Figure 1 summarizes the strategy used for comparing the effects of in vitro infection and persistent in vivo infection on the behavior of CD4 + and CD8 + cells. T-cell limiting dilution cloning of PBMCs from the 4 TSP/HAM patients allowed us to clone uninfected and naturally infected CD4 + and CD8 + cells from the same infected individuals . This permitted us to enrich our previously published library of in vivo derived clones . PBMCs from Patient 1 have been previously assayed for clonal expansion and 3’ flanking sequence analyses on several occasions [4,29-31]. IPCR products from 4 clones generated by limiting dilution cloning of patient 1 PBMCs were sequenced and, for 1 CD4 + clone, the 3 ’ provirus integration site sequence matched that identi- fied in PBMCs collected 7 years earlier (Figure 2). This indicates that the present cloning strategy allows for the analysis of in vivo infected and persistently expanded clones. In vitro HTLV-1 cellular infection was per- formed herein by co-culturing PBMCs isolated from healthy adult donors, seronegative for HTLV-1/2, HIV, HBV, and HCV, with lethally irradiated MT2 . Cells were next cloned and cultured as PBMCs from HAM/ TSP, and all generated clones were assayed for HTLV-1 infection, tax expression, CD4 + and CD8 + expression, cell cycling and apoptosis, as shown in Figure 1 and as detailed in the Methods section. Clonal efficiency was identical for in vivo- and in vitro- derived cells. Table 1 represents the distribution of analyzed T cell clones according to the route of infection. All 152 clones har- bored distinct and unique TCR, as evidenced by multi- plex PCR-gamma-DGGE . Infected and uninfected clones were not immortalized and required IL-2 and sti- mulation with PHA and feeder cells at 14-day intervals for continued growth. MT2 cells harbor 18 integrated proviruses per cell , and its level of tax expression was measured as 25274.3 arbitrary units (AU). At day 7 of co-culture of fresh PBMCs with irradiated MT2 cells, inverse PCR failed to detect any MT2 specific HTLV-1 integration site; at this time point, the proviral copies detected corresponded to newly infected cells. There- fore, subsequently cloned CD8 + and CD4 + cells corre- sponded to bona fide newly infected cells in vitro.
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physiological regulator of the cell cycle, its abundance in normal LPTs suggests a central role in mucosal immunity and immune tolerance. Upholding this con- cept is the observation that p53 is downregulated in LPTs from patients with Crohn disease, a condition where tolerance is lost, and that this downregulation is associated with faster cell cycling and increased number of cell divisions (A. Sturm, unpublished obser- vations). This supports the key role of cell cycle regu- lation in immunity, tolerance, and autoimmunity (3). In conclusion, in contrast to PBTs, activation of LPTs leads to an inherently slower progression through the cell cycle, resulting in delayed replication that is not explained by their memory status. In particular, CD2 stimulation of LPTs induces Rb phosphorylation together with remarkably high levels of the inhibitor p53, whose blockade leads to a dramatic acceleration of LPT cell cycling. Since LPTs must limit their reactiv- ity to maintain local immune homeostasis, the p53- dependent slow cycling of LPTs likely reflects the need for a strict control in response to the gut’s heavy anti- genic load. In this regard, p53 behaves similarly to the lung Krüpple-like transcription factor and Tob antiproliferative protein, which also contribute to maintenance of T cell quiescence through active and highly regulated processes (55, 56). The results of this study show that p53 plays a previously unrecognized role as a negative regulator of mucosal immunity by restraining a uniquely tuned but effective mucosal T cell replication, which may prevent excessive reactivity and contribute to maintaining tolerance.
