use of blood glucose or cho lesterol lowering medications or supplements, corticoster oid use in the preceding 12 weeks. NSAID use 3 days week in the preceding 4 weeks. or a history of chronic ill ness. This study was approved by the Copernicus Group Independent Review Board and was conducted based on good clinical selleck compound practice guidelines. Informed written con sent was obtained from each participant before enroll ment in the study. Study design This study was a randomized, 12 week, open label, 2 arm trial conducted at the Functional Medicine Research Center in Gig Harbor, WA Inhibitors,Modulators,Libraries from June 15, 2006 through November 20, 2006. Subjects who satisfied the inclusion criteria were randomized to 1 of 2 arms using a commercial software program, subjects were stratified by sex.

Participants from both arms were instructed to follow a modified Med iterranean style, low glycemic load diet and were provided with dietary guidelines, including a list of allow able foods, suggested Inhibitors,Modulators,Libraries serving sizes and recipes. Subjects were asked to consume the diet until satisfied. Modified Mediterranean style low glycemic load diet The rationale for defining this dietary program as modi fied Mediterranean style, low glycemic load is that it includes a variety of low glycemic phytochemically rich foods. Not all Mediterranean style diets are low in glyc emic load, and not all low glycemic load diets are phytochemically Inhibitors,Modulators,Libraries diverse. thus, we chose to leverage the benefits of both by combining them together. Specifically, Inhibitors,Modulators,Libraries the diet used in this study is distinguishable from the classic Mediterranean diet in that it is limited in the number of servings of alcohol and, in particular, whole grain.

Inhibitors,Modulators,Libraries Alcohol intake was kept to a minimum an optional 1 glass of red wine daily for all subjects. In the Mediterranean diet, several serv ings of grain are often advocated. however, based on the available literature and our experience with this dietary program in the past decade, we decided to limit whole grains to 1 serving daily. Riccardi et al. suggested that the standard Mediterranean diet may not be beneficial for individuals with insulin resistance due to the high carbo hydrate content. Additionally, in our own use of this pro gram, we found that reducing grain intake lowers cravings in many subjects. Moreover, Mediterranean like food items such as pizza and hard toasted bread have been shown to have glycemic responses similar to white bread.

Thus, one of the primary stipulations selleck chemicals llc for foods in this die tary program was to ensure that all items included were low in glycemic load. The glycemic index of foods most commonly eaten was 55, with occasional selection from a small category of phy tochemical rich vegetables with a moderate glycemic index. This diet is also notable in that it omits all forms of sweeteners except low glycemic agave nectar syrup and stevia.

We have previously characterized the expression of OPH in LNCaP, RWPE 1, COS 7 and COS 7 OPH cell lines. Moreover, Kumar et al. have Oligomycin A IC50 characterized the degree of Akt activation in RWPE 1, LNCaP, DU145 and PC3 cells as well as the basal levels of oxidative stress. We found that S NPAA was the most effective prodrug in its ability to deplete GSH, cause oxidative stress, induce apoptosis, and de crease cell viability, particularly in cell lines overex pressing OPH. Methods Materials Reduced glutathione, digitonin, dimethyl sulfoxide, 2,2,2 trichloroacetic acid, 2,4 dinitro phenylhydrazine, 5,5 dithiobionitrobenzoic acid and diisopropyl fluorophosphate were purchased from Sigma Chemical Company.

DMEM, KSFM and growth factors, and RPMI 1640 cell medium, penicillin streptomycin solution, and genet icin and KB plus DNA ladder, Celltracker Inhibitors,Modulators,Libraries blue, 10kD spin columns, and EnzChek Caspase 3 assay kit were purchased from Invitrogen. BCA kit and the anti DYKDDDDK antibody were purchased from Pierce. Celltiter Inhibitors,Modulators,Libraries 96 AQueous One MTS kit, described as the MTS viability assay in experiments, was purchased from Promega and contained CellTiter96 Aqueous One Solution composed of a tetrazolium compound phenyl N acetyl L alaninate was synthesized as previously described. R NPAA, S NQM, and R NQM were synthesized with the fol lowing modifications. R enantiomers were synthesized using N acetyl D alanine in place of N acetyl L alanine. The naphthyl core of NQM Inhibitors,Modulators,Libraries prodrugs were synthesized by re placing 4 phenol with 4 1 naphthol.

Cell culture and lysates Tumorigenic cell lines LNCaP, DU 145, and PC 3 and the non tumorigenic cell line RWPE 1, and COS 7 cells were purchased from American Type Culture Collection, cultured according to ATCCs in structions and supplemented with 100 U ml penicillin and 100 mg ml streptomycin. Inhibitors,Modulators,Libraries Cells were detached from the 75 cm3 cell culture flasks after reaching 80% confluence by washing the cells with PBS followed by the addition of 0. 25% trypsin. The detached cells were centrifuged at Inhibitors,Modulators,Libraries 500 g for 5 min and washed with PBS to remove trypsin. Cells were centrifuged a second time and pellets stored at ?80 C. Cell pellets of each cell line were lysed using 2% digitonin in PBS on ice with vortexing every two min. After 10 min of incubation on ice, the ly selleck compound sates were centrifuged at 18,000 g for 5 min at 4 C and the supernatant collected. Protein concentrations were determined with the BCA kit using the manufac turers instructions. Semi purified OPH from rat liver OPH was semi purified from 100 g of rat liver using the method described by Stone et al. The pooled semi purified rOPH was analyzed by mass spectroscopy as described by Stone et al. to verify that no other esterases or proteases were present.

The Orbitrap was used to collect MS scans. All data were con verted from raw files to the. dta format using ExtractMS version 2. 0 and searched against human reference database fairly downloaded from the National Center for Biotechnology Information using the SEQUEST Sorcerer algo rithm. Searching parameters included mass tolerance of precursor ions and product ion, partial tryptic restriction, with a dynamic mass shift for oxidized Met, two maximal modification sites and a maximum of two missed cleavages. Only b and y ions were considered during the database match. To eval uate the false discovery rate, all original protein sequences were reversed to generate a decoy database that was concatenated to the original database. The FDR was estimated by the number of decoy matches and total number of assigned matches.

FDR 2 nd nt, assuming mismatches in the original database were the same as in the decoy database. To remove false positive matches, assigned peptides were grouped by a Inhibitors,Modulators,Libraries combination of trypticity and precursor ion charge state. Each group was first filtered by mass accuracy and by dynamically increasing correlation coefficient and Cn values to reduce theoretical protein FDR by the above measure to less than 1%. All MS MS spectra Inhibitors,Modulators,Libraries for proteins identified by a single peptide were manually inspected as described previously. If peptides were shared by mul tiple members of a protein family, the matched members were clustered into a single group. On the basis of the principle of parsimony, the group was represented by the protein with the greatest number of assigned peptides.

Inhibitors,Modulators,Libraries All identified Inhibitors,Modulators,Libraries proteins and ungrouped are provided respectively in Table S1 and Table S2 in Additional File 1. Quantification of peptides and proteins was based on the comparison of paired pep tides from AD and control samples. Ion current intensities for identified peptides were extracted in MS survey scans of high resolution and a ratio of the peak intensities for the peptide precursor ion was calculated using in house DQuan software as described previously. Establishing candidate biomarkers with statistical and pathway analysis Statistical analysis to evaluate the significance of the pro tein changes was performed as previously described with modifications. Relative differences in protein levels Inhibitors,Modulators,Libraries were derived from extracted ion intensities for all identi fied peptides and expressed as signal to noise ratios. A ratio of ion intensities for the peptide precursor ions from AD and control pools were calculated, log2 transformed, and averaged to obtain a protein ratio across samples. A null experiment was represented Regorafenib 755037-03-7 by a comparison of log2 transformed protein ratios for control replicates.

Weil left a model that was both compelling and competitive. Ponatinib dna Aggression was a byproduct of the organization Weil helped create. it was also part of a trend in AIDS services. This imprint was left on a culture of harm reduction as the movement churned forward. It also became Inhibitors,Modulators,Libraries part of the culture and organizational prac tices of the organizations born of the social movement that created harm reduction. Many lamented that harm reduction was increasingly a part of the system of non profits funded by governments, philanthropy, and modern capitalism. Capitalism is a system which encourages people to view each other in competitive ways, notes another anonymous author de scribing the occupational culture of harm reduction. The violence of late capitalism finds its way into the very organizations used to challenge it.

Within agencies there is as much jockeying for position, staking out personal turf, gossip and bitter interpersonal strife as might be found in any office in corporate America perhaps even more so notes an anonymous worker in a harm Inhibitors,Modulators,Libraries reduction agency. After all, the writer confesses, those in harm reduction work in a space where, suffering, injustice, and misery are a lot of the population we serve is what is constantly before our eyes. It wreaks havoc with the psyche of even the most dedicated and in control of us to witness this day after day. While some thrive in the field. many others stumble. Some workers leave of their own volition. others are forced out when they fail to keep up or are unable to han dle their drug use or the stress of the ongoing pain.

I called someone I knew from the field Inhibitors,Modulators,Libraries and asked for help noted one former worker at Cardens funeral, musing about how isolated he felt after he was dismissed from a position in a harm reduction agency. Maybe I need de tox he asked one former colleague, who responded. Youre fucked up. No one was really there for him, he explained. Durkheim suggests Inhibitors,Modulators,Libraries that such a lack of soli darity may be a contributing factor to social isolation. these broken social bonds wear on people, causing stress on minds and bodies. Alienation takes many forms. A worker loses a job and needs support. The worker feels isolated from old colleagues. One colleague confessed that losing that job was the most painful thing that ever hap pened to her. Her whole sense of self was lost.

Turnover Inhibitors,Modulators,Libraries is not just part of the work, it is part of life for those in non profit, AIDS service and harm reduction organizations. That does not mean the workers selleck do not still need help. Many do not get it or even know how to ask for it. After all, harm reduction is a business involving con tract management, constant deliverables, and competition for dwindling funding contracts. This is something funders and contract managers remind subcontracting agencies all the time.

Despite the observation that prohibitin is upregulated in both http://www.selleckchem.com/products/dorsomorphin-2hcl.html cell lines following lovastatin treatment, an expected down regulation of E2F1 only occurred in Rb positive MDAMB231 cells. Therefore, while acting synergistically Inhibitors,Modulators,Libraries with Rb in the suppression of E2F1, prohibitin does not seem to impair E2F1 expression alone. As for the down stream targets in the E2F mediated pathway, we identi fied changes in both MCM7 and MSH2. Although MCM7 belongs to the cell cycle DNA check points, MSH2 is a representative member of MMR sys tems. The expression of both of these was significantly suppressed by lovastatin. Interestingly, the suppression occurred in both cell lines, suggesting that it may not be mediated exclusively through E2F1 reduction, and that perhaps other regulatory pathways are also affected by lovastatin.

Statin treated breast cancer cells die through apoptosis. It was therefore not surprising that a large Inhibitors,Modulators,Libraries number of identified proteins was associated with the programmed cell death pathway. In addition Inhibitors,Modulators,Libraries to prohibi tin, RhoB and cofilin 1 2, there was also suppression of TRAP 1 and Ku70 expression. Both of these proteins protect the cells from apoptosis and oxidative stress. These data comply with previous reports sug gesting that increased oxidative stress may be a cause of statin induced cytotoxicity in breast cancer. Recently, it has been shown that fluvastatin and simvas tatin enhance NO levels and increase iNOS RNA and protein expression in breast cancer MCF 7 cells, indicat ing that iNOS mediated NO is responsible, in part, for the Inhibitors,Modulators,Libraries proapoptotic, tumoricidal, and antiproliferative effect of statins.

Furthermore, the cell death of MCF 7 cells incubated with N acetyl L cysteine plus statins could almost be reversed, supporting our results that oxidative stress plays an important role Inhibitors,Modulators,Libraries in the cell death induced by statins. In terms of metabolic changes, the downregulation of glycolytical this research enzymes triosephosphate isomerase, alpha enolase and dihydrolipoamide acetyltransferase and tri carboxylic acid cycle enzymes such as SDHA represent potential pathways by which lovastatin may induce cell death through the suppression of energy producing pathways. Glycolysis is the primary energy producing pathway in cancer cells and is therefore a highly valu able target in anti cancer therapy. The changes in enzyme expressions correlate with the NMR based metabolic profiles, decreased production of de novo 13 C lactate, 13C alanine and C4 glutamate and accumu PTEN through an Akt dependant pathway. The lation of intracellular glucose. Due to its close relation to anaerobic glycolysis, we chose to investigate the role of the protein kinase Akt. A downregulation of the active p Akt form was detected in both cell lines.

