Perceiving molecular evolution processes in Escherichia coli by comprehensive metabolite and gene expression profiling

Transcript and metabolite abundance changes were analyzed in evolved and ancestor strains of Escherichia coli in three different evolutionary condition

Integrative Functional Genomic Analyses Implicate Specific Molecular Pathways and Circuits in Autism

SummaryGenetic studies have identified dozens of autism spectrum disorder (ASD) susceptibility genes, raising two critical questions: (1) do these genetic loci converge on specific biological processes, and (2) where does the phenotypic specificity of ASD arise, given its genetic overlap with intellectual disability (ID)? To address this, we mapped ASD and ID risk genes onto coexpression networks representing developmental trajectories and transcriptional profiles representing fetal and adult cortical laminae. ASD genes tightly coalesce in modules that implicate distinct biological functions during human cortical development, including early transcriptional regulation and synaptic development. Bioinformatic analyses suggest that translational regulation by FMRP and transcriptional coregulation by common transcription factors connect these processes. At a circuit level, ASD genes are enriched in superficial cortical layers and glutamatergic projection neurons. Furthermore, we show that the patterns of ASD and ID risk genes are distinct, providing a biological framework for further investigating the pathophysiology of ASD

Timing and Form of Federal Regulation: The Case of Climate

BACKGROUND: Common genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2METHODS: To identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calcium-dependent co-regulationRESULTS: We identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca(2+) signaling in TS patients leads to the observed molecular dysregulationCONCLUSIONS: We applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca(2+) signaling dysregulation on transcriptio

Proteome, transcriptome and metabolome plasticity in closely related strains of Escherichia coli - K12 and its diversity during molecular evolution processes

Vijayendran C. Proteome, transcriptome and metabolome plasticity in closely related strains of Escherichia coli - K12 and its diversity during molecular evolution processes. Bielefeld (Germany): Bielefeld University; 2007.Escherichia coli, originally known as Bacterium coli commune, was identified in 1885 by the German pediatrician, Theodor Escherich. They are Gram-negative, straight and rod-shaped bacteria from the Enterobacteriaceae family. Due to its rapid growth rate, simple nutritional requirements and established genetic manipulation techniques, E. coli has become a model organism in the field of basic biomolecular sciences for understanding various biological phenomena. As a result, well established information about E. coli's genetics and several completed genome sequences are available. Among them, the genome sequences of two closely related K-12 non-pathogenic strains, MG1655 and W3110 have been accurately determined. Resequencing of PCR products of selected regions indicates that there is only eight true insertion/deletion or base differences between the two strains in addition to the 13 sites where differences are due to insertion sequences, defective prophages and two sites due to the W3110 inversion between the ribosomal RNA genes rrnD and rrnE. The rate of nucleotide changes between both the strains is estimated to be relatively low with almost identical genome structures. Hence the following important questions arise: - Is this high degree of similarity at the nucleotide level reflected in the metabolic phenotype? - Do sub-strains with almost identical genome structures exhibit similar behaviour in cellular metabolism? - How do global aspects of cell metabolism, protein synthesis and gene expression differ among closely related sub-strains of the same species, revealing possible complexities of cellular metabolism? To address these queries, we analyzed the growth behaviour in strictly controlled conditions and analysed the global proteome and transcriptome pools of these closely related E. coli sub-strains W3110 and MG1655. We applied the conventional 2-dimensional polyacrylamide gel electrophoresis for global proteomic profiling which is still the major method for global proteome analysis. Global changes in the gene expression levels were analysed using microarrays, thus providing quantitative information about the gene expression levels. Being the most extensively studied model organism, E. coli is frequently used in molecular evolutionary studies. A few potential reasons for this are: its capacity to propagate and reproduce quickly, facilitating the evolution experiments for many generations in a short time span, and capability to store the evolved and ancestor strains, allowing for direct comparison between them. As a result, several studies have used gene expression and proteome profiling methods to study molecular evolution, but these studies were confined to a single type of evolution process and were focused on a single molecular aspect that characterizes a cell (transcript or protein abundance). Metabolome profiling has been frequently applied for obtaining quantitative information on metabolites for studies on mutational or environmental effects, but not in an evolutionary context. In our study, we depicted a complete picture of molecular evolution processes in the laboratory among the two strains MG1655 and DH10B under three different evolutionary conditions in all three functional levels of the cell (transcriptome, proteome and metabolome). These data sets obtained from the three functional levels would be of vital importance for viewing a global picture of the experimental sample in question. To eliminate the possibility of the strain-dependent phenomenon of evolution and to examine the parallelism of the laboratory evolution processes, we examined all the evolutionary processes in two strains. The major questions that arose during our study were: - What are the transcriptome, proteome and metabolome changes occurring during the excess-nutrient adaptive evolution process? - Which genes, proteins and metabolites are vitally involved in the prolonged stationary phase evolution process? - What are the transcript, protein and metabolite changes occurring due to the pleiotropic effects due to environmental shift? - To what extent are the changes occurring during these evolutionary processes seen in both strains? - Among both the strains, is the path of evolution similar in these evolutionary processes (parallelism)? By global protein profiling technologies and integrating the multidimensional datasets generated, we aimed to find vital genes, proteins and metabolites involved in the evolutionary processes in three conditions in two E. coli K-12 strains. These generated datasets from all the three functional levels would be an initial resource for the systems biology of microbial evolution