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Streptomyces staurosporeus, is a relatively broad, nonspecific protein kinase antagonist, originally isolated in an effort to define inhibitors of protein kinase C (PKC). 7-OH staurosporine (UCN-01) was defined as a more selective, but not specific, PKC antagonist. Two prominent effects of UCN- 01 have emerged in preclinical studies in vitro: induction of cell-cycle arrest, and abrogation of the checkpoint to cell-cycle progression induced by DNA damaging agents. UCN-01 inhibited cell growth in several in vitro and in vivo human tumor preclinical models (Akinaga et al., 2000); however, antiproliferative activity on the part of UCN-01 cannot be explained solely by inhibition of PKC. First, in cell-cycle analyses UCN-01 inhibits Rb + cells at G1/S phase of the cell cycle (Akiyama et al., 1997). In addition, cells treated with various concentrations of UCN-01 showed decreased pRb phosphorylation in a dose-dependent manner (Chen et al., 1999). These results suggest that CDK2- or CDK4-regulated steps are targets for UCN-01-induced cell-cycle arrest. UCN-01 abrogates the DNA damage-induced checkpoints to cell-cycle progression in G2 (Bunch et al., 1996; Wang et al., 1996) and in S phase (Shao et al., 1997). It is noteworthy that these effects were apparent at drug concentrations that appeared to have little direct effect on cell proliferation or that caused enhanced cytotoxicity by clonogenic or proliferation assays. In addition, they provided a mechanistic framework for prior observations that DNA-damaging agents such as mitomycin (Akinaga et al., 1993) could greatly potentiate UCN-01 action. In contrast to animal studies, UCN-01 displayed strong binding to human plasma proteins, apparently to the 1-acid glycoprotein (AAG) in initial human phase I clinical trials (Sausville et al., 2001; Fuse et al., 1998). One partial response occurred in a patient with melanoma, and a protracted (>4 year) period of stabilization of minimal residual disease was observed in a patient with alk(+) anaplastic large cell lymphoma.
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Ngn2 protein level is regulated by ubiquitin-mediated proteolysis, and the rate of proteolysis is sensitive to cell cycle stage; Xenopus Ngn2 [xNgn2, also known as XNgnR1 (Nieber et al., 2009)], the frog homologue of mammalian Ngn2 (Ma et al., 1996; Nieber et al., 2009), has a shorter half-life in mitosis than in interphase (Vosper et al., 2009). Accumulation of cyclin-dependent kinase inhibitors (cdkis), which occurs upon cell cycle lengthening and exit, promotes neurogenesis mediated by Ngn2 (Vernon et al., 2003). Surprisingly, this particular effect of cdkis is independent of their ability to inhibit cdk kinase activity, but instead results from an independent role for cdkis in stabilising the Ngn2 protein (Vernon et al., 2003; Nguyen et al., 2006). In addition, it is also possible that Ngn2 protein undergoes other post-translational modifications, including phosphorylation, that might be used to coordinate its activity with cell cycle progression.
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Martin Raff (University College, London, UK) addressed how cell growth and proliferation are coordinated in mitotic mammalian cells, and specifically whether mammalian cells have cell-size checkpoints that are analogous to those in yeast (checkpoints arrest the cell cycle if proper cell-cycle events are not completed). Experiments in which the cell-cycle and growth rate of rat Schwann cells were modulated in culture by extracellular factors indicate that these cells may not have such checkpoints (Conlon and Raff, 2003). Instead, cell size at cell division appears to depend on how fast the cells progress through the cell cycle and how fast they grow, which in turn depends on extracellular signals that control cell cycle progression, cell growth, or both. By contrast, Jim Umen (Salk Institute, La Jolla, CA, USA) has found that, in the unicellular green alga Chlamydomonas reinhardtii, the activity of the retinoblastoma-like protein Mat3/Rb is sensitive to a minimum Fig. 2. Examples of negative regulators of proliferation. (A) In the Drosophila eye, Shar-pei (Salvador) controls cell number by inhibiting cell proliferation and promoting cell death. This is indicated by the increase of interommatidial cells in the shar- pei/salvador mutant compared with wild type. (B) In the mouse cochlea, hair cells (brackets) re-enter the cell cycle in the absence of p19 Ink4d . Myosin VIIa is a marker of postmitotic, differentiated hair
In conclusion, our study exhibited the expres- sion patterns and correlation of RNF43 and p53 pathway proteins involved in cell cycling. The most probable mechanism of RNF43- regulated p53 cell cycling pathway was shown in Figure 2. Briefly, RNF43 inhibited p53 in a MDM2 dependent or independent manner, and then down-regulated the expression of p21. Down-regulated p21 induced the activity of Cyclin D1 and the phosphorylation of pRB- S780. These data provide valuable information for better understanding of the association of RNF43 and p53 in HCC.
