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1.  EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability 
Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure to elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring following in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retains high levels of catalytic activity after exposure to temperatures as high as 95°C for several hours. Furthermore, it exhibits very good stability against exposure to high concentrations of a variety of organic solvents. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modeling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold that seems to include a “subdomain insertion”, which is similar to the one present in its closest homolog of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a range of organic solvents and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications.
PMCID: PMC5110521  PMID: 27899916
hyperthermostability; esterase; Dictyoglomus; functional genomics; biocatalysis; biotechnology
2.  Combinatorial approaches for inverse metabolic engineering applications 
Traditional metabolic engineering analyzes biosynthetic and physiological pathways, identifies bottlenecks, and makes targeted genetic modifications with the ultimate goal of increasing the production of high-value products in living cells. Such efforts have led to the development of a variety of organisms with industrially relevant properties. However, there are a number of cellular phenotypes important for research and the industry for which the rational selection of cellular targets for modification is not easy or possible. In these cases, strain engineering can be alternatively carried out using “inverse metabolic engineering”, an approach that first generates genetic diversity by subjecting a population of cells to a particular mutagenic process, and then utilizes genetic screens or selections to identify the clones exhibiting the desired phenotype. Given the availability of an appropriate screen for a particular property, the success of inverse metabolic engineering efforts usually depends on the level and quality of genetic diversity which can be generated. Here, we review classic and recently developed combinatorial approaches for creating such genetic diversity and discuss the use of these methodologies in inverse metabolic engineering applications.
PMCID: PMC3962077  PMID: 24688681
inverse metabolic engineering; genetic engineering; microbes; genetic screening; mutagenesis
3.  Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate 
PLoS ONE  2016;11(1):e0146454.
With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.
PMCID: PMC4704807  PMID: 26741138
4.  Integrative workflows for metagenomic analysis 
The rapid evolution of all sequencing technologies, described by the term Next Generation Sequencing (NGS), have revolutionized metagenomic analysis. They constitute a combination of high-throughput analytical protocols, coupled to delicate measuring techniques, in order to potentially discover, properly assemble and map allelic sequences to the correct genomes, achieving particularly high yields for only a fraction of the cost of traditional processes (i.e., Sanger). From a bioinformatic perspective, this boils down to many GB of data being generated from each single sequencing experiment, rendering the management or even the storage, critical bottlenecks with respect to the overall analytical endeavor. The enormous complexity is even more aggravated by the versatility of the processing steps available, represented by the numerous bioinformatic tools that are essential, for each analytical task, in order to fully unveil the genetic content of a metagenomic dataset. These disparate tasks range from simple, nonetheless non-trivial, quality control of raw data to exceptionally complex protein annotation procedures, requesting a high level of expertise for their proper application or the neat implementation of the whole workflow. Furthermore, a bioinformatic analysis of such scale, requires grand computational resources, imposing as the sole realistic solution, the utilization of cloud computing infrastructures. In this review article we discuss different, integrative, bioinformatic solutions available, which address the aforementioned issues, by performing a critical assessment of the available automated pipelines for data management, quality control, and annotation of metagenomic data, embracing various, major sequencing technologies and applications.
PMCID: PMC4237130  PMID: 25478562
metagenomics; bioinformatics; distributed computing; cloud computing; workflow engines
5.  HuR-Regulated mRNAs Associated with Nuclear hnRNP A1-RNP Complexes 
Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicating their concerted function in mRNA metabolism.
PMCID: PMC3821614  PMID: 24152440
RNA-binding proteins (RBPs); ribonucleoprotein (hnRNP) complexes; post-transcriptional regulation; mRNA processing; RNA-immunoprecipitation (RIP)-Chip technology; mouse embryonic fibroblasts (MEFs)
6.  Application of an Integrative Computational Framework in Trancriptomic Data of Atherosclerotic Mice Suggests Numerous Molecular Players 
Advances in Bioinformatics  2012;2012:453513.