Impact of profiling technologies in the understanding of recombinant protein production

Vijayendran C, Flaschel E. Impact of profiling technologies in the understanding of recombinant protein production. Adv Biochem Eng Biotechnol. 2010;121:45-70.Since expression profiling methods have been available in a high throughput fashion, the implication of these technologies in the field of biotechnology has increased dramatically. Microarray technology is one such unique and efficient methodology for simultaneous exploration of expression levels of numerous genes. Likewise, two-dimensional gel electrophoresis or multidimensional liquid chromatography coupled with mass spectrometry are extensively utilised for studying expression levels of numerous proteins. In the field of biotechnology these highly parallel analytical methods have paved the way to study and understand various biological phenomena depending on expression patterns. The next phenomenological level is represented by the metabolome and the (metabolic) fluxome. However, this chapter reviews gene and protein profiling and their impact on understanding recombinant protein production. We focus on the computational methods utilised for the analyses of data obtained from these profiling technologies as well as prominent results focusing on recombinant protein expression with Escherichia coli. Owing to the knowledge accumulated with respect to cellular signals triggered during recombinant protein production, this field is on the way to design strategies for developing improved processes. Both gene and protein profiling have exhibited a handful of functional categories to concentrate on in order to identify target genes and proteins, respectively, involved in the signalling network with major impact on recombinant protein production

2DBase: 2D-PAGE database of Escherichia coli

Vijayendran C, Burgemeister S, Friehs K, Niehaus K, Flaschel E. 2DBase: 2D-PAGE database of Escherichia coli. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 2007;363(3):822-827.We present a web-based integrated proteome database, termed 2DBase of Escherichia coli which was designed to store, compare, analyse, and retrieve various information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry. The main objectives of this database are (1) to provide the features for query and data-mining applications to access the stored proteomics data (2) to efficiently compare the specific protein spots present in the comparable proteome maps and (3) to analyse the data with the integrated classification for cellular functions of gene products of E. coli. This database currently contains 12 gels consisting of 1185 protein spots information in which 723 proteins were identified and annotated. Individual protein spots in the existing gels can be displayed, queried, analyzed, and compared in a tabular format based on various functional categories enabling quick and subsequent analyses. Our database satisfies the requirement to be a federated 2-DE database by accomplishing various tasks through a web interface providing access to a relational database system. The 2DBase of E. coli database can be accessed at http://2dbase.techfak.uni-bielefeld.de/

The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12

Vijayendran C, Polen T, Wendisch VF, Friehs K, Niehaus K, Flaschel E. The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12. Journal of Biotechnology. 2007;128(4):747-761.The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional a gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. C Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading. (c) 2007 Elsevier B.V. All rights reserved

Long non-coding RNA TUG1 is downregulated in Friedreichs ataxia.