A major advance emerging from work in our laboratory and those of others is that although IL-2 and IL-15 share two receptor subunits and some functions, they also provide distinct and at times contrasting contributions to the life and death of lymphocytes [15–19]. Although IL-2 is an important growth and survival factor, it also plays a critical role in Fas-mediated activation-induced cell death (AICD) of CD4 T cells . Receptor-mediated stimulation of CD4 T cells by antigen at high concentration (or by CD3 plus CD28) induces the expression of IL-2 and the IL-2 receptor, which in turn interact to yield T-cell activation and T-cell cycling. Antigen stimulation of the cycling T cells at this stage through the T-cell antigen receptor increases the transcription and surface expression of the death-effector molecule Fas ligand (FasL). The interaction of FasL with Fas then leads to death of the self-reactive T cells . My colleagues and I showed that IL-15, in contrast, acts to extend the survival of lymphocytes, both by acting as a growth factor and by inhibiting IL-2-medi- ated AICD of CD4 T cells . In ex vivo studies, CD4 +
remaining T cells undergo extensive compensatory expansion in order to reconstitute the immune system. This process, termed homeostatic proliferation, relies on stimulation through the TCR– self peptide–MHC complex (22–24) and so results in a population skewed toward increased recognition of self antigen, as seen in this study. In addition, rapidly expanding T cells acquire the phenotype and functional characteristics of memory cells, including reduced dependence on costimulation, the ability to respond to lower doses of antigen than naive cells, and the rapid secretion of inflamma- tory cytokines on restimulation, so further promoting the break- down of self tolerance (25–28). Yet, despite these changes, autoim- munity is not an inevitable consequence of lymphopenia. Indeed, as with our patients, most lymphopenic subjects do not develop autoimmunity, suggesting that additional “cofactors” are required. We have already disproved the suggestion that those who develop autoimmunity have selective depletion of Tregs (15). Another pos- sibility arises from the demonstration that excessive cell cycling and reduced T cell survival, driven by the overproduction of IL-21, underlie the development of diabetes in the NOD mouse (14). Here, we demonstrate for what we believe to be the first time in humans that overproduction of IL-21 is also the “second hit” required in the development of secondary autoimmunity, following otherwise suc- cessful treatment of multiple sclerosis with alemtuzumab.
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at a more moderate electrolyte condition of pH 12. Symmetric cell cycling of 1,2- (Figure S14A) and 1,8-DBEAQ (Figure S14B) at pH 12 showed higher temporal fade rates—greater than 0.1% and 10%/day, respectively—than 2,6-DBEAQ at the same pH (<0.01%/day, Figure S15) and were therefore not tested in full cell exper- iments. Theoretical calculations suggest that these isomers are more thermodynam- ically susceptible to hydroxide or water-induced g -hydroxybutyrate cleavage than 2,6-DBEAQ (see Table S4). Given the 0.6 M solubility measured for 2,6-DBEAQ at pH 12 (Table S2), subsequent cell tests were performed at 0.5 M in order to examine the performance of a full cell with reasonable energy density. From the Pourbaix di- agram of 2,6-DBEAQ (Figure S16A), its reduction potential becomes pH indepen- dent above pH 11.5 and is not expected to change during cell cycling. Polarization and capacity utilization measurements with a negolyte containing 0.5 M 2,6-DBEAQ are shown in Figure 3. Polarization studies (Figure 3A) at room temperature showed a near-linear relationship between current density and voltage at currents close to the open-circuit voltage (OCV), which increased from 0.97 V at 10% SOC to 1.12 V at 100% SOC (Figure 3B). Between 80% and 90% of the polarization ASR is ac- counted for by the high-frequency resistance measured using electrochemical impedance spectroscopy (EIS), which largely reflects membrane resistance. A peak galvanic power density of 0.24 W/cm 2 was realized at 100% SOC (Figure S17). This power density is about half of that previously reported in a 2,6-DHAQ-ferrocy- anide cell, 4 owing to the higher OCV of the latter (1.20 V as opposed to 1.05 V at 50% SOC) and smaller ASR (0.858 U cm 2 as opposed to 1.2 U cm 2 at 50% SOC). When voltage and current have a linear, ohmic relationship, the peak galvanic power den- sity is given by p max = V OC 2 =r, where V OC is the open-circuit potential and r is the ASR.
