The relationships between total serum IgE levels and gene expression patterns in peripheral blood CD4+ T cells  are not known.Peripheral blood CD4+ T cells from 223 participants from the Childhood Asthma Management Program (CAMP) with simultaneous measurement of IgE. Total RNA was isolated, and expression profiles were generated with Illumina HumanRef8 v2 BeadChip arrays. Modeling of the relationship between genome-wide gene transcript levels and IgE levels was performed in all subjects, and stratified by sex.2) in IgE measurement . Sex stratified analyses revealed that the correlation between IL17RB and IgE was restricted to males only . Significant correlation between gene transcript abundance and IgE level was not found in females. Additionally we demonstrated substantial sex-specific differences in IgE when considering multi-gene models, and in canonical pathway analyses of IgE level.Among all subjects, significant evidence for association between gene transcript abundance and IgE was identified for a single gene, the interleukin 17 receptor B (IL17RB), explaining 12% of the variance (rOur results indicate that IL17RB may be the only gene expressed in CD4+ T cells whose transcript measurement is correlated with the variation in IgE level in asthmatics. These results provide further evidence sex may play a role in the genomic regulation of IgE. Total serum immunoglobulin E (IgE) is a risk factor for both the development of  and diseSex is a critical determinant of IgE level, with males having a stronger tendency towards higher total IgE than females . Sex-relBased on these findings we hypothesized that genome-wide CD4+ T cell microarray analyses would provide unique insights into genes that correlate with the variability in IgE level, and that sex would be an important modifier of these correlations. To test these hypotheses we explored the relationship between total serum IgE levels and genome-wide gene expression transcript abundance in a cohort of 223 young adults with asthma, both in all subjects and within each sex specifically.6 total cells. Pilot studies confirmed CD4+ cell yields of ~4 × 106 at ≥ 95% purity per collection. Total RNA was extracted using the RNeasy Mini Protocol   5-17) of the number of transcripts that should overlap by chance alone. At a more liberal threshold for inclusion of the results from females (p < 0.1), only 38 of 1205 unique transcripts (3.1%) overlap. At a more conservative threshold (p < 0.01) there were no transcripts common to both males and females.2 of 0.09 in the 89 CAMP females).We considered whether our findings of association in males but not females could be attributable to differences in sample size. Though power was somewhat lower in females due to the smaller number of subjects , power was very high (>91%) to detect correlations half as strong as those observed among males , hexose-6-phosphate dehydrogenase (H6PD), IL17RB, poly(A) binding protein interacting protein 1 (PAIP1), and PVALB  could both confirm our findings of association from single gene regressive strategies, and could identify additional determinants of total serum IgE levels. Among all subjects, the optimal multivariate model included 17 transcripts (including IL17RB), of which 16 map to unique HUGO gene identifiers . Notably, there were no significant canonical pathways in common between males and females. Additional information about the canonical pathway analyses, including lists of differentially expressed genes within the pathways are included in Additional Files To better understand the combinatorial effect of multiple gene transcripts on IgE regulation we performed canonical pathway analyses. Of the 763 transcripts demonstrating nominally significant (p < 0.05) correlation with total serum IgE, 652 85%) map to a unique HUGO gene id and were used for IPA canonical pathway analysis. Among all subjects, enrichment for six pathways was observed (Fisher's exact p < 0.05) including the antigen presentation pathway, propanoate metabolism, interferon signaling, primary immunodeficiency signaling, the coