The load was set according to each subject’s mass [21]. The test was a 30-second WAnT followed by 5 min of rest and then eight 10-s intervals of all-out cycling. Each interval was separated by 2 min of rest. The resistance for the WAnT and intervals was set at 0.10 kP/kg body mass. Performance Measures Peak power during the WAnT was defined as the highest mechanical power output elicited during each 30 s test. Mean power was calculated based on the average mechanical power produced during the test. Additionally, average peak power output and average mean power output were both calculated across the WAnT and all 8 intervals.

buy Saracatinib Biochemical Measures Capillary blood samples (5 μL) were taken from the fingertip during the baseline resting blood draw and at 0, 5, and 10 min post-exercise Nutlin-3a solubility dmso in order to determine peak blood lactate values and clearance. The Lactate Pro (Arkray, Japan) portable analyzer was used to determine whole blood lactate content. Before (t0), immediately after (t1), 30 min post (t2), and 60 min post (t3) each WAnT + interval session, blood samples were collected via an indwelling cannula inserted into an antecubital

vein using a vacutainer system (Becton Dickinson, Rutherford, NJ). Approximately 10 mL were collected in a serum separator tube and 10 mL in an EDTA coated tube. After removing a 1 mL aliquot of whole blood for hemoglobin and hematocrit analysis to account for plasma volume changes, an additional 300 μL aliquot (2 × 100 μL for GSSG; 2 × 50 μL for GSH) was obtained for GSH/GSSG analysis. 1-methyl-2-vinylpyridium (M2VP) was added to the tubes containing samples for GSSG analysis. Plasma for 8-isoprostane assay was obtained by centrifugation

of whole blood in the EDTA tubes at 3000 × g 10 min at 4°C with 1 mL aliquots placed in microvials pre-coated with 200-μg of butylatedhydroxytoluene (BHT). The serum separator tubes were left to stand for 30 min to facilitate clotting before being centrifuged at 3500 × g for 15 min at 4°C in order to obtain serum for IL-6 and CORT analysis. Aliquots of blood, serum, and plasma were stored at -80°C until analysis of the dependent measures. All assays were performed in duplicate and assays for each measure were run in one batch. Pembrolizumab Total and oxidized glutathione were analyzed using a commercially-available EIA kit (Bioxytech® GSH/GSSG-412, OxisResearch, Portland, OR). The within assay coefficient of variation (CV) for GSH was ± 7.3% and for GSSG was ± 8.6%. Similarly, IL-6 was determined via ELISA using commercial kits (IBL, Hamburg, Germany). Within assay CV for IL-6 was ± 6.9%. Serum CORT was analyzed using RIA (MP Biomedicals, Irvine, CA), and the within assay CV was ± 6.2%. In order to analyze plasma free 8-iso PGF2α, plasma from the EDTA tubes was first purified by diluting the sample in a 1:5 ratio with Eicosanoid Affinity Column Buffer (Cayman Chemical, Ann Arbor, MI).

Reef health and productivity may be compromised in such settings by the steep slopes and thick soils of high island interiors, where extreme rainfall can trigger high runoff, landslides, and debris flows (e.g., Harp et al. 2004). Larger islands may also have major rivers, creating flood hazards and delivering large quantities of sediment, which can dominate coastal morphology in the vicinity of their outlets (e.g., Mimura and Nunn 1998; Kostaschuk et al. 2001). Near-atolls, Pexidartinib supplier atolls, and reef islands Atolls are more or less

annular reef and reef-island systems found predominantly in oceanic mid-plate settings, where they rest on the peaks of submarine volcanic edifices (Fig. 2). Darwin (1842) referred to barrier reefs surrounding volcanic islands

as an intermediate stage in the development of atolls through long-term subsidence and reef growth. Others have referred to such ‘near-atolls’ as ‘almost-atolls’ (Stoddart 1975). Aitutaki in the southern Cook Islands is a good example (Fig. 4), with a 17 km2 central volcanic upland rising to 120 m ASL and two very small volcanic islands in the southeastern lagoon (Forbes 1995). The total area inside the surrounding reef is more than 70 km2 (by contrast Chuuk is more than 2,800 km2). Aitutaki is subject to moderately frequent storms (de Scally 2008), during which the reef takes the brunt of deepwater wave energy, but combined surge and setup with overtopping allows some wave energy to penetrate across the reef flat and lagoon to form GSK-3 inhibitor review a high berm on the western side of the island (Forbes 1995; Allen 1998). Fig. 4 Near-atoll of Aitutaki, southern CYTH4 Cook Islands, showing central volcanic core and two small volcanic outliers, surrounded by a barrier reef and lagoon with partial rim of reef islands (from

Forbes 1995). Broken line Reef. Reproduced with permission from the Secretariat of the Pacific Community, New Caledonia Atolls lack an emergent volcanic core and are characterized by very low maximum elevations, limited land area, and thin freshwater lenses (McLean and Woodroffe 1994). With long-term subsidence typical of many atolls (Scott and Rotondo 1983), the volcanic peak is submerged and capped by limestone (Fig. 2). With fluctuating sea levels over glacial-interglacial cycles, most present-day atolls have been exposed subaerially during glacial lowstands, experiencing solution and denudation (Woodroffe 2002). Reefs are reactivated when sea levels rise again. Depending on rates of SLR and coral productivity, reefs may keep up with sea level, fall behind (becoming submerged), or catch up (if the rate of SLR diminishes or productivity increases) (Neumann and Macintyre 1985).

However, results were mainly based on clinical assessments while concomitant

endoscopic and histopathologic features of the radiation-induced damage in bowel mucosa were not described [6–10]. The aim of this study was to assess the efficacy of subcutaneous amifostine in preventing radiation colitis in patients irradiated for pelvic neoplasms, by combining clinical, endoscopic and histopathologic data. Methods Study and Patients This randomised phase II exploratory clinical trial was activated in May 2001 and conducted in an Academic Hospital FK506 ic50 [University General Hospital]. The procedures followed were in accordance with the Helsinki Declaration (1964, amended in 1975, 1983, 1989, 1996 and 2000) of the World Medical Association. Institutional review boards and the ethics committee of our University Hospital approved the trial protocol with and patient informed consent. Patients with pelvic malignancies were considered for participation into this trial if they fulfilled a list of eligibility criteria [see below] Depsipeptide and signed an informed consent. Enrolled patients were randomly assigned to receive daily amifostine (subcutaneously, 500 mg flat dose) before radiotherapy (A) or radiotherapy alone (R). Sigmoidoscopy and blinded biopsies were scheduled

for all patients prior to initiation of treatment and twice following completion of radiotherapy. Study endpoints The primary study endpoint was to determine the efficacy of amifostine in preventing radiation-induced colitis (RC) by using combined clinical, endoscopic and histopathologic data from patients irradiated to the pelvis. The secondary endpoints of the study were the assessment of agreement between clinical, endoscopic and histopathologic data during radiotherapy and post-radiotherapy period and the evaluation of amifostine-related toxicity. Eligibility Non-specific serine/threonine protein kinase criteria The study enrolled patients with primary pelvic or metastatic

to the pelvis malignancies who were referred for adjuvant, radical or palliative radiotherapy but not for re-irradiation. All patients recruited in the study were older than 18 years, had a World Health Organization (WHO) performance status 0-2 and a life expectancy of more than 6 months. Pregnant or lactating women, patients with severe infections or severe psychiatric or neurologic illnesses were excluded. Patients with decreased hematologic reserves, with major organ failure, severe electrolyte or metabolic abnormalities were also excluded. In patients with haemoglobin levels below 11 g/dl before radiotherapy, subcutaneous erythropoietin was administered. Patients with hypertension controlled with medication were eligible for amifostine administration. Patients with asymptomatic low blood pressure were included. Patients with symptomatic hypotension were excluded.

