Short tandem repeat profiling provides an international reference standard for human cell lines

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines

Background: MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. Methods: An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. Results: Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. Conclusions: The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance. Keywords Akt, breast cancer cells, endocrine resistance, insulin, MAPK, MCF-7 cells, mTOR, oestrogen, oestrogen-deprived, PI3K, picropodophyllin, rapamycin, T-47-D cells, ZR-75-1 cells

Using learning networks to understand complex systems: a case study of biological, geophysical and social research in the Amazon

Developing high-quality scientific research will be most effective if research communities with diverse skills and interests are able to share information and knowledge, are aware of the major challenges across disciplines, and can exploit economies of scale to provide robust answers and better inform policy. We evaluate opportunities and challenges facing the development of a more interactive research environment by developing an interdisciplinary synthesis of research on a single geographic region. We focus on the Amazon as it is of enormous regional and global environmental importance and faces a highly uncertain future. To take stock of existing knowledge and provide a framework for analysis we present a set of mini-reviews from fourteen different areas of research, encompassing taxonomy, biodiversity, biogeography, vegetation dynamics, landscape ecology, earth-atmosphere interactions, ecosystem processes, fire, deforestation dynamics, hydrology, hunting, conservation planning, livelihoods, and payments for ecosystem services. Each review highlights the current state of knowledge and identifies research priorities, including major challenges and opportunities. We show that while substantial progress is being made across many areas of scientific research, our understanding of specific issues is often dependent on knowledge from other disciplines. Accelerating the acquisition of reliable and contextualized knowledge about the fate of complex pristine and modified ecosystems is partly dependent on our ability to exploit economies of scale in shared resources and technical expertise, recognise and make explicit interconnections and feedbacks among sub-disciplines, increase the temporal and spatial scale of existing studies, and improve the dissemination of scientific findings to policy makers and society at large. Enhancing interaction among research efforts is vital if we are to make the most of limited funds and overcome the challenges posed by addressing large-scale interdisciplinary questions. Bringing together a diverse scientific community with a single geographic focus can help increase awareness of research questions both within and among disciplines, and reveal the opportunities that may exist for advancing acquisition of reliable knowledge. This approach could be useful for a variety of globally important scientific questions.

Between the lines: Konrad Wolf's 'Mama, ich lebe' and the dilemmas of betrayal

The article offers a close reading of Konrad Wolf’s anti-fascist Second World War film 'Mama, ich lebe' (DEFA, 1977). 'Mama, ich lebe', like all East German films about the Nazi past, deals with the re-founding of post-war Germany. Unlike the usual approach which focused on political redemption of the past crimes, Wolf’s approach explores rupture and failure of political agency as the pre-condition for a new beginning. The rupture is effected by the defection of four Wehrmacht soldiers who decide to cooperate with the Soviet enemy. Their betrayal of the national collective is ethically motivated and arises from their responsibility for the Soviet ‘other’. Its radicalness opens up a moment of utopian freedom and conciliation for the traitors. Yet the back side of betrayal is insecurity and confliction with regard to their role and roots. While the four meet their role as traitors with self-deception about their ambivalent position, they are eventually forced to acknowledge their position as one of self-defeat. Their ‘ethical betrayal’ (Parikh 2009) does therefore not lead to utopian fulfilment but to the traitors’ expiatory sacrifice as the only form of accountability and self-justification. In Wolf’s film antifascism as a tale of political redemption is thus revised and becomes a tale of necessary individual atonement.

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.

The glacial question, unsolved: a specimen commentary on lines 1-31

A commentary on J.H. Prynne's poem 'The Glacial Question, Unsolved', from The White Stones (1969)

HPMA copolymer-aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer–AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM–Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 × 10− 8 M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2–0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [3H]androstenedione both AGM and HPMA copolymer–GFLG–AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

A review of Holocene rainforest ecotonal dynamics at opposite ends of the Amazon - Bolivia versus Colombia

In this review paper, the aim is to compare and contrast fossil pollen evidence for Holocene rainforest ecotonal dynamics at opposite ends of the Amazon basin – the southern ecotone in NE lowland Bolivia versus the northern ecotone in lowland Colombia. During the Holocene, tropical South America experienced major changes in precipitation (Silva Dias et al. 2009). Consideration of Amazonian rainforest dynamics over this time-frame may therefore provide important insights into rainforest responsiveness to climate change.

Generation of nonvernal-obligate, faster-cycling Noccaea caerulescens lines through fast neutron mutagenesis

Noccaea caerulescens (formerly Thlaspi caerulescens) is a widely studied metal hyperaccumulator. However, molecular genetic studies are challenging in this species because of its vernal-obligate biennial life cycle of 7-9 months. Here, we describe the development of genetically stable, faster cycling lines of N. caerulescens which are nonvernal-obligate. A total of 5500 M(0) seeds from Saint Laurent Le Minier (France) were subjected to fast neutron mutagenesis. Following vernalization of young plants, 79 of plants survived to maturity. In all, 80 000 M(2) lines were screened for flowering in the absence of vernalization. Floral initials were observed in 35 lines, with nine flowering in < 12 wk. Two lines (A2 and A7) were selfed to the M(4) generation. Floral initials were observed 66 and 87 d after sowing (DAS) in A2 and A7, respectively. Silicle development occurred for all A2 and for most A7 at 92 and 123 DAS, respectively. Floral or silicle development was not observed in wild-type (WT) plants. Leaf zinc (Zn) concentration was similar in WT, A2 and A7 lines. These lines should facilitate future genetic studies of this remarkable species. Seed is publicly available through the European Arabidopsis Stock Centre (NASC).

Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates, and not by zinc-transporter activity

Zinc (Zn)-deficient soils constrain rice (Oryza sativa) production and cause Zn malnutrition. The identification of Zn-deficiency-tolerant rice lines indicates that breeding might overcome these constraints. Here, we seek to identify processes underlying Zn-deficiency tolerance in rice at the physiological and transcriptional levels. A Zn-deficiency-tolerant line RIL46 acquires Zn more efficiently and produces more biomass than its nontolerant maternal line (IR74) at low Zn(ext) under field conditions. We tested if this was the result of increased expression of Zn(2+) transporters; increased root exudation of deoxymugineic acid (DMA) or low-molecular-weight organic acids (LMWOAs); and/or increased root production. Experiments were performed in field and controlled environment conditions. There was little genotypic variation in transcript abundance of Zn-responsive root Zn(2+)-transporters between the RIL46 and IR74. However, root exudation of DMA and LMWOA was greater in RIL46, coinciding with increased root expression of putative ligand-efflux genes. Adventitious root production was maintained in RIL46 at low Zn(ext), correlating with altered expression of root-specific auxin-responsive genes. Zinc-deficiency tolerance in RIL46 is most likely the result of maintenance of root growth, increased efflux of Zn ligands, and increased uptake of Zn-ligand complexes at low Zn(ext); these traits are potential breeding targets.

A GCSS model intercomparison for a tropical squall line observed during toga-coare. II: Intercomparison of single-column models and a cloud-resolving model

This paper presents single-column model (SCM) simulations of a tropical squall-line case observed during the Coupled Ocean-Atmosphere Response Experiment of the Tropical Ocean/Global Atmosphere Programme. This case-study was part of an international model intercomparison project organized by Working Group 4 ‘Precipitating Convective Cloud Systems’ of the GEWEX (Global Energy and Water-cycle Experiment) Cloud System Study. Eight SCM groups using different deep-convection parametrizations participated in this project. The SCMs were forced by temperature and moisture tendencies that had been computed from a reference cloud-resolving model (CRM) simulation using open boundary conditions. The comparison of the SCM results with the reference CRM simulation provided insight into the ability of current convection and cloud schemes to represent organized convection. The CRM results enabled a detailed evaluation of the SCMs in terms of the thermodynamic structure and the convective mass flux of the system, the latter being closely related to the surface convective precipitation. It is shown that the SCMs could reproduce reasonably well the time evolution of the surface convective and stratiform precipitation, the convective mass flux, and the thermodynamic structure of the squall-line system. The thermodynamic structure simulated by the SCMs depended on how the models partitioned the precipitation between convective and stratiform. However, structural differences persisted in the thermodynamic profiles simulated by the SCMs and the CRM. These differences could be attributed to the fact that the total mass flux used to compute the SCM forcing differed from the convective mass flux. The SCMs could not adequately represent these organized mesoscale circulations and the microphysicallradiative forcing associated with the stratiform region. This issue is generally known as the ‘scale-interaction’ problem that can only be properly addressed in fully three-dimensional simulations. Sensitivity simulations run by several groups showed that the time evolution of the surface convective precipitation was considerably smoothed when the convective closure was based on convective available potential energy instead of moisture convergence. Finally, additional SCM simulations without using a convection parametrization indicated that the impact of a convection parametrization in forced SCM runs was more visible in the moisture profiles than in the temperature profiles because convective transport was particularly important in the moisture budget.

Pre-Columbian raised-field agriculture and land use in the Bolivian Amazon

We present an integrated palaeoecological and archaeobotanical study of pre-Columbian raised-field agriculture in the Llanos de Moxos, a vast seasonally inundated forest–savanna mosaic in the Bolivian Amazon. Phytoliths from excavated raised-field soil units, together with pollen and charcoal in sediment cores from two oxbow lakes, were analysed to provide a history of land use and agriculture at the El Cerro raised-field site. The construction of raised fields involved the removal of savanna trees, and gallery forest was cleared from the area by AD 310. Despite the low fertility of Llanos de Moxos soils, we determined that pre-Columbian raised-field agriculture sufficiently improved soil conditions for maize cultivation. Fire was used as a common management practice until AD 1300, at which point, the land-use strategy shifted towards less frequent burning of savannas and raised fields. Alongside a reduction in the use of fire, sweet potato cultivation and the exploitation of Inga fruits formed part of a mixed resource strategy from AD 1300 to 1450. The pre-Columbian impact on the landscape began to lessen around AD 1450, as shown by an increase in savanna trees and gallery forest. Although agriculture at the site began to decline prior to European arrival, the abandonment of raised fields was protracted, with evidence of sweet potato cultivation occurring as late as AD 1800.