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Although we have demonstrated that hematopoietic Cbl/Cbl-b deletion promotes MPD, a systematic analysis of DKO vs. single KO mice in HSC regulation was not carried out . Therefore, to firmly establish that Cbl and Cbl-b are redundant in the regulation of HSCs, we examined the occurrence of lethal MPD in 8-week old mice of the following strains: Cbl-KO , Cbl-b-KO , hematopoietic Cbl/Cbl-b-DKO (MMTV-Cre-driven Cbl deletion on a Cbl-b null background [11, 21], and wild type (WT; control) (genotypes in Supplementary Table 1). Based on peripheral blood (PB) observations of increased total white cell counts, monocytes and granulocytes, extra- medullary hematopoiesis and death of animals (Figure S1A- S1C), development of MPD required the deletion of both alleles of Cbl and Cbl-b. Analysis of BM hematopoietic compartments demonstrated that expansion of HSCs and specific progenitor compartments, including common myeloid progenitors (CMP), granulocyte/macrophage progenitors (GMP) and common lymphoid progenitor (CLP) but not megakaryocyte/erythrocyte progenitors (MEP), was only seen in DKO mice (Figure S1D). Lack of MPD in Cbl flox/flox , Cbl-b WT/del , MMTV-Cre mice (referred to
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There is emerging evidence to suggest that in some cases, macrophages play an active role in inducing programmed cell death. This was first appreciated after macrophage ablation experiments resulted in the persistence of viable cells in ocular vessel networks that failed to undergo scheduled regression (Lang and Bishop, 1993). The proposal that macrophages can actively signal cell death has received support from a number of systems including C. elegans, where caspase (CED-3) partial function combined with recognition pathway mutants demonstrate that recognition is a back- up stimulus for apoptosis (Hoeppner et al., 2001; Reddien et al., 2001). Although there is currently no mechanistic parallel apparent, discovery of macrophage-induced (Lang and Bishop, 1993; Diez- Roux and Lang, 1997) and phagocyte-enhanced (Hoeppner et al., 2001; Reddien et al., 2001) apoptosis in mice and worms may suggest an activity functionally conserved in metazoans.
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The human breast cancer cell lines, MCF-7 and MDA-MB-231 (ATCC), were maintained in Dulbecco’s Modified Eagle’s Media (DMEM, 4500 mg/L glucose, Sigma Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% (v/v) penicillin-streptomycin (PS; Thermo Fisher Scientific). The human ovarian cancer cell line, OVCAR-3 (ATCC), was cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) media supplemented with 10% (v/v) FBS, 1% (v/v) PS, and 0.001% (w/v) bovine insulin (Sigma Al- drich). For cell growth analysis, cells were seeded at a density of 1 × 10 5 cells per well in 6-well plates or 35 mm dishes. Prior to counting, cells were singularized using 0.25% trypsin-EDTA (Thermo Fisher Scientific) and treated with a 1:1 ratio of Trypan blue (Thermo Fisher Scientific), after which the live cell number was determined using a Countess II FL automated cell coun- ter (Thermo Fisher Scientific).
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Objective measures of road traffic volume were esti- mated using a GIS (ESRI ArcGIS 9.2). We calculated a proxy measure of traffic volume (Road Traffic Volume Index Score - RTVIS) for each participant using four different distance based buffers around each indivi- duals home postcode (0.5 km, 1 km, 2 km, 3.2 km). It was calculated by computing the total lengths of four different types of road (principal roads or motorways, A-roads (major roads), B-roads (minor or local roads) and unclassified roads) within these buffers (centred on participants’ homes) and weighting these based on the average road speed for each classification . Scores were divided into quartiles and the lowest quar- tile was used as the reference group. Using a variety of distance buffers allowed us to examine any potential differences in the associations between cycling beha- viour and traffic volume at different proximities, as there is currently uncertainty about size of the area from home which influences commuting or leisure related activities. The choice of the largest radius size reflected the UK government ’ s aim to encourage a shift from car use to walking or cycling for short jour- neys under 2 miles (3.2 km) . We estimated that a 2 mile cycle journey (at 8 mph) should take an adult approximately 15 minutes.
tance evaluation of SCCCs and RCCCs in indicated models. (A–C) Analysis of apoptosis (A and C) and proportion of SCCCs (B and C) after chemo- therapy exposure. OX, oxaliplatin; DTIC, light-activated dacarbazine; TMZ, temozolomide. Apoptosis measurements: SW1222 RCCC vehicle (VEH) vs. SCCC OX (P ≤ 0.01); RCCC OX vs. SCCC VEH (P ≤ 0.0001); MMLN9 RCCC DTIC vs. SCCC VEH (P ≤ 0.0001); e216 RCCC TMZ vs. SCCC VEH (P ≤ 0.0001). (D) Immunofluorescence of caspase-3 (CASP3) (n = 6 per group) treated or not treated with oxaliplatin. Arrowheads, SCCCs; asterisk, apoptotic areas. Scale bar: 100 μm. (E) qPCR of indicated genes. Data are represented as mean ± SD of triplicates. ND, not detected; r.u., relative units. (F) Apop- tosis flow cytometric evaluation in RCCCs and SCCCs from CRC-SW1222- H2BeGFP cells growing as MTs. FTC, fumitremorgin C. Apoptosis measure- ments: RCCC VEH/FTC vs. RCCC OX/OX+FTC (P ≤ 0.0001); RCCC VEH/FTC vs. SCCC VEH/FTC (P ≤ 0.01); RCCC VEH/FTC vs. SCCC OX+FTC (P ≤ 0.001); RCCC OX/OX+FTC vs. SCCC VEH/FTC/OX/OX+FTC (P ≤ 0.0001); SCCC VEH/ FTC vs. SCCC OX+FTC (P ≤ 0.0001); SCCC OX vs. SCCC OX+FTC (P ≤ 0.001). (G) Drug sensitivity of cancer cell lines according to PanC-SCCC signature scores. Adjusted Wilcoxon test. (H) Disease-free survival of chemo-treated high-risk stage II/III colon cancer patients (GSE39582, n = 151) according to CRC-SCCC signature score. HR, hazard ratio. Cox proportional hazards model. (A–C and F) Data are represented as mean ± SEM. (A, B, E, and F) Data were obtained from triplicates of 3 independent experiments. (A–C, E, and F) Blue bars, RCCCs; green bars, SCCCs. (A and F) 1-way ANOVA. (B, C, and E) 2-tailed Student’s t test. (A–C and E–G) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
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History of cycling in Iran returns to one decade before Second World War, when bicycle entered Iran for the first time. Although at early years it was an expensive vehicle allocated to reach people, but gradually it changed to a popular and affordable type of transportation for all groups of people.
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Four critical observations lead us to believe that HSET might have additional roles in driving tumor progression, independent of its centrosome clustering/spindle pole focusing role in mitosis, viz., (i) elevated ‘nuclear’ expression of HSET predominantly in the interphase cells within high grade tumors as revealed by immunohistochemical staining suggests that HSET may perform critical mitosis-independent functions in aggressive tumors or plausibly lead to more aggressive phenotypes within tumors; (ii) overexpression of HSET results in accelerated G2 and M phases. Faster mitoses can conceivably arise from a severely compromised SAC function that presumably allows HSET-overexpressing cells to rapidly traverse mitosis in the presence of aberrations including chromosome attachment errors. However, we are aware of the caveat that this mitotic role of HSET does not provide an alibi for the observed faster progression through the G2-phase upon HSET overexpression; (iii) HSET OE in HeLa cells leads to faster cell kinetics and enhanced overall proliferation (Fig. 5.4.5A,B,C), and (iv) HSET OE leads to the upregulation of the expression of phospho-survivin, Bcl-2, HIF1α, Aurora B and Mad1, and presumably upregulates the signaling pathways that lie downstream of these key regulatory factors. Furthermore, since fewer than 3 percent of HeLa cells possess amplified centrosomes (our unpublished observations), we believe that the pro-proliferative role of HSET that we have demonstrated in our study in HeLa cells provides strong evidence for a centrosome clustering- independent activity of HSET.
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ABSTRACT The marine subsurface sediment biosphere is widely inhabited by bacteria affiliated with the class Dehalococcoidia (DEH), phylum Chloroflexi, and yet little is known regarding their metabolisms. In this report, genomic content from a single DEH cell (DEH-C11) with a 16S rRNA gene that was affiliated with a diverse cluster of 16S rRNA gene sequences prevalent in marine sediments was obtained from sediments of Aarhus Bay, Denmark. The distinctive gene content of this cell suggests meta- bolic characteristics that differ from those of known DEH and Chloroflexi. The presence of genes encoding dissimilatory sulfite reductase (Dsr) suggests that DEH could respire oxidized sulfur compounds, although Chloroflexi have never been implicated in this mode of sulfur cycling. Using long-range PCR assays targeting DEH dsr loci, dsrAB genes were amplified and sequenced from various marine sediments. Many of the amplified dsrAB sequences were affiliated with the DEH Dsr clade, which we pro- pose equates to a family-level clade. This provides supporting evidence for the potential for sulfite reduction by diverse DEH species. DEH-C11 also harbored genes encoding reductases for arsenate, dimethyl sulfoxide, and halogenated organics. The re- ductive dehalogenase homolog (RdhA) forms a monophyletic clade along with RdhA sequences from various DEH-derived con- tigs retrieved from available metagenomes. Multiple facts indicate that this RdhA may not be a terminal reductase. The presence of other genes indicated that nutrients and energy may be derived from the oxidation of substituted homocyclic and heterocyclic aromatic compounds. Together, these results suggest that marine DEH play a previously unrecognized role in sulfur cycling and reveal the potential for expanded catabolic and respiratory functions among subsurface DEH.