Friedreichs ataxia is a neurodegenerative disorder caused by reduced frataxin levels. It leads to motor and sensory impairments and has a median life expectancy of around 35 years. As the most common inherited form of ataxia, Friedreichs ataxia lacks reliable, non-invasive biomarkers, prolonging and inflating the cost of clinical trials. This study proposes TUG1, a long non-coding RNA, as a promising blood-based biomarker for Friedreichs ataxia, which is known to regulate various cellular processes. In a previous study using a frataxin knockdown mouse model, we observed several hallmark Friedreichs ataxia symptoms. Building on this, we hypothesized that a dual-source approach-comparing the data from peripheral blood samples from Friedreichs ataxia patients with tissue samples from affected areas in Friedreichs ataxia knockdown mice, tissues usually unattainable from patients-would effectively identify robust biomarkers. A comprehensive reanalysis was conducted on gene expression data from 183 age- and sex-matched peripheral blood samples of Friedreichs ataxia patients, carriers and controls and 192 tissue data sets from Friedreichs ataxia knockdown mice. Blood and tissue samples underwent RNA isolation and quantitative reverse transcription polymerase chain reaction, and frataxin knockdown was confirmed through enzyme-linked immunosorbent assays. Tug1 RNA interaction was explored via RNA pull-down assays. Validation was performed in serum samples on an independent set of 45 controls and 45 Friedreichs ataxia patients and in blood samples from 66 heterozygous carriers and 72 Friedreichs ataxia patients. Tug1 and Slc40a1 emerged as potential blood-based biomarkers, confirmed in the Friedreichs ataxia knockdown mouse model (one-way ANOVA, P ≤ 0.05). Tug1 was consistently downregulated after Fxn knockdown and correlated strongly with Fxn levels (R 2 = 0.71 during depletion, R 2 = 0.74 during rescue). Slc40a1 showed a similar but tissue-specific pattern. Further validation of Tug1s downstream targets strengthened its biomarker candidacy. In additional human samples, TUG1 levels were significantly downregulated in both whole blood and serum of Friedreichs ataxia patients compared with controls (Wilcoxon signed-rank test, P < 0.05). Regression analyses revealed a negative correlation between TUG1 fold-change and disease onset (P < 0.0037) and positive correlations with disease duration and functional disability stage score (P < 0.04). This suggests that elevated TUG1 levels correlate with earlier onset and more severe cases. This study identifies TUG1 as a potential blood-based biomarker for Friedreichs ataxia, showing consistent expression variance in human and mouse tissues related to disease severity and key Friedreichs ataxia pathways. It correlates with frataxin levels, indicating its promise as an early, non-invasive marker. TUG1 holds potential for Friedreichs ataxia monitoring and therapeutic development, meriting additional research

Inducible and reversible phenotypes in a novel mouse model of Friedreich's Ataxia.

Friedreich's ataxia (FRDA), the most common inherited ataxia, is caused by recessive mutations that reduce the levels of frataxin (FXN), a mitochondrial iron binding protein. We developed an inducible mouse model of Fxn deficiency that enabled us to control the onset and progression of disease phenotypes by the modulation of Fxn levels. Systemic knockdown of Fxn in adult mice led to multiple phenotypes paralleling those observed in human patients across multiple organ systems. By reversing knockdown after clinical features appear, we were able to determine to what extent observed phenotypes represent reversible cellular dysfunction. Remarkably, upon restoration of near wild-type FXN levels, we observed significant recovery of function, associated pathology and transcriptomic dysregulation even after substantial motor dysfunction and pathology were observed. This model will be of broad utility in therapeutic development and in refining our understanding of the relative contribution of reversible cellular dysfunction at different stages in disease