Model qualification Selleck KU-60019 of the final model, using a visual predictive check (VPC) and a numerical

predictive check (NPC), showed that the model was a good description of the data (figure 8). Fig. 8 (a) Visual predictive check; (b) numerical predictive check (upper prediction interval limit); and (c) numerical predictive check (lower prediction interval limit). In graph (), the thick solid dataline shows the median of the observed data, and the dark gray shading shows the model-predicted 95% confidence interval around the median. The dotted datalines are the limits between which 95% of the observed data are found, and the light gray shading shows the model-predicted 95% confidence intervals around those limits. In graphs (b) and (c), the thin solid datalines and white datapoints show the ratios between the actual and expected numbers of points for (b) the upper prediction interval and (c) the lower prediction interval indicated on the x-axes, and the light gray shading shows Decitabine cell line the uncertainty of the model around the ratio of 1. The dashed datalines are identity lines, with no difference between the actual and expected numbers. Sample time optimization was performed using the WinPOPT library two-compartment model with first-order absorption. This is a simpler model than

the final population pharmacokinetic model, adjusted to reflect the structure of the library model prior to performing the sample time optimization. The absorption process was simplified from the sequential zero- then first-order process to a first-order process only, and the IOV terms for D1, Frel, and ka were also removed. The actual

parameter values used for the sample time optimization are presented in table IX. The simplified model retained the influence of dose on ka, thus the value for ka (0.403/hour) is that calculated for a 50 mg dose. The results of sample time optimization are shown in table X. Table IX GLPG0259 parameter estimates used for sample time optimization Table X GLPG0259 parameter estimates used for sample time optimization The gold-standard design (six samples per subject after Cytidine deaminase both the 7th and 84th doses) criterion value was set at 100%. Further, the imprecision in the estimated CL/F value under this design was only 4.2%, indicating that the design was able to estimate CL/F well. The poor design (a single sample per subject after each of the 7th, 14th, 28th, 56th, and 84th doses, at 2 hours postdose) gave a criterion value that was 0.026% of that for the gold-standard design, and CL/F was estimated extremely imprecisely. Design no. 4, where a single sample was taken per subject but at different times per visit and always in the afternoon (thus at 5, 6, 7, 8, and 9 hours postdose across the visits) gave rise to a criterion ratio of 4.1%, and CL/F was estimated with 64.4% imprecision. Thus design no. 4 was not very good but was a considerable improvement over the poor design. Design no. 5 was similar to design no.

Oligonucleotide primers targeting the 5′ and 3′ flanking regions of VNTR loci were used for amplification (Table 1). The

following conditions were used: PCR reactions were performed in 15 μl containing 2 ng DNA, 1 × PCR Reaction Buffer, 1.5 mM MgCl2, 1 Unit of Taq DNA polymerase (Qiagen, KU-60019 price Courtaboeuf, France), 200 μM of each dNTP, 0.3 μM of each flanking primer (Eurogentec, Angers, France). Amplification was performed with a PTC 200 thermocycler (Biorad, Marnes-la-Coquette, France) using the following conditions: initial denaturation cycle for 5 min at 94°C, 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 58°C and elongation for 45 s at 72°C plus a final elongation step for 10 min at 72°C. For the analysis of all markers, 3 μl of PCR products were separated in a 2% agarose gel using agarose for routine use (Eurogentec, Angers, France). Electrophoresis Hormones antagonist was performed in 20 cm-wide gels made in 0.5 × TBE buffer (Sigma), run at 8 V/cm. For each PCR run the reference strain Mu50 was included. The 100-bp

ladder DNA size marker was from MBI Fermentas (Euromedex, Souffelweyersheim, France). The gels were stained after the run in 0.5-1.0 g/ml ethidium bromide for 15 to 30 min, then rinsed with water and photographed under ultraviolet illumination (Vilber-Lourmat, Marne la Vallée, France). The amplicon size was determined using the dedicated tool Gelcompar MG-132 concentration in the BioNumerics software (Applied Math, Sint-Martens-Latem, Belgium). The MLVA genotype of an isolate with 14 VNTRs (MLVA-14) is expressed as its allelic profile corresponding to the number of repeats at each VNTR in the order Sa0122 (alias spa), Sa0266 (alias coa), Sa0311, Sa0704, Sa1132, Sa1194, Sa1291 (alias SIRU13), Sa1729, Sa1866, Sa2039, Sa0906, Sa1213, Sa1425 and Sa1756 (alias SIRU15). The genotype of the Mu50 strain deduced from its genomic sequence is 10 6 3 4 6 7 4 5 3 3 3 5 4 1. Clustering analyses were performed using the categorical coefficient (also called Hamming’s distance) and UPGMA. A cut off value of 45%

was previously shown [21] to define clusters which correspond to MLST clonal complexes and is therefore used in this study to identify CCs. The isolates in these CCs differ at a maximum of three VNTRs out of 14. Lineages are arbitrarily defined as groups of isolates for which the genotype between two isolates differs at a maximum of 2 VNTRs (cut-off 80%). The minimum spanning tree was produced in BioNumerics, scaling with member count. The polymorphism index of individual or combined VNTR loci was calculated using the Hunter-Gaston diversity index (HGDI) [36], an application of the Simpson’ s index of diversity [37] is 0.9965 [21]. Spa typing The spa tandem repeat was amplified using the primers for Sa0122, and the amplicons were purified by polyethylene glycol (PEG) precipitation and sequenced (MWG Biotech).

This clearly shows that both Crp and IclR regulate the aceBAK operon independently. Under glucose abundant conditions, deleting arcA does not have a major effect on glyoxylate pathway fluxes (wild type vs. ΔarcA and ΔiclR vs. ΔarcAΔiclR), despite the fact that ArcA is a known repressor of the aceBAK operon [57]. This is in stark contrast with the glyoxylate pathway fluxes under

Selleck CDK inhibitor glucose limiting conditions. Here, arcA deletion reduces the bypass activity but only in a ΔiclR genetic environment. This is illustrated by the AceA/(AceA + Icd) flux ratio, which decreases from 55% in the wild type to 34% in the ΔarcAΔiclR strain). However, the regulatory mechanism behind this remains unclear and needs to be resolved. Compared to the wild type, the ΔarcA strain has a similar overall flux distribution which was also found by Nanchen et al. [23], but contradicts the data

obtained by Nizam et al. [58] Physiological comparison between E. coli K12 ΔarcAΔiclR and E. coli BL21 As explained in the previous sections the double knockout strain E. coli K12 ΔarcAΔiclR shows an improved formation of biomass under both glucose abundant and limiting conditions (see Figure 1), with the most distinct effect under glucose abundant conditions (50% increase). This is mainly check details attributed to a reduced acetate and CO2 formation. After investigation of the intracellular fluxes (Figure 5A), the higher biomass yield under batch conditions can be explained by the activity of the glyoxylate pathway and the concomitant lower CO2 loss in the TCA. Furthermore, as a result of arcA deletion, repression on TCA cycle genes is removed, resulting in a higher TCA flux and a lower acetate formation. Also a slight increase in glycogen content was noticed in this strain under both growth conditions as shown in Table 3. Many of these characteristics

are also attributed to E. coli BL21 (DE3) and therefore metabolic flux ratios and netto fluxes were determined for this strain as well and compared with E. coli K12 ΔarcAΔiclR as illustrated in Figure 6 and 7, respectively. Small differences are observed in the OAA from PEP fraction, but this does not seem to influence the metabolic fluxes profoundly as almost all fluxes do not significantly differ between the two Unoprostone strains. Figure 6 Comparison of origin of metabolic intermediates in E. coli MG1655 Δ arcA Δ iclR and E. coli BL21 (DE3) under glucose abundant conditions. Standard deviations are calculated on different samples originating from different cultivations. The serine through EMP and the pyruvate through ED results were obtained from experiments using 50% 1-13C glucose and 50% naturally labeled glucose. To determine the remaining values a mixture of 20% U-13C glucose and 0 naturally labeled glucose was used. To determine the fractions resulting in the formation of OAA a Monte-Carlo approach was applied.

