Phytoremediation of light non-aqueous phase liquids

Non-aqueous phase liquids (NAPLs) are persistent sources of contamination in the ground, providing a long-term supply of dissolved phase contamination and taking significant periods to dissipate naturally. Light NAPLs (LNAPLs) take the form of a separate phase within the ground, often as individual ganglia in pore spaces within the capillary zone such that the contaminated region is diffuse and comprised of many unconnected small contaminant sources. Consequently, remedial action is challenging and success may be limited to ex-situ remediation techniques. The ability of plants to phytoremediate dissolved-phase contamination is well known, but the impact of LNAPLs on plant growth and subsequent contaminant behaviour is largely unknown. Experimental work with ryegrass (Lolium perenne) is presented, exploring the impact of the physical presence of an LNAPL (mineral oil) on plant growth, root distribution and oil removal. The presence of the oil was found to significantly impact root biomass and distribution, leading to zones of increased root growth alongside decreased shoot growth. Significant removal of the LNAPL was noted in both hydroponic conditions and planted soil

Enhanced genome assembly and a new official gene set for Tribolium castaneum

Background. The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. Results. Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. Conclusions. The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS).\ud \ud METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response.\ud \ud RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay.\ud \ud CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS

Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin

To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin–streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice

Enhanced genome assembly and a new official gene set for Tribolium castaneum.

BACKGROUND:The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS:Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS:The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis