Cardiac gene expression profiling – the quest for an atrium-specific biomarker

Biomarkers are gaining increasing interest to predict risk but also to aid in diagnostics. Tissue-specific biomarkers are of utmost importance to detect diseases of respective organs. As of yet there are no atriumspecific biomarkers for risk stratification of atrial disease, such as atrial fibrillation. Bioinformatics such as mRNA microarrays can help to detect tissue-enriched and possibly tissue-specific expressed genes that can be targets for biomarkers. We describe an approach to identify genes preferably expressed in atrial cardiomyocytes compared with ventricular cardiomyocytes by RNA microarray and confirmed by quantitative real-time polymerase chain reaction. By this approach we identified several atrium-enriched genes but also ventricle-enriched genes. As expected atrial natriuretic peptide (ANP) mRNA showed higher expression in atrial cardiomyocytes while with adrenergic stimulation expression was almost as high in ventricular as in atrial cells. Brain-type natriuretic peptide (BNP), however, was not different between atrial and ventricular cells giving a possible explanation for increased levels of NT-proBNP in atrial fibrillation patients. Interesting identified candidates are serpine1 and ltbp2 as atrium-enriched genes whereas alpha-adrenergic receptor subtype 1b and S100A1 expression was significantly higher in ventricular cells. The identified genes need to be confirmed in human tissue and might ultimately be tested as potential biomarkers for atrial stress. (Neth Heart J 2010;18:610–4.)     

Estimating demographic parameters for loggerhead sea turtles using mark–recapture data and a multistate model

The survival for adult loggerhead sea turtles from a saturation tagging study on Bald Head Island, NC, USA, was estimated using a multistate model with unobservable states to relax assumptions that are violated when survival is estimated from multistate models and produce more accurate estimates of survival, recapture, and breeding transition probabilities. The influence of time, trap dependence, and low site fidelity to the study nesting beach on survival and recapture were examined. The best model given the data included an imprecise site-fidelity effect on survival, constrained the reproductive cycle to 4 years, and contained a time effect on recapture rates. The estimate of annual survival for adult females was of 0.85, producing the highest estimate in the literature for loggerhead sea turtles. Multistate models should be applied to other nesting beach data for sea turtles to improve survival estimates and in turn the ability to model and manage populations.     

Attenuator LRR – a regulatory tool for modulating gene expression in Gram-positive bacteria

With the rapid development of synthetic biology in recent years, particular attention has been paid to RNA devices, especially riboswitches, because of their significant and diverse regulatory roles in prokaryotic and eukaryotic cells. Due to the limited performance and context-dependence of riboswitches, only a few of them (such as theophylline, tetracycline and ciprofloxacin riboswitches) have been utilized as regulatory tools in biotechnology. In the present study, we demonstrated that a ribosome-dependent ribo-regulator, LRR, discovered in our previous work, exhibits an attractive regulatory performance. Specifically, it offers a 60-fold change in expression in the presence of retapamulin and a low level of leaky expression of about 1–2% without antibiotics. Moreover, LRR can be combined with different promoters and performs well in Bacillus thuringiensis, B. cereus, B. amyloliquefaciens, and B. subtilis. Additionally, LRR also functions in the Gram-negative bacterium Escherichia coli. Furthermore, we demonstrate its ability to control melanin metabolism in B. thuringiensis BMB171. Our results show that LRR can be applied to regulate gene expression, construct genetic circuits and tune metabolic pathways, and has great potential for many applications in synthetic biology.     

Laboratory information management system for membrane protein structure initiative – from gene to crystal

Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.     

YIpDCE1 – an integrating plasmid for dual constitutive expression in yeast

YIpDCE1 (Dual Constitutive Expression), a novel Saccharomyces cerevisiae integrating plasmid, constitutively expresses two genes under the control of separate phosphoglycerate kinase promoters. YIpDCE1 contains the complete ADE2 gene which can be used as a marker for selecting integrants at mutant ade2 loci commonly present in laboratory yeast strains. The YIpDCE1 plasmid can be inserted into the ade2-101 locus of the HF7c strain used in two hybrid screens. Thus it could be useful for analysis of two hybrid interactions that occur in the context of additional protein components (e.g. modifying enzymes such as kinases or phosphatases, or multimeric complexes consisting of three or four distinct protein components). YIpDCE1 has been used to create strains simultaneously overexpressing the permease (ftr1) and oxidase (fet3) components of the yeast high-affinity iron uptake system. This confers constitutive high-affinity iron uptake on the transformed strains, bypassing the normal regulatory mechanisms.     