In the case of the mutants d8-60a, d8-60b, d8-60c, all three generated identical length PCR products by this method indicating identical deletion

end points. Membrane protein analysis The outer membrane proteins (OMPs) were extracted as previously described [35] using equal number of cells Acalabrutinib research buy (equivalent to 5 ml of cells diluted to an OD600 of 5.0). The membrane pellet was resuspended in 200 μl of SDS sample buffer containing 5 mM tributylphosphine and 20 mM acrylamide for reduction and alkylation of proteins [36]. The solubilized proteins were diluted 1:5 in SDS sample buffer and 5 μl subject to polyacrylamide gel electrophoresis using a Criterion XT precast gel (4-12% Bis-Tris; Bio-Rad). Protein gels were stained with Flamingo protein stain (Bio-Rad) and imaged using a Pharos FX Plus Molecular Imager (Bio-Rad).

Flamingo stained protein gels were post-stained with colloidal Coomassie G-250 stain and proteins of interest excised for identification by find more LC-MS/MS as previously described [37]. PEAKS software (Bioinformatics Solutions Inc.) was used to directly search peptides against a protein sequence FASTA output derived from the V. rotiferianus DAT722 genome [12]. The highest PEAKS score (percentage based on a p-value < 0.05) was taken as the closest peptide match. The full sequence of identified proteins is given in the additional file 1. Acknowledgements This work was supported by a grant from the National Health and Medical Research Council of Australia. ML is supported by an ithree Institute Postdoctoral Fellowship. Electronic supplementary material Additional file 1: lists the full sequence of outermembrane proteins that showed changes in concentration between wild type DAT722 and the mutant d8-60a under particular growth conditions. Proteins were identified

via LC-MS/MS analysis as described in the methods. (DOC 48 KB) References 1. Hall RM, Brookes DE, Stokes HW: Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point. Mol Microbiol 1991, 5:1941–1959.PubMedCrossRef 2. Boucher Y, Labbate M, Koenig JE, Stokes HW: Integrons: mobilizable platforms that promote genetic diversity Amino acid in bacteria. Trends in Microbiol 2007, 15:301–309.CrossRef 3. Labbate M, Case RJ, Stokes HW: The integron/gene cassette system: an active player in bacterial adaptation. In Horizontal gene transfer. Edited by: Gogarten MB, Gogarten JP, Olendzenski LC. Humana Press; 2009:103–125.CrossRef 4. Thompson FL, Iida T, Swings J: Biodiversity of vibrios. Microbiol Mol Biol Rev 2004, 68:403–431.PubMedCrossRef 5. Meibom KL, Blokesch M, Dolganov NA, Wu C-Y, Schoolnik GK: Chitin induces natural competence in Vibrio cholerae . Science 2005, 310:1824–1827.PubMedCrossRef 6.

J Bacteriol 1998, 180:5567–5573.PubMed 62. Palma M, Zurita J, Ferreras JA, Worgall S, Larone DH, Shi L, Campagne F, Quadri LE: Pseudomonas aeruginosa SoxR does not conform to the archetypal paradigm for SoxR-dependent regulation of the bacterial oxidative stress adaptive response. Infect Immun 2005, Y 27632 73:2958–2966.PubMedCrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions LQ conceived the study. ET, SC, PM, UE and SA carried out the experiments. LQ, ET, SC, and DC analyzed results and drafted the manuscript. All authors read and approved the final manuscript.”
“Background