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The data used in this paper is taken from the 2011 and 2006 census of Ireland (CSO, 2011, 2006). The data examined in this paper represent individual’s regular trips to work; by the mode of transport they state that they use the most often. While this data is not ideal, especially when examining cycling, due to the seasonal variations in cycling, it is the only data source available in Ireland that can compare changes in modal share over time. The data set has 1.8 million individuals work trips in 2006 and 1.7 million individuals work trips in 2011, in Ireland. The data provided from the Irish census is for all individuals resident in Ireland and the data is provided at an individual level (which is anonymised). The data analysed in this paper relates to all those individuals that are in employment at the time of the census. This makes the data used in this research more robust than that used in other countries, as it is not a sample of the population. It should be noted that the results presented in this paper relate to the commute to work. Typically these trips account for a quarter of all trips (CSO, 2009).
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Included studies were assessed for quality using the criteria de- scribed in the section ’Assessment of risk of bias in included studies’. The results are presented narratively in the table ’ Characteristics of included studies’. The results are also presented graphically across all studies (Figure 2) and for individual studies (Figure 3). The majority of studies were at high or unclear risk of bias for selection bias and bias due to confounding. In terms of how well intervention and control areas were matched and their prox- imity to each other, six (29%) studies were rated as well matched and proximal, 13 (62%) were rated as poorly matched and prox- imity was either unknown or distal, and for two (10%) studies it was unclear. In terms of detection bias, the majority of studies (n = 19, 90%) analysed routinely collected collision data taken from databases maintained by organisations external to the study team. For three (14%) studies the source of collision data was unclear. In terms of attrition bias, over half the studies (n = 14, 67%) used data collection periods both before and after installation of the cycling infrastructure of at least one year. The length of time of data collection pre- and post-installation of infrastructure varied widely from one to five years pre-instalment to one to 9.5 years post-instalment. For four studies (19%) the data collection periods were unclear and three studies (14%) used data collection periods after the intervention of less than one year. In terms of selective reporting, one study excluded data from the analysis collected at an intervention site which had not been successful in reducing col- lisions (Mountain 2005); and a second study reported little data making it difficult to draw conclusions (Buckley 2000).
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Although many studies have suggested the potential of CSCs as the seeds for distal metastasis, few studies have directly tested the metastatic capability of putative CSCs in vivo. Collective evidence from a few studies that directly tested the in vivo metastasis using sorted CSCs suggests that the CSC phenotype alone may exhibit inva- sive property in vitro but is not sufficient to determine or predict in vivo metastasis. Here, we show that a non- adherent, stem-like, and metastatic CSC-enriched subpo- pulation could be isolated by exposing human metastatic breast cancer cell lines to cycles of chronic hypoxia fol- lowed by reoxygenation. Since very few studies have demonstrated the formation of macro-metastasis from low numbers of sorted CSCs and currently proposed CSC markers might not be sufficient to identify all stem cell populations , we believe that our study presents a promising approach to isolate stem-like and metastatic breast CSCs as opposed to the cell-sorting strategy based on putative stem cell surface markers. Also, it will be of great interest to investigate the possibility that repetitive cycles of hypoxia/reoxygenation lead to the selective expansion of a pre-existing metastatic CSC subpopula- tion. Our results demonstrated the possibility of isolating highly metastatic breast CSCs using the hypoxia/reoxy- genation regimen we established. With the recent success of identifying selective inhibitors targeting CSCs, we believe that the newly isolated cycling hypoxia-selected subpopulation may present a new opportunity for chemi- cal screening and discovery of compounds with selective toxicity for metastatic breast CSCs.
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