Direct screening for high-level expression of an introduced α-amylase gene in plants

A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature -amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for -amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlated with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.     

inGeno – an integrated genome and ortholog viewer for improved genome to genome comparisons

Background  

Systematic genome comparisons are an important tool to reveal gene functions, pathogenic features, metabolic pathways and genome evolution in the era of post-genomics. Furthermore, such comparisons provide important clues for vaccines and drug development. Existing genome comparison software often lacks accurate information on orthologs, the function of similar genes identified and genome-wide reports and lists on specific functions. All these features and further analyses are provided here in the context of a modular software tool "inGeno" written in Java with Biojava subroutines.     

Integrative factor analysis — An unsupervised method for quantifying cross-study consistency of gene expression data

Integrative analyses of multiple gene expression studies are frequently performed. In the setting of two studies, integrative correlation (IGC) can be used to assess the consistency of co-expression of a given gene. For three or more studies, an extension of IGC gives a global score per gene. We propose to extend IGC and use factor analysis to assess the study-specific consistency of co-expression of genes when there are three or more studies, possibly on different platforms. Our method is able to identify studies whose expression patterns are different from others. Filtering genes based on our score is shown to improve the concordance of association with phenotype across studies.     

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

Background  

Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors.     

Gene expression in reproductive organs of tsetse females – initial data in an approach to reduce fecundity

Background

Tsetse flies are vectors of African trypanosomes, and their vectorial capacity results in a major public health emergency and vast economic losses in sub-Saharan Africa. Given the limited ability of trypanosome prevention and eradication, tsetse vectors remain major targets of control efforts. Larvae of all three instars are developed in mothers’ uteri, nourished through milk, and ‘larviposited’ shortly before pupation. The past few years have witnessed the emergence of approaches based on knockdown of genes involved in milk production, resulting in a significant reduction of fecundity.

Results

In order to identify further genes applicable in the control of tsetse flies, we determined the expression of protein-coding genes in ovaries and uteri from both virgin and heavily pregnant Glossina morsitans morsitans females. Comparison of expression profiles allowed us to identify candidate genes with increased expression in pregnant individuals. Lists with the highest increases include genes involved in oocyte and embryonic development, or nourishment. Maximum ovarian fold change does not exceed 700, while the highest uterine fold change reaches to more than 4000. Relatively high fold changes of two neuropeptide receptors (for corazonin and myosuppressin) propose the corresponding genes alternative targets.

Conclusions

Given the higher fold changes in the uterus, targeting gene expression in this tissue may result in a more evident reduction of fecundity. However, ovaries should not be neglected, as manifested by several genes with top fold changes involved in early developmental stages. Apart from focusing on the highest fold changes, neuropeptide receptors with moderate increases in expression should be also verified as targets, given their roles in mediating the tissue control. However, this data needs to be considered initial, and the potential of these genes in affecting female fecundity needs to be verified experimentally.

     

SC²ATmd: a tool for integration of the figure of merit with cluster analysis for gene expression data

Standard and Consensus Clustering Analysis Tool for Microarray Data (SC2ATmd) is a MATLAB-implemented application specifically designed for the exploration of microarray gene expression data via clustering. Implementation of two versions of the clustering validation method figure of merit allows for performance comparisons between different clustering algorithms, and tailors the cluster analysis process to the varying characteristics of each dataset. Along with standard clustering algorithms this application also offers a consensus clustering method that can generate reproducible clusters across replicate experiments or different clustering algorithms. This application was designed specifically for the analysis of gene expression data, but may be used with any numerical data as long as it is in the right format. AVAILABILITY: SC2ATmd may be freely downloaded from http://www.compbiosci.wfu.edu/tools.htm.     

Rhizocompetence and antagonistic activity towards genetically diverse Ralstonia solanacearum strains – an improved strategy for selecting biocontrol agents

Bacterial wilt caused by Ralstonia solanacearum is a serious threat for agricultural production in China. Eight soil bacterial isolates with activity against R. solanacearum TM15 (biovar 3) were tested in this study for their in vitro activity towards ten genetically diverse R. solanacearum isolates from China. The results indicated that each antagonist showed remarkable differences in its ability to in vitro antagonize the ten different R. solanacearum strains. Strain XY21 (based on 16S rRNA gene sequencing affiliated to Serratia) was selected for further studies based on its in vitro antagonistic activity and its excellent rhizocompetence on tomato plants. Under greenhouse conditions XY21 mediated biocontrol of tomato wilt caused by seven different R. solanacearum strains ranged from 19 to 70 %. The establishment of XY21 and its effects on the bacterial community in the tomato rhizosphere were monitored by denaturing gradient gel electrophoresis of 16S rRNA gene fragments PCR-amplified from total community DNA. A positive correlation of the in vitro antagonistic activities of XY21 and the actual biocontrol efficacies towards seven genetically different R. solanacearum strains was found and further confirmed by the efficacy of XY21 in controlling bacterial wilt under field conditions.     

Robust computational reconstitution – a new method for the comparative analysis of gene expression in tissues and isolated cell fractions

Background  

Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis.