Genomic Characterisation of Small Cell Lung Cancer Patient-Derived Xenografts Generated from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Specimens

Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63–188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.     

An extinct nestorid parrot (Aves,Psittaciformes, Nestoridae) from the Chatham Islands,New Zealand

We describe an extinct parrot from late Quaternary fossil bone deposits on the Chatham Islands, located c. 800 km east of mainland New Zealand. Mitochondrial DNA analyses and osteological characters confirm that the Chatham Islands parrot was a sister taxon to the New Zealand kaka (Nestor meridionalis Gmelin, 1788). The relatively large femur : humerus length ratio and broad pelvis of the Chatham Islands parrot indicate that it had a more terrestrial habit than the kaka. Stable dietary isotope analyses (δ ¹⁵N and δ ¹³C) of Chatham Islands parrot bones suggest that the species may have been mainly herbivorous, although further analyses are required to confirm this. The presence of Chatham Islands parrot bones in early midden deposits shows that the species persisted into the post‐settlement era, and became extinct possibly as a result of habitat loss, hunting pressure, and rat predation following initial Polynesian settlement of the islands (sometime between the 13^th and 16^th centuries AD). © 2014 The Linnean Society of London     

Implementing Individual Producer Responsibility for Waste Electrical and Electronic Equipment through Improved Financing

Under the European Union (EU) Waste Electrical and Electronics Equipment (WEEE) Directive, producers are responsible for financing the recycling of their products at end of life. A key intention of such extended producer responsibility (EPR) legislation is to provide economic incentives for producers to develop products that are easier to treat and recycle at end of life. Recent research has shown, however, that the implementation of EPR for WEEE has so far failed in this respect. Current WEEE systems calculate their prices according to simple mass‐based allocation of costs to producers, based on broad collection categories containing a mixture of different product types and brands. This article outlines two alternative approaches, which instead calculate charges for products sold by producers by classifying them according to their eventual end‐of‐life treatment requirements and cost. Worked examples indicate that these methods provide both effective and efficient frameworks for financing WEEE, potentially delivering financial incentives to producers substantial enough to affect their potential profitability and, as a likely consequence, the decisions relating to the design of their products. In particular they fulfill three important criteria required by the WEEE Directive: they can financially reward improved design, allocate costs of historic waste proportionately (on the basis of tonnes of new products sold), and provide sufficient financial guarantees against future waste costs and liabilities. They are also relatively practical for implementation because they are based solely on cost allocation and financing. Further research and investigation would be worthwhile to test and verify this approach using real‐world data and under various scenarios.     

Application of palaeogenetic techniques to historic mollusc shells reveals phylogeographic structure in a New Zealand abalone

Natural history collections worldwide contain a plethora of mollusc shells. Recent studies have detailed the sequencing of DNA extracted from shells up to thousands of years old and from various taphonomic and preservational contexts. However, previous approaches have largely addressed methodological rather than evolutionary research questions. Here, we report the generation of DNA sequence data from mollusc shells using such techniques, applied to Haliotis virginea Gmelin, 1791, a New Zealand abalone, in which morphological variation has led to the recognition of several forms and subspecies. We successfully recovered near-complete mitogenomes from 22 specimens including 12 dry-preserved shells up to 60 years old. We used a combination of palaeogenetic techniques that have not previously been applied to shell, including DNA extraction optimized for ultra-short fragments and hybridization-capture of single-stranded DNA libraries. Phylogenetic analyses revealed three major, well-supported clades comprising samples from: (1) The Three Kings Islands; (2) the Auckland, Chatham and Antipodes Islands; and (3) mainland New Zealand and Campbell Island. This phylogeographic structure does not correspond to the currently recognized forms. Critically, our nonreliance on freshly collected or ethanol-preserved samples enabled inclusion of topotypes of all recognized subspecies as well as additional difficult-to-sample populations. Broader application of these comparatively cost-effective and reliable methods to modern, historical, archaeological and palaeontological shell samples has the potential to revolutionize invertebrate genetic research.     

Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.