Cloning and characterization of excision repair genes

For all living organisms, it is of vital importance to maintain intact the genetic information stored in the nucleotide sequence of DNA. Numerous environmental and genotoxic agents can affect the DNA and lead to, for example, mutagenesis or carcinogenesis. Study of the mechanism of UV-carcinogenesis has become even more pressing given recent concerns about atmospheric ozone depletion (Mimms, 1994; Watson, 1995), because such atmospheric alterations would result in increased UVB at the earth's surface. Carcinogenesis appears to be a multistep process through which normal cells progress from benign, through transitional stages to the fully malignant forms of cancer by the gradual accumulation of genetic errors (Bishop, 1995). Therefore, normal cells have an intricate quality control mechanism that recognizes and mends damage to the DNA helix

Gene expression and functional annotation of the human and mouse choroid plexus epithelium

Background: The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods: We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results: Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion: Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and man differ with respect to transport and metabolic functions

IReport: A generalised Galaxy solution for integrated experimental reporting

Background: Galaxy offers a number of visualisation options with components, such as Trackster, Circster and Galaxy Charts, but currently lacks the ability to easily combine outputs from different tools into a single view or report. A number of tools produce HTML reports as output in order to combine the various output files from a single tool; however, this requires programming and knowledge of HTML, and the reports must be custom-made for each new tool.Findings: We have developed a generic and flexible reporting tool for Galaxy, iReport, that allows users to create interactive HTML reports directly from the Galaxy UI, with the ability to combine an arbitrary number of outputs from any number of different tools. Content can be organised into different tabs, and interactivity can be added to components. To demonstrate the capability of iReport we provide two publically available examples, the first is an iReport explaining about iReports, created for, and using content from the recent Galaxy Community Conference 2014. The second is a genetic report based on a trio analysis to determine candidate pathogenic variants which uses our previously developed Galaxy toolset for whole-genome NGS analysis, CGtag. These reports may be adapted for outputs from any sequencing platform and any results, such as omics data, non-high throughput results and clinical variables.Conclusions: iReport provides a secure, collaborative, and flexible web-based reporting system that is compatible with Galaxy (and non-Galaxy) generated content. We demonstrate its value with a real-life example of reporting genetic trio-analysis

In silico analysis of the molecular machinery underlying aqueous humor production: Potential implications for glaucoma

Background: The ciliary body epithelia (CBE) of the eye produce the aqueous humor (AH). The equilibrium between the AH production by the CBE and the outflow through the trabecular meshwork ultimately determines the intraocular pressure (IOP). An increased IOP is a major risk factor for primary open angle glaucoma (POAG). This study aims to elucidate the molecular machinery of the most important function of the CBE: the AH production and composition, and aims to find possible new molecular clues for POAG and AH production-lowering drugs.Methods: We performed a gene expression analysis of the non-pigmented (NPE) and pigmented epithelia (PE) of the human CBE of post mortem eyes. We used 44 k Agilent microarrays against a common reference design. Functional annotations were performed with the Ingenuity knowledge database.Results: We built a molecular model of AH production by combining previously published physiological data with our current genomic expression data. Next, we investigated molecular CBE transport features which might influence AH composition. These features included caveolin- and clathrin vesicle-mediated transport of large biomolecules, as well as a range of substrate specific transporters. The presence of these transporters implies that, for example, immunoglobins, thyroid hormone, prostaglandins, cholesterol and vitamins can be secreted by the CBE along with the AH. In silico, we predicted some of the molecular apical interactions between the NPE and PE, the side where the two folded epithelia face each other. Finally, we found high expression of seven POAG disease genes in the plasma membrane of extracellular space of the CBE, namely APOE, CAV1, COL8A2, EDNRA, FBN1, RFTN1 and TLR4 and we found possible new targets for AH lowering drugs in the AH.Conclusions: The CBE expresses many transporters, which account for AH production and/or composition. Some of these entries have also been associated with POAG. We hypothesize that the CBE may play a more prominent role than currently thought in the pathogenesis of POAG, for example by changing the composition of AH

Towards a rational design of faecal transplant analogues

Faecal transplants (microbiota transfer) have shown to be promising therapies having a wide range of therapeutic applications. However, current safety considerations hamper further valorisation. As such, well designed faecal transplant analogues provide an interesting alternative to minimize possible safety aspects. However, to date little knowledge on how to rationally design such analogues exists. Here, we show by applying first order basic graph theory that such analogues dedicated to restoring a specific physiological functionality (a microbial guild) should consist of 5–6 species to maximize stability, efficiency, and minimize safety issues and production costs

The Coevolution of Phycobilisomes: Molecular Structure Adapting to Functional Evolution

Phycobilisome is the major light-harvesting complex in cyanobacteria and red alga. It consists of phycobiliproteins and their associated linker peptides which play key role in absorption and unidirectional transfer of light energy and the stability of the whole complex system, respectively. Former researches on the evolution among PBPs and linker peptides had mainly focused on the phylogenetic analysis and selective evolution. Coevolution is the change that the conformation of one residue is interrupted by mutation and a compensatory change selected for in its interacting partner. Here, coevolutionary analysis of allophycocyanin, phycocyanin, and phycoerythrin and covariation analysis of linker peptides were performed. Coevolution analyses reveal that these sites are significantly correlated, showing strong evidence of the functional and structural importance of interactions among these residues. According to interprotein coevolution analysis, less interaction was found between PBPs and linker peptides. Our results also revealed the correlations between the coevolution and adaptive selection in PBS were not directly related, but probably demonstrated by the sites coupled under physical-chemical interactions

Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors

Enzymatic photoreactivation is a DNA repair mechanism that removes UV- induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light. Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes. We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes. The human gene product is more similar to plant blue-light receptors within class I ph

ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS

Background: Sequence analysis of immunoglobulin heavy chain (IGH) gene rearrangements and frequency analysis is a powerful tool for studying the immune repertoire, immune responses and immune dysregulation in health and disease. The challenge is to provide user friendly, secure and reproducible analytical services that are available for both small and large laboratories which are determining VDJ repertoire using NGS technology. Results: In this study we describe ImmunoGlobulin Galaxy (IGGalaxy)- a convenient web based application for analyzing next-generation sequencing results and reporting IGH gene rearrangements for both repertoire and clonality studies. IGGalaxy has two analysis options one using the built in igBLAST algorithm and the second using output from IMGT; in either case repertoire summaries for the B-cell populations tested are available. IGGalaxy supports multi-sample and multi-replicate input analysis for both igBLAST and IMGT/HIGHV-QUEST. We demonstrate the technical validity of this platform using a standard dataset, S22, used for benchmarking the performance of antibody alignment utilities with a 99.9 % concordance with previous results. Re-analysis of NGS data from our samples of RAG-deficient patients demonstrated the validity and user friendliness of this tool. Conclusions: IGGalaxy provides clinical researchers with detailed insight into the repertoire of the B-cell population per individual sequenced and between control and pathogenic genomes. IGGalaxy was developed for 454 NGS results but is capable of analyzing alternative NGS data (e.g. Illumina, Ion Torrent). We demonstrate the use of a Galaxy virtual machine to determine the VDJ repertoire for reference data and from B-cells taken from immune deficient patients. IGGalaxy is available as a VM for download and use on a desktop PC or on a server