Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets

Aerodynamic investigations of ventilated brake discs.

The heat dissipation and performance of a ventilated brake disc strongly depends on the aerodynamic characteristics of the flow through the rotor passages. The aim of this investigation was to provide an improved understanding of ventilated brake rotor flow phenomena, with a view to improving heat dissipation, as well as providing a measurement data set for validation of computational fluid dynamics methods. The flow fields at the exit of four different brake rotor geometries, rotated in free air, were measured using a five-hole pressure probe and a hot-wire anemometry system. The principal measurements were taken using two-component hot-wire techniques and were used to determine mean and unsteady flow characteristics at the exit of the brake rotors. Using phase-locked data processing, it was possible to reveal the spatial and temporal flow variation within individual rotor passages. The effects of disc geometry and rotational speed on the mean flow, passage turbulence intensity, and mass flow were determined. The rotor exit jet and wake flow were clearly observed as characterized by the passage geometry as well as definite regions of high and low turbulence. The aerodynamic flow characteristics were found to be reasonably independent of rotational speed but highly dependent upon rotor geometry

Proteomic Insights into the Hidden World of Phloem Sap Feeding

The physical interface between a phloem-feeding insect and its host plant is a single cell buried deep within the plant tissue. As such, the molecular interactions between these notorious agricultural pests and the crop plants upon which they feed are diffi cult to study. ‘Omic’ technologies have proved crucial in revealing some of the fascinating detail of the molecular interplay between these partners. Here we review the role of proteomics in identifying putative components of the secreted saliva of phloem-feeding insects, particularly aphids, and discuss the limited knowledge concerning the function of these proteins

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

A Functional Genomics Approach Identifies Candidate Effectors from the Aphid Species Myzus persicae (Green Peach Aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)–mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes

International laboratory comparison of influenza microneutralization assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) influenza viruses by CONSISE

The microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HAMNassay protocols to enable better correlation of these assays in the future

Semiconductor dosimetry of epithermal neutron beams for Boron neutron capture therapy

Boron neutron capture therapy (BNCT) is a binary targeted therapy that uses suitably designed pharmaceuticals to deliver (superscript 10)B to tumor cells. The region is then irradiated with neutrons and neutron capture by the (superscript 10)B nucleus leads to the emission of an alpha particle and lithium ion. These have very short ranges similar to the dimensions of a biological cell and therefore the technique could have potential for selective killing of tumour cells. In order to achieve adequate neutron fluxes at the site of the tumour epithermal (0.5 eV � 10s of keV) neutron beams are used. A review of the general details of BNCT is presented in this thesis. This thesis investigates the use of two semiconductor devices for measuring the neutron and gamma dose components involved in epithermal neutron beams used for BNCT. The silicon lattice in PIN diodes undergoes displacement damage when irradiated with neutrons. This leads to a change in the forward bias voltage of the diode that is proportional to the neutron dose received. To verify that the energy dependence of this effect follows the published silicon displacement damage KERMA (Kinetic Energy Released per Mass of Absorber) data measurements were performed using quasi-monoenergetic neutrons obtained from a Van de Graff accelerator (Ansto) in the energy range from 90 keV � 890 keV. These measurements were in agreement with the published data for silicon displacement damage KERMA. A sensitivity factor for the diodes was also derived from these measurements. The thermal neutron sensitivity of the PIN diodes was then determined using the TC-10 thermal neutron column on the Moata reactor at Ansto. The sensitivity results were in general agreement with the Van de Graff derived sensitivity factor. Since the silicon damage KERMA is not the same as the tissue KERMA function PIN diodes are not intrinsically tissue equivalent. A Monte Carlo (MNCP) ideal beam study was undertaken to see if for some limited energy range tissue dose could be parameterised in terms of silicon damage dose and foil activation. This was found to be approximately true for neutron energies from thermal to 100 keV. Coefficients are given that allow tissue dose to be determined on the basis of a single PIN diode and activation foil measurement in spectra where the maximum neutron energy is 100 keV or less. MOSFETS can be used as gamma radiation dosimeters by measuring the change in threshold voltage (simplistically understood as the potential applied to the gate to initiate current flow from source to drain electrodes) that occurs when they are exposed to radiation. The MOSFETs used in this study were characterised using a Varian 2100C medical linac beam and low energy x-rays from a superficial x-ray unit. The sensitivity of the MOSFETs was measured with different potentials applied to the gate during irradiation. Depth dose profiles in 6 MV x-ray beams were measured and found to be in good agreement with both ionisation chamber measurements and MCNP simulations. This good agreement was also obtained for the buildup region. Although the silicon oxide layer of the MOSFET is not intrinsically very sensitive to neutron irradiation the presence of encapsulating materials leads to the generation of secondary photons and electrons which lead to shifts in threshold voltage and therefore confound gamma ray measurements in mixed neutron / gamma fields. To determine the energy dependant neutron response function of the MOSFET a detailed MCNP simulation was used. A lithiated shield was also incorporated into this model. The calculated neutron response functions were used to correct for neutron contributions to MOSFET measurements in mixed fields. MOSFET thermal neutron responses were measured using a series of measurements with MOSFETS both with and without lithiated covers exposed in the the Moata thermal neutron column at Ansto. The measurements were repeated with various gate potentials. The gamma doses measured were consistent with gamma doses measured using paired ionisation chambers. The Petten HB11 facility is briefly described as are phantoms and MCNP models fabricated by S Wallace for an associated work. Measurements using PIN diodes and MOSFETs in phantoms exposed in the HB11 beam are described. Foil activation data is compared to MCNP calculations to validate the MCNP models used. This thesis presents a number of results that have been recalculated in more detail and with various parameters changed. In particular the effect of variations in phantom hydration have been incorporated as have response functions for MOSFET detectors and associated shields. PIN diode measurements in a Perspex cube phantom exposed in the HB11 beam show good agreement with MCNP calculated silicon displacement dose. Similarly good agreement is obtained for a cylindrical phantom filled with tissue equivalent gel when the hydrogen content of the gel in the original model is corrected for dehydration. Measurements in a more complex skull phantom show larger discrepancies between the experimental results and a MCNP simulation especially at depth. The discrepancies range from 25 � 300% in absolute terms but are only 2-3% of the maximum silicon dose. MOSFET measurements performed in the Perspex cube phantom using lithiated covers show excellent agreement with ionisation chamber measurements (also with lithiated covers). Measurements in a cylinder phantom and head phantom using lithium/perpex covered MOSFETs are compared with Monte Carlo calculations of induced gamma dose. In this case the measured gamma doses at approximately 2 cm depth appear to be too low. Further investigation involving a detailed MCNP simulation including the lithiated MOSFET covers in the model indicated that the covers suppress the thermal neutron flux at the measurement point and therefore the gamma dose is also reduced. Measurements at greater depths show a similar effect but to a lesser extent. The epithermal neutron beam at the Brookhaven Medical Research Reactor (BMRR) is described is described. PIN diode and MOSFET measurement results in a Perspex cube phantom are also presented. Reasonable agreement between calculated and measured PIN diode results is observed. MOSFET measurements show good agreement with the known percentage depth dose curve for the total gamma dose. However there is a discrepancy in the absolute magnitude of the measured gamma doses. It is proposed that this is also due to thermal neutron flux depression arising from the use of relatively thick lithiated neutron shields around the MOSFETS. In summary; It is demonstrated that PIN diodes could be useful for verifying treatment planning dose distributions in epithermal neutron beams. This includes the possibility of on line real-time measurements. They could also be used in conjunction with an activation foil to yield tissue equivalent dose measurements where the maximum neutron energy is less than 100 keV. Lithium shielded MOSFET measurements can be reconciled with calculated gamma dose distributions when the effect of flux depression is taken into account. However the perturbations introduced by the shield mean that the measured dose does not represent the dose at the measurement point in the absence of the shield. In order to use MOSFETs for gamma dosimetry in epithermal neutron beams different encapsulation is required to minimise neutron response and eliminate the need for lithiated covers. It is suggested that MCNP simulations of MOSFETs similar to the models in this thesis would provide an adequate tool for optimising the appropriate encapsulation

A method for measuring tissue-equivalent dose using a pin diode and activation foil in epithermal neutron beams with En \u3c 100 keV

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