Concrete corrosion of wastewater collection systems is a significant cause of deterioration and premature failure. In the U.S., costs associated with maintaining an estimated 800,000 miles of wastewater collection infrastructure are approximately $4.5 billion per year [1]. Many systems may be beyond their design life and must be replaced because they cannot be rehabilitated [2]. Failure to adequately address the deteriorating infrastructure networks threatens our environment, public health, and safety. In wastewater collection systems microbial-induced concrete corrosion (MICC) may occur in areas under higher concentrations of hydrogen sulfide (H2S) [3–5]. The primary source of sulfur is sulfate (SO4 2-) which can

be reduced by sulfate-reducing bacteria (SRB) to hydrogen sulfide (H2S) under anaerobic conditions. H2S is transferred across the air-water interface to the sewer atmosphere where chemoautotrophic B-Raf assay bacteria on the pipe surface, including sulfide-oxidizing bacteria (SOB), convert the H2S to biogenic sulfuric acid (H2SO4). Biogenic sulfuric acid (H2SO4) can be generated by various microbial Acyl CoA dehydrogenase species [6–9]. While many of the microorganisms and general mechanism involved in MICC has been known for decades, and recent studies using molecular-based approaches have more accurately described the microbial ecology of these engineered systems [6, 8, 9], a better understanding

of the metabolic processes and functional capabilities is needed to develop new approaches to mitigate MICC and its associated effects. The objective of this study was to characterize the microbial community of concrete wastewater biofilms and their functional capability based on molecular analyses of metagenome libraries and to compare it with 16S rRNA gene sequences from previously generated clone libraries [7–11]. Specifically, we sampled biofilms from two sections of a severely corroded concrete wastewater pipe to obtain a better understanding of microbial community colonization processes and mechanisms of concrete deterioration. To our knowledge this is the first published report utilizing metagenomics to elucidate microbial community functional capabilities involved in MICC in wastewater collection systems.

1) 0 Plante 2003 H&E +SS+IHC NVP-LDE225 order 70 IA-IIA 8 (13.1) 3 (37.5) Dargent 2003 H&E +SS+IHC 70 IA1-IIB 19

(30.2) 9 (47.4) Hubalewska 2003 H&E +SS+IHC 37 I-IIA 5 (13.5) na Pijpers 2004 H&E +SS+IHC 34 early 12 (36.3) 4 (33) Silva 2005 H&E +SS+IHC 56 IA2-IIA 17 (32.7) 3 (17.6) Rob 2005 H&E +SS+IHC 183 IA2-IB2 35 (21.9) na Angioli 2005 H&E +SS+IHC 37 IB1 6 (23) 0 Di Stefano 2005 H&E +SS+IHC 50 IA2-IIA 9 (20) 2 (22.2) Frumovitz 2006 H&E +SS+IHC 50 IA2-IB1 9 (18.8) na Wang 2006 H&E +SS+IHC+CK19PCR 46 early 18 (39) 7 (38.9) Yuan 2007 H&E +SS+IHC 81 IB1-IIA 17(20.9) 4 (23.5) Coutant 2007 H&E +SS+IHC+HPV DNA 59 IA-II 15 (25.4) 3 (20) Lee 2007 H&E +HPV DNA 57 IB-IIA 11 (19.3) na Hauspy 2007 H&E +SS+IHC 39 IA1-IIA 2 (5.2) na Bats 2007 H&E +SS+IHC 25 IA2-IA1 3 (12) 1 (33) Total     908   187 (20.6) 36 (19.2) SLN: sentinel lymph node; H&E: hematein eosin staining; IHC: immunohistochemy; SS: serial sectioning; HPV: human papilloma virus; na: not available Four studies have performed a histological analysis of lymph nodes using H&E and IHC [32–35]. In the series of Kraft et al including 54 patients, overall rate of macrometastases was 42% but there was no mention

of the rate of micrometastases [35]. In the three remaining studies including 65 patients, the rate of macrometastases varied from 10% to 18.2% but none of the studies reported detecting micrometastases. Although the total number of patients included in these series was low, it is possible to suggest that H&E and IHC are insufficient CCI-779 to detect

