Gene expression profiling to characterize sediment toxicity – a pilot study using Caenorhabditis elegans whole genome microarrays

BACKGROUND: Traditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants. RESULTS: In this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment. CONCLUSION: This study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments

Publisher Correction: Deep learning enables fast and dense single-molecule localization with high accuracy

In the version of this Article initially published, Jacob H. Macke and Jonas Ries were not listed as corresponding authors. Their contact information and designation as corresponding authors are now included. The error has been corrected in the online version of the Article

Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage ϕBT1

We have established the integrase of the Streptomyces phage ϕBT1 as a tool for eukaryotic genome manipulation. We show that the ϕBT1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in Schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. We show that the ϕBT1 integrase can be used in conjunction with Cre recombinase to promote the iterative integration of transgenic DNA. We describe five cycles of iterative integration of a candidate mouse centromeric sequence 80 kb in length into a human mini-chromosome within a human-Chinese hamster hybrid cell line. These results establish the generality of the iterative site-specific integration technique

Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate

Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids

Generation of Long Insert Pairs Using a Cre-LoxP Inverse PCR Approach

Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput. We developed a new approach, Cre-LoxP Inverse PCR Paired-End (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) the use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends and to improve the self-ligation efficiency. We have successfully created CLIP-PE libraries up to 22 kb that are rich in informative read pairs and low in small fragment background. These libraries have demonstrated the ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms

Treatment of non-small cell lung cancer with intensity-modulated radiation therapy in combination with cetuximab: the NEAR protocol (NCT00115518)

BACKGROUND: Even today, treatment of Stage III NSCLC still poses a serious challenge. So far, surgical resection is the treatment of choice. Patients whose tumour is not resectable or who are unfit to undergo surgery are usually referred to a combined radio-chemotherapy. However, combined radio-chemotherapeutic treatment is also associated with sometimes marked side effects but has been shown to be more efficient than radiation therapy alone. Nevertheless, there is a significant subset of patients whose overall condition does not permit administration of chemotherapy in a combined-modality treatment. It could be demonstrated though, that NSCLCs often exhibit over-expression of EGF-receptors hence providing an excellent target for the monoclonal EGFR-antagonist cetuximab (Erbitux(®)) which has already been shown to be effective in colorectal as well as head-and-neck tumours with comparatively mild side-effects. METHODS/DESIGN: The NEAR trial is a prospective phase II feasibility study combining a monoclonal EGF-receptor antibody with loco-regional irradiation in patients with stage III NSCLC. This trial aims at testing the combination's efficacy and rate of development of distant metastases with an accrual of 30 patients. Patients receive weekly infusions of cetuximab (Erbitux(®)) plus loco-regional radiation therapy as intensity-modulated radiation therapy. After conclusion of radiation treatment patients continue to receive weekly cetuximab for 13 more cycles. DISCUSSION: The primary objective of the NEAR trial is to evaluate toxicities and feasibility of the combined treatment with cetuximab (Erbitux(®)) and IMRT loco-regional irradiation. Secondary objectives are remission rates, 3-year-survival and local/systemic progression-free survival

Cisgenesis and intragenesis as new strategies for crop improvement

Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation

Murid Herpesvirus-4 Exploits Dendritic Cells to Infect B Cells

Dendritic cells (DCs) play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4), infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells