Microbiological influences on fracture surfaces of intact mudstone and the implications for geological disposal of radioactive waste

The significance of the potential impacts of microbial activity on the transport properties of host rocks for geological repositories is an area of active research. Most recent work has focused on granitic environments. This paper describes pilot studies investigating changes in transport properties that are produced by microbial activity in sedimentary rock environments in northern Japan. For the first time, these short experiments (39 days maximum) have shown that the denitrifying bacteria, Pseudomonas denitrificans, can survive and thrive when injected into flow-through column experiments containing fractured diatomaceous mudstone and synthetic groundwater under pressurized conditions. Although there were few significant changes in the fluid chemistry, changes in the permeability of the biotic column, which can be explained by the observed biofilm formation, were quantitatively monitored. These same methodologies could also be adapted to obtain information from cores originating from a variety of geological environments including oil reservoirs, aquifers and toxic waste disposal sites to provide an understanding of the impact of microbial activity on the transport of a range of solutes, such as groundwater contaminants and gases (e.g. injected carbon dioxide)

Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG.

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features

Hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival

Chronic myeloid leukemia (CML) stem/progenitor cells (SPC) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPC maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase dependent pathway mediated by the up-regulation of hsa-mir183, the down-regulation of its direct target EGR1 and, as a consequence, up-regulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPC. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPC, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication

Reconstruction of cell population dynamics using CFSE

Background: Quantifying cell division and death is central to many studies in the biological sciences. The fluorescent dye CFSE allows the tracking of cell division in vitro and in vivo and provides a rich source of information with which to test models of cell kinetics. Cell division and death have a stochastic component at the single-cell level, and the probabilities of these occurring in any given time interval may also undergo systematic variation at a population level. This gives rise to heterogeneity in proliferating cell populations. Branching processes provide a natural means of describing this behaviour. Results: We present a likelihood-based method for estimating the parameters of branching process models of cell kinetics using CFSE-labeling experiments, and demonstrate its validity using synthetic and experimental datasets. Performing inference and model comparison with real CFSE data presents some statistical problems and we suggest methods of dealing with them. Conclusion: The approach we describe here can be used to recover the (potentially variable) division and death rates of any cell population for which division tracking information is available

Generation of Breast Cancer Stem Cells through Epithelial-Mesenchymal Transition

Recently, two novel concepts have emerged in cancer biology: the role of so-called “cancer stem cells” in tumor initiation, and the involvement of an epithelial-mesenchymal transition (EMT) in the metastatic dissemination of epithelial cancer cells. Using a mammary tumor progression model, we show that cells possessing both stem and tumorigenic characteristics of “cancer stem cells” can be derived from human mammary epithelial cells following the activation of the Ras-MAPK pathway. The acquisition of these stem and tumorigenic characters is driven by EMT induction

Is there a cloud in the silver lining for imatinib?

Imatinib mesylate (Gleevec® or Glivec®), a small molecule tyrosine kinase inhibitor for the treatment of chronic myeloid leukaemia, has been said to herald the dawn of a new er-a of rationally designed, molecularly targeted oncotherapy. Lurking on the same new horizon, however, is the age-old spectre of drug resistance. This review sets the intoxicating clinical perspective against the more sobering laboratory evidence of such divergent mechanisms of imatinib resistance as gene amplification and stem cell quiescence. Polychemotherapy has already been considered to combat resistance, but a more innovative, as yet unformulated, approach may be advocated

Molecular properties of CD133+ glioblastoma stem cells derived from treatment-refractory recurrent brain tumors

Glioblastoma multiforme (GBM) remains refractory to conventional therapy. CD133+ GBM cells have been recently isolated and characterized as chemo-/radio-resistant tumor-initiating cells and are hypothesized to be responsible for post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM cells that resist treatment, we isolated CD133+ GBM cells from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM cells sorted from the CD133+ GBM spheres express SOX2 and CD44 and are capable of clonal self-renewal and dividing to produce fast-growing CD133− progeny, which form the major cell population within GBM spheres. Intracranial injection of purified CD133+, not CD133− GBM daughter cells, can lead to the development of YKL-40+ infiltrating tumors that display hypervascularity and pseudopalisading necrosis-like features in mouse brain. The molecular profile of purified CD133+ GBM cells revealed characteristics of neuroectoderm-like cells, expressing both radial glial and neural crest cell developmental genes, and portraying a slow-growing, non-differentiated, polarized/migratory, astrogliogenic, and chondrogenic phenotype. These data suggest that at least a subset of treated and recurrent GBM tumors may be seeded by CD133+ GBM cells with neural and mesenchymal properties. The data also imply that CD133+ GBM cells may be clinically indolent/quiescent prior to undergoing proliferative cell division (PCD) to produce CD133− GBM effector progeny. Identifying intrinsic and extrinsic cues, which promote CD133+ GBM cell self-renewal and PCD to support ongoing tumor regeneration may highlight novel therapeutic strategies to greatly diminish the recurrence rate of GBM

The in vitro activity of the tyrosine kinase inhibitor STI571 in BCR-ABL positive chronic myeloid leukaemia cells: synergistic interactions with anti-leukaemic agents.

Chronic myeloid leukaemia is typically characterised by the presence of dysregulated BCR-ABL tyrosine kinase activity, which is central to the oncogenic feature of being resistant to a wide range of cytotoxic agents. We have investigated whether the inhibition of this tyrosine kinase by the novel compound STI571 (formerly CGP57148B) would render K562, KU812 cell lines and chronic myeloid leukaemia-progenitor cells sensitive to induction of cell kill. Proliferation assays showed STI571 to be an effective cytotoxic agent in chronic myeloid leukaemia-derived cell lines (IC(50) on day 5 of 4.6 microg ml(-1) and 3.4 microg ml(-1) for K562 and KU812 respectively) and in leukaemic blast cells (per cent viability on day 3 at 4 microg ml(-1): 55.5+/-8.7 vs 96.4+/-3.7%). STI571 also appeared to specifically target bcr-abl expressing cells, as results from colony forming assays using the surviving cell fraction from STI571-treated peripheral CD34(+) chronic myeloid leukaemia blast cells, indicated a reduction in the expansion of colonies of myeloid lineage, but no effect on normal colony formation. Our data also showed synergy between STI571 and other anti-leukaemic agents; as an example, there were significant increases in per cent cell kill in cell lines cultured with both STI571 and etoposide compared to the two alone (per cent cell kill on day 3: 73.7+/-11.3 vs 44.5+/-8.7 and 17.8+/-7.0% in cultures with STI571 and etoposide alone respectively; P<0.001). This study confirms the central oncogenic role of BCR-ABL in the pathogenesis of chronic myeloid leukaemia, and highlights the role of targeting this tyrosine kinase as a useful tool in the clinical management of the disease

Microbiological effects on transport processes (BioTran) : data production from column experiments containing Sherwood Sandstone (October 2010-July 2011)

The BioTran project was initiated to examine the effects of microbes on sub-surface transport processes, especially in the context of geological disposal of radioactive waste. Early work focussed on hard rock environments using dioritic material from the Äspö Underground Research Laboratory (URL). In 2008-2009, the scope of Biotran broadened to encompass batch experiments on mudstones, host rocks relevant to radioactive waste management in Japan. The 2009-2010 BioTran work programme used column experiments to investigate the effects of microbes on fractured mudstone. This work was undertaken on behalf of the Japanese Atomic Energy Authority (JAEA) and part-funded by the Ministry of Economy, Trade and Industry of Japan (METI), Japan