Atherosclerosis is a multifactorial disease involving a lot of genes and proteins recruited throughout its manifestation. The present study aims to exploit bioinformatic tools in order to analyze microarray data of atherosclerotic aortic lesions of ApoE knockout mice, a model widely used in atherosclerosis research. In particular, a dynamic analysis was performed among young and aged animals, resulting in a list of 852 significantly altered genes. Pathway analysis indicated alterations in critical cellular processes related to cell communication and signal transduction, immune response, lipid transport, and metabolism. Cluster analysis partitioned the significantly differentiated genes in three major clusters of similar expression profile. Promoter analysis applied to functional related groups of the same cluster revealed shared putative cis-elements potentially contributing to a common regulatory mechanism. Finally, by reverse engineering the functional relevance of differentially expressed genes with specific cellular pathways, putative genes acting as hubs, were identified, linking functionally disparate cellular processes in the context of traditional molecular description.
PMCID: PMC3502768  PMID: 23193398
7.  Escherichia coli genome-wide promoter analysis: Identification of additional AtoC binding target elements 
BMC Genomics  2011;12:238.
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 E. coli activates the expression of atoDAEB operon genes, and the subsequent catabolism of short-chain fatty acids, upon acetoacetate induction. Transcriptome and phenotypic analyses suggested that atoSC is also involved in several other cellular activities, although we have recently reported a palindromic repeat within the atoDAEB promoter as the single, cis-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 E. coli genome.
Through the implementation of a computational de novo motif detection workflow, a set of candidate motifs was generated, representing putative AtoC binding targets within the E. coli 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 in vivo 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.
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.
PMCID: PMC3118216  PMID: 21569465
8.  A transcriptomic computational analysis of mastic oil-treated Lewis lung carcinomas reveals molecular mechanisms targeting tumor cell growth and survival 
BMC Medical Genomics  2009;2:68.
Mastic oil from Pistacia lentiscus variation chia, a blend of bioactive terpenes with recognized medicinal properties, has been recently shown to exert anti-tumor growth activity through inhibition of cancer cell proliferation, survival, angiogenesis and inflammatory response. However, no studies have addressed its mechanisms of action at genome-wide gene expression level.
To investigate molecular mechanisms triggered by mastic oil, Lewis Lung Carcinoma cells were treated with mastic oil or DMSO and RNA was collected at five distinct time points (3-48 h). Microarray expression profiling was performed using Illumina mouse-6 v1 beadchips, followed by computational analysis. For a number of selected genes, RT-PCR validation was performed in LLC cells as well as in three human cancer cell lines of different origin (A549, HCT116, K562). PTEN specific inhibition by a bisperovanadium compound was applied to validate its contribution to mastic oil-mediated anti-tumor growth effects.
In this work we demonstrated that exposure of Lewis lung carcinomas to mastic oil caused a time-dependent alteration in the expression of 925 genes. GO analysis associated expression profiles with several biological processes and functions. Among them, modifications on cell cycle/proliferation, survival and NF-κB cascade in conjunction with concomitant regulation of genes encoding for PTEN, E2F7, HMOX1 (up-regulation) and NOD1 (down-regulation) indicated some important mechanistic links underlying the anti-proliferative, pro-apoptotic and anti-inflammatory effects of mastic oil. The expression profiles of Hmox1, Pten and E2f7 genes were similarly altered by mastic oil in the majority of test cancer cell lines. Inhibition of PTEN partially reversed mastic oil effects on tumor cell growth, indicating a multi-target mechanism of action. Finally, k-means clustering, organized the significant gene list in eight clusters demonstrating a similar expression profile. Promoter analysis in a representative cluster revealed shared putative cis-elements suggesting a common regulatory transcription mechanism.
Present results provide novel evidence on the molecular basis of tumor growth inhibition mediated by mastic oil and set a rational basis for application of genomics and bioinformatic methodologies in the screening of natural compounds with potential cancer chemopreventive activities.
