their predictive accuracy was only moderate (AUC: 0.67, 95% CI: 0.6-0.8 and 0.68, 95% CI: 0.6 - 0.8, for crown-rump length and nuchal translucency discrepancy respectively CONCLUSIONS: First trimester detection of structural abnormalities in twin pregnancies is possible in 27.3% (95% CI: 15.0-42.8) of cases. The likelihood for first trimester detection of structural anomalies in twins was maximum for cranial vault, midline brain and abdominal wall defects. Monochorionicity and increasing discrepancy in crown-rump length and nuchal translucency were associated with fetal structural abnormalities, although their predictive performance was only moderately good. This article is protected by copyright. All rights reserved

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.publishedVersio

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.publishedVersio

Systematic review of first trimester ultrasound screening in detecting fetal structural anomalies and factors affecting screening performance.

OBJECTIVES: To determine the sensitivity and specificity of first trimester ultrasound for the detection of fetal abnormalities; and to establish which factors might impact this screening. METHODS: Systematic review and meta-analysis of all relevant publications assessing the diagnostic accuracy of first trimester 2D (transabdominal and transvaginal) ultrasound in the detection of congenital fetal anomalies prior to 14 weeks gestation was performed. The reference standard used was the detection of abnormalities at birth or postmortem. Factors that may impact detection rates were evaluated including population characteristics, gestation, healthcare setting, ultrasound modality, use of an anatomical checklist for first trimester anomaly detection and what types of malformations were included in the study. In an effort to reduce the impact of study heterogeneity on results of the meta-analysis, data from the studies were analyzed within subgroups of major anomalies versus all types of anomalies; and low risk / unselected populations versus high risk populations. RESULTS: An initial electronic search identified 2,225 citations, from which a total of 30 relevant studies, published between 1991 and 2015, were selected for inclusion. For low risk or unselected populations (19 studies, 115,731 fetuses) the pooled estimate for detection of major abnormalities was 46.10% (95% C.I. 36.88-55.46). The detection rate for all abnormalities in low risk or unselected populations was 32.35% (95% C.I. 22.45-43.12), in 14 studies (97,976 fetuses); while the detection rate in high risk populations for the presence of all types of anomalies (six studies, 2,841 fetuses) was 61.18% (95% C.I. 37.71-82.19). Of the factors examined impacting detection rates there was a statistically significant relationship between the use of an anatomical protocol during first trimester anomaly screening and sensitivity for the detection of fetal anomalies in all subgroups (P < 0.0001). CONCLUSION: Detection rates for first trimester anomalies range from 32% in low risk, to over 60% in high risk groups. This demonstrates that first trimester ultrasound has the potential to identify a large proportion of fetuses affected with structural anomalies. The use of a standardized anatomical protocol improves the sensitivity of first trimester ultrasound screening for all anomalies and major anomalies in populations of varying risk. The development and introduction of international protocols with standard anatomical views should be undertaken, in order to optimize rates of first trimester anomaly detection

Escherichia coli genome-wide promoter analysis: Identification of additional AtoC binding target elements

<p>Abstract</p> <p>Background</p> <p>Studies on bacterial signal transduction systems have revealed complex networks of functional interactions, where the response regulators play a pivotal role. The AtoSC system of <it>E. coli </it>activates the expression of <it>atoDAEB </it>operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that <it>atoSC </it>is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the <it>atoDAEB </it>promoter as the single, <it>cis</it>-regulatory binding site of the AtoC response regulator. In this work, we used a computational approach to explore the presence of yet unidentified AtoC binding sites within other parts of the <it>E. coli </it>genome.</p> <p>Results</p> <p>Through the implementation of a computational <it>de novo </it>motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the <it>E. coli </it>genome. In order to assess the biological relevance of the motifs and to select for experimental validation of those sequences related robustly with distinct cellular functions, we implemented a novel approach that applies Gene Ontology Term Analysis to the motif hits and selected those that were qualified through this procedure. The computational results were validated using Chromatin Immunoprecipitation assays to assess the <it>in vivo </it>binding of AtoC to the predicted sites. This process verified twenty-two additional AtoC binding sites, located not only within intergenic regions, but also within gene-encoding sequences.</p> <p>Conclusions</p> <p>This study, by tracing a number of putative AtoC binding sites, has indicated an AtoC-related cross-regulatory function. This highlights the significance of computational genome-wide approaches in elucidating complex patterns of bacterial cell regulation.</p

Zea mays iRS1563: A Comprehensive Genome-Scale Metabolic Reconstruction of Maize Metabolism

The scope and breadth of genome-scale metabolic reconstructions have continued to expand over the last decade. Herein, we introduce a genome-scale model for a plant with direct applications to food and bioenergy production (i.e., maize). Maize annotation is still underway, which introduces significant challenges in the association of metabolic functions to genes. The developed model is designed to meet rigorous standards on gene-protein-reaction (GPR) associations, elementally and charged balanced reactions and a biomass reaction abstracting the relative contribution of all biomass constituents. The metabolic network contains 1,563 genes and 1,825 metabolites involved in 1,985 reactions from primary and secondary maize metabolism. For approximately 42% of the reactions direct literature evidence for the participation of the reaction in maize was found. As many as 445 reactions and 369 metabolites are unique to the maize model compared to the AraGEM model for A. thaliana. 674 metabolites and 893 reactions are present in Zea mays iRS1563 that are not accounted for in maize C4GEM. All reactions are elementally and charged balanced and localized into six different compartments (i.e., cytoplasm, mitochondrion, plastid, peroxisome, vacuole and extracellular). GPR associations are also established based on the functional annotation information and homology prediction accounting for monofunctional, multifunctional and multimeric proteins, isozymes and protein complexes. We describe results from performing flux balance analysis under different physiological conditions, (i.e., photosynthesis, photorespiration and respiration) of a C4 plant and also explore model predictions against experimental observations for two naturally occurring mutants (i.e., bm1 and bm3). The developed model corresponds to the largest and more complete to-date effort at cataloguing metabolism for a plant species