When Kaplan Meier analysis was performed using relapse as an endpoint, patients with breast tumors having high expression of these genes showed a significantly poorer outcome. To further assess the relevance and applicability of this 14 3 3 signature, we selected ER positive tumors from three other independent breast tumor microarray datasets with clinical information selleck products available. For all these three datasets no information on hormonal treatment was reported. Figure 2a shows the heat maps and den drograms for expression of the 14 3 3 signature genes from these three studies. The red group ing in the dendrogram represents breast tumors with high expression of the signature genes. The Wang et al. dataset includes data from 209 patients, and used the same Inhibitors,Modulators,Libraries Hu133A Affymetrix microarray platform used by Frasor et al.

All 29 genes from the 14 3 3 sig nature were retrieved and used for data mining. Unsu pervised clustering analysis identified the red group Panel I as a poor prognosis group driven by high expression of the Inhibitors,Modulators,Libraries signature genes. In a similar fashion, we analyzed the ER positive Inhibitors,Modulators,Libraries breast Inhibitors,Modulators,Libraries tumors included in the vant Veer data set. Given the different microarray platform used, a reduced number of genes were retrieved, 17 out of the 29 genes in the 14 3 3 gene sig nature. The signature genes not retrieved by our analysis were not present on those arrays. However, the subset of patients characterized by high expression of the 14 3 3 signature showed a significantly earlier relapse. We also examined the dataset of Sorlie et al. which used cDNA Stanford arrays con taining 8,102 genes.

Expression Inhibitors,Modulators,Libraries data for 19 genes of the gene signature were recovered and used for the analysis. The findings confirmed once again that overexpression of the 14 3 3 signature was significantly associated with a poorer disease free survival. Breast cancer subtypes and the 14 3 3 gene signature We next examined the distribution of the five major breast cancer molecular subtypes in the set of patients that showed high expression of the 14 3 3 gene signa ture and a poor clinical outcome in the different clinical studies by using a centroid mediated clustering algo rithm. All datasets showed enrichment for luminal B subtypes in tumors with elevated expression of the 14 3 3 signature genes, ranging from 53 to 58% of all tumors. In addition, 7 to 21% of total ER positive breast cancers showing high expression of the 14 3 3 gene signature were represented by the basal breast cancer subtype. For comparison we also classified tumors characterized by low expression of the 14 3 3 gene signature, and found that luminal A fda approved was the most abundantly represented molecular subtype in the differ ent datasets.

Slides were treated with 3% hydrogen peroxide to reduce endogenous peroxidase activity and washed with PBS containing 0. 5% Tween 20. Proteins of interest were detected using specific antibodies diluted in PBS Tween 20 and visualized using the EnVision Dual Link Labelled Polymer Kit following Ruxolitinib JAK the manufacturers instructions. Images were captured using the Aperio ScanScope scanner. Reduced representation bisulfite deep sequencing Analysis of CpG island methylation by reduced repre sentation bisulfite deep sequencing was determined as described previously. Briefly, DNA extracted from cell lines was fragmented using endonuclease MspI, followed by QIAquick purification. Digested DNA was then treated according to the Illumina protocol, separated by 2% agarose gel and purified using the QIAquick Gel Extraction Kit.

The purified DNA was modified and purified using the EpiTect Bisulfite Kit. The bisulfite converted DNA was then amplified by PCR. The amplification con ditions were as follows, 5 min at 95 C, 30 s at 98 C, then 66 cycles, followed by 5 min at 72 C. The PCR product was puri fied using the MinElute PCR Purification Kit, and the concentration of a final library was measured Inhibitors,Modulators,Libraries using the Agilent 2100 Bioanalyzer. The library was sequenced on an Illumina Genome Analyzer IIx sequencing instru ment according to standard Illumina cluster generation and sequencing protocols. Methylated C base was mea sured by counting the C C T ratio. Summarized methy lation data on the PRKD1 promoter CpG island were obtained by averaging all CpG sites.

These data repre sent the percentage of methylated CpGs over total num ber of CpGs in the island. The differentially methylated CpG islands were identified using the limma software package as described Inhibitors,Modulators,Libraries for analysis of gene expression. A P value cutoff of 0. 05 was applied for significantly meth ylated CpG islands. Bisulfite conversion Inhibitors,Modulators,Libraries and methylation specific PCR Genomic DNA was isolated from cell lines and tumor samples using the QIAamp DNA Mini Kit according to the manufacturers instructions. Genomic DNA was then modified with a sodium bisulfite solution using the EZ DNA Methylation Kit and amplified by PCR using the GC Rich PCR Amplification Advantage GC 2 Polymerase Mix and PCR Kit. In situ methylation specific PCR In situ methylation specific PCR was per formed as described previously.

Paraffin embedded Inhibitors,Modulators,Libraries sections were digested with pepsin for 20 min, washed in water for 1 min and air dried. Sections were then placed in 3 M bisulfite Inhibitors,Modulators,Libraries solu tion, heated at 94 C for 3 min and incubated at 50 C for 15 h. The in situ MSP PCR step selleckchem was performed as follows, denaturation at 94 C for 1 min, amplification for 35 cycles using AmpliTaq Gold 360 DNA Polymerase Kit. diluted with in situ hybridization buffer. The PCR product and the probe were codenatured at 95 C for 8 min and hybridized at 37 C for 15 h. Sections were then washed in 0.

Our results show that in RKO this particular cancer cell trait is modulated by and dependent sellckchem on B RafV600E and that targeting mutant BRAF is sufficient to restore sensitivity to caspase dependent apoptosis after serum withdrawal via p53 independent PUMA induction. Complementing and extending previous studies, we thus provide evidence from an endogenous and quantitative genetic model of BRAF mutant colorectal cancer cells, thereby ruling out the occurrence of artifacts caused by unspecific cellular response or incomplete knockdown in RNAi setups and, likewise, avoiding inter species bias potentially experienced in mouse models of colorectal cancer. Pharmacogenetic characterization Hyperactivated Raf Mek Erk signaling has been sug gested to mediate resistance towards drug induced cell death.

However, data from prostate cancer cells transfected with mutant BRAF showed that there might be tumor entity dependent differences. Our Inhibitors,Modulators,Libraries model system of corresponding tumor cells is ideally suited to determine the B RafV600E specific effects of a com prehensive panel of widely used chemotherapeutic agents including crosslinking agents, a taxane, a topoisomerase II inhibitor, and the nucleic acid metabolism in hibitor 5 fluorouracil. We found that the BRAF mutational status did not have a detectable impact on chemosensitivity towards any of these agents. These findings Inhibitors,Modulators,Libraries suggest that B RafV600E does not significantly contribute to resistance towards conventional chemotherapeutics in colorectal cancer cells and are in accordance with previous studies suggesting Inhibitors,Modulators,Libraries the Raf Mek Erk cascade to play a minor role in chemoresistance.

Taken together with the observed differential sensitivity of BRAF mutant cells towards Inhibitors,Modulators,Libraries starvation induced apoptosis, these results further dissect the distinct apoptosis pathways in our model, i. e. serum starvation versus chemotherapeutic agents. wild type clones express the epidermal growth factor re ceptor at comparable levels. To test whether loss of mutant BRAF might reconstitute respon siveness to the inhibition of EGFR, cells were treated with the monoclonal antibody cetuximab. However, no difference in proliferation was observed between BRAF wild type and mutant cells, while cetuximab sufficiently inhibited growth of the control cell line Lim1215. All cells revealed a similar slight decrease in the prolifer ation index down to 0.

6 at Inhibitors,Modulators,Libraries very high concentrations of cetuximab. This modest effect might be due to unspecific toxicity or to dilution, rather than to a spe cific anti proliferative effect of cetuximab, since at 0. 8 g L the antibody solution accounts for 16% of the culture medium. These findings are in line with previous studies showing that resistance against EGFR targeted treatment frequently occurs in selleck compound BRAF mutant tumors. Next, we investigated the impact of the BRAF V600E mutation on several established B Raf inhibitors.

Soft agar growth was measured in PrECs after co culture with EVs from prostate cancer patients selleck chemicals Calcitriol 18 and 19. EVs from patients 18 or 19 significantly increased soft agar growth in non malignant PrECs. A portion of the sample used for soft agar cloning was analyzed by mass spectrometry. Table 2 shows a partial list of the proteins identified in PrECs exposed to tumor derived EVs from patients 18 and 19 as well as the log2 relative expression of each protein. Some 14 3 3 isoforms are associated with increased ma lignancy and are therapeutic targets and our analysis revealed an increase of 14 3 3 zeta delta which was confirmed by Western blot analysis. Also of note is the increase in pRKIP when patient 18 and 19 EVs were co cultured with PrECs in reference to the levels of RKIP in PrECs alone.

RKIP has been shown to regulate Inhibitors,Modulators,Libraries apoptosis and cell survival in prostate cancer. Western blot analysis revealed that RKIP Inhibitors,Modulators,Libraries was phos phorylated after co culture of patient 18 and 19 EVs with PrECs. This result would explain, in part, our data in Figure 4 because pRKIP antagonizes the function of RKIP and allows for Raf MAPK signaling to occur. This pathway promotes oncogenesis and cell pro liferation and, presumably, soft agar growth. In our analysis of the total proteome content of PrECs exposed to EVs derived from patient 18, we identified 36 protein groups in PrECs alone and 44 protein groups in PrECs with Patient 18 EVs. From these, 8 protein groups were unique to PrECs and 16 were unique in Patient 18 EVs with 28 common protein groups. Expos ure of PrECs with EVs from Patient 19 yielded similar results.

For Inhibitors,Modulators,Libraries example, Macrophage migration inhibitory factor and Peptidyl prolyl cis trans isomerase A were found to be unique in both Pa tient 18 EVs and Patient 19 EVs when compared to PrECs alone. Analysis of proteome content between patients 18 and 19 yielded Inhibitors,Modulators,Libraries minimal differences between the numbers of protein groups identified in each sample indicating low patient heterogeneity. We examined the EV content of 3 additional Gleason grade 8 patients. The Venn diagram shows that there are 222 common pro teins between these patients. The bar graph shows the functionalities listed by IPA based on the ProteoIQ pro tein relative expression values. The relative expression value of each protein in addition to the presence or ab sence of key proteins associated with that term both contribute Inhibitors,Modulators,Libraries to the significance value assigned to each function or pathway.

The terms presented in the bar graphs were selected because they are related to cancer and or are important topics discussed in the paper. In addition, we compared patients 18 and 19, with pa tients 13, 14, and 16, and determined the common pro teins between these 5 Gleason grade 8 patients. These Bioactive compound 71 common proteins between the 5 Gleason grade 8 pa tients were then further filtered according to GO anno tations which are related to apoptotic and cell survival pathways.

It is worth noting though that all four studies found no association with cardioembolic stroke, consistent with our results. There is a potential explanation, speculative at this stage, which might explain the stronger association between air pollution and mild stroke, and which does not require considering the small vessel disease hypothesis. It may be that thrombus formation triggered by air pollutants is smaller, less dense or more easily broken down. In addition, atheromatous plaques induced by air pollutants may be more modest in size. With this hypothesis, the air pollution effect would not Inhibitors,Modulators,Libraries need to be preferentially associated with small vessel disease stroke as ischemic stroke caused by air pollutants could affect large and small arteries alike but the resulting clinical picture would tend to be that of a mild stroke.

We previously observed that air pollution exposure was more strongly associated with stroke mortality than with stroke hospital admissions. Others have also reported a stronger association with fatal than with non fatal stroke. This suggests that air pollution ought to be more strongly associated with severe stroke and is therefore not consistent Inhibitors,Modulators,Libraries with the results we have observed. However, a possible alternative explanation is that whilst air pollution is more strongly associated with mild stroke, exposure to high pollution reduces survival after stroke and it is the latter which accounts for the stronger association seen with stroke mortality. In this regard, we have previously reported a strong independent adverse effect of outdoor air pollution exposure on survival after stroke.

Internationally significant novelty The internationally significant novelty of our study results from two aspects. Firstly, air pollution is a widespread environmental hazard, with increasing levels of road traffic related air pollution encountered in many high and middle income countries across the globe. Secondly, Inhibitors,Modulators,Libraries ours is the first study to examine the chronic effects of outdoor Inhibitors,Modulators,Libraries air pollution on the incidence Inhibitors,Modulators,Libraries of ischemic stroke subtypes. Limitations There are a number of potential limitations to our study. The ecological study design is susceptible to ecological bias, which is the situation where the association seen at the area level is different from that which exists at the individual level. Ecological bias cannot be ruled out in our study.

However, we used a small area level ecological study design which would have mitigated ecological bias as small areas tend to be relatively more homogenous in terms of population characteristics and exposure to environmental pollutants. In addition, we compared effects on different stroke subtypes and severity within the same ecological study and any bias inherent in the study Sunitinib design might be expected to have similar effects on the different severity and subtype groups examined.