PMCID: PMC2801511  PMID: 20003503
9.  Gene ARMADA: an integrated multi-analysis platform for microarray data implemented in MATLAB 
BMC Bioinformatics  2009;10:354.
The microarray data analysis realm is ever growing through the development of various tools, open source and commercial. However there is absence of predefined rational algorithmic analysis workflows or batch standardized processing to incorporate all steps, from raw data import up to the derivation of significantly differentially expressed gene lists. This absence obfuscates the analytical procedure and obstructs the massive comparative processing of genomic microarray datasets. Moreover, the solutions provided, heavily depend on the programming skills of the user, whereas in the case of GUI embedded solutions, they do not provide direct support of various raw image analysis formats or a versatile and simultaneously flexible combination of signal processing methods.
We describe here Gene ARMADA (Automated Robust MicroArray Data Analysis), a MATLAB implemented platform with a Graphical User Interface. This suite integrates all steps of microarray data analysis including automated data import, noise correction and filtering, normalization, statistical selection of differentially expressed genes, clustering, classification and annotation. In its current version, Gene ARMADA fully supports 2 coloured cDNA and Affymetrix oligonucleotide arrays, plus custom arrays for which experimental details are given in tabular form (Excel spreadsheet, comma separated values, tab-delimited text formats). It also supports the analysis of already processed results through its versatile import editor. Besides being fully automated, Gene ARMADA incorporates numerous functionalities of the Statistics and Bioinformatics Toolboxes of MATLAB. In addition, it provides numerous visualization and exploration tools plus customizable export data formats for seamless integration by other analysis tools or MATLAB, for further processing. Gene ARMADA requires MATLAB 7.4 (R2007a) or higher and is also distributed as a stand-alone application with MATLAB Component Runtime.
Gene ARMADA provides a highly adaptable, integrative, yet flexible tool which can be used for automated quality control, analysis, annotation and visualization of microarray data, constituting a starting point for further data interpretation and integration with numerous other tools.
PMCID: PMC2771024  PMID: 19860866
10.  KEGGconverter: a tool for the in-silico modelling of metabolic networks of the KEGG Pathways database 
BMC Bioinformatics  2009;10:324.
The KEGG Pathway database is a valuable collection of metabolic pathway maps. Nevertheless, the production of simulation capable metabolic networks from KEGG Pathway data is a challenging complicated work, regardless the already developed tools for this scope. Originally used for illustration purposes, KEGG Pathways through KGML (KEGG Markup Language) files, can provide complete reaction sets and introduce species versioning, which offers advantages for the scope of cellular metabolism simulation modelling. In this project, KEGGconverter is described, implemented also as a web-based application, which uses as source KGML files, in order to construct integrated pathway SBML models fully functional for simulation purposes.
A case study of the integration of six human metabolic pathways from KEGG depicts the ability of KEGGconverter to automatically produce merged and converted to SBML fully functional pathway models, enhanced with default kinetics. The suitability of the developed tool is demonstrated through a comparison with other state-of-the art relevant software tools for the same data fusion and conversion tasks, thus illustrating the problems and the relevant workflows. Moreover, KEGGconverter permits the inclusion of additional reactions in the resulting model which represent flux cross-talk with neighbouring pathways, providing in this way improved simulative accuracy. These additional reactions are introduced by exploiting relevant semantic information for the elements of the KEGG Pathways database. The architecture and functionalities of the web-based application are presented.
KEGGconverter is capable of producing integrated analogues of metabolic pathways appropriate for simulation tasks, by inputting only KGML files. The web application acts as a user friendly shell which transparently enables the automated biochemically correct pathway merging, conversion to SBML format, proper renaming of the species, and insertion of default kinetic properties for the pertaining reactions. The tool is available at:
PMCID: PMC2764712  PMID: 19814801

Results 1-10 (10)