micrometastases. Thirteen studies have used the combination of H&E, serial sectioning and IHC [10, 19, 28, 36–44]. In four of the thirteen studies no attempt to evaluate the presence of micrometastases was noted. In the remaining nine studies involving 356 patients the rate of macrometastases varied between 7.1% C1GALT1 and 36.3% with a mean value of 25.8% (92/356). Among patients with lymph node metastases, the percentage of women with micrometastases ranged from 0% and 47.4% with a mean value of 28.3%. Therefore, at least one quarter of patients with lymph node metastases exhibited micrometastases. Few data are available on the contribution of molecular biology to detect micrometastases. In Wang et al’s series, the combination of H&E, serial sectioning, IHC and CK-19 expression by RT-PCR detected macrometastases in 18 out of 46 patients (39%) with lymph node metastases and micrometastases in 7 out of the 18 patients (38.9%) with macrosmetastases [45]. For Coutant et al, HPV DNA analysis in conjuction with H&E, serial sectioning and IHC detected macrometastases in 15 out of 59 patients including three with micrometastases (20%) [29].

To determine if the appearance of the cells in the dormant clones was due to cortically rearranged F-actin, a characteristic of non-transformed mammary epithelial cells [33], we stained them with phallacidin. Figures 1 a and b demonstrate Temsirolimus nmr that 74.1 + 7.8% of these very large, quiescent cells have parallel bundles of cortical actin as compared with 33.0 + 11.5% of cells in growing colonies. This difference is significant at p < 0.01 (Student’s t test). Analogously,

MCF-10A non-transformed, immortalized mammary epithelial cells incubated on fibronectin also have a cortical actin distribution. To test the hypothesis that the re-differentiated state depends on outside-in signaling through re-expressed integrin α5β1, we incubated the cells growing on fibronectin with blocking antibodies to this integrin. Figure 2 a demonstrates that cortical rearrangement of F-actin requires binding of integrin α5β1 by fibronectin, while blocking antibody to integrin α2β1, also upregulated in these dormant cells [3], has no effect and acts as a negative control. The increase in the percent cells with cortical actin from 28.6 + 0.9%

X-396 in vivo of growing cells to 67.9 + 6.6% of dormant cells and the decrease back to 21.6 + 8.5% due to blocking of integrin α5β1 binding are statistically significant (p < 0.005, p < 0.001, respectively, Fig. 2b). Antibody to integrin α2β1 had no effect with 66.0 + 13.2% of the cells having cortical actin (p > 0.05). Other characteristics of these dormant cells, including increased nuclear size (Fig. 2c) and increased cytoplasm to nucleus ratios (Fig. 2d) were also partially reversed by blocking

antibody to integrin α5β1. The mean longitudinal nuclear axis increased from 15.4 + 2.0 μm in growing cells to 26.7 + 3.7 μm in dormant cells (p < 0.001) and was reversed to 19.8 + 4.0 with blocking antibody to integrin α5β1 (p < 0.001). Blocking antibody to integrin α2β1 did not have an affect. Similarly, the mean square of the cytoplasm to nucleus ratios increased from 4.6 + 1.9 in growing cells to 17.2 + 10.7 in dormant cells and was reversed to 9.48 + 5.6 with blocking Tau-protein kinase antibody to integrin α5β1 (p < 0.001). Blocking antibody to integrin α2β1 did not have an affect on this characteristic either. Fig. 1 Cortical actin stabilization in dormant breast cancer cells. a MCF-7 cells incubated with or without FGF-2 10 ng/ml on fibronectin-coated cover slips for 6 days at clonogenic density were stained with BODIPY-Phallacidin (green actin staining) and DAPI (blue nuclear staining) and photographed at 400 x magnification (see Materials and Methods). A 20 μM size bar is included in all fluorescence photographs in all figures. MCF-10A cells were incubated on fibronectin-coated slides and stained in a similar manner as controls and demonstrate morphological similarity with dormant MCF-7 cells. Arrows indicate prominent places of cortical actin redistribution around the perimeter of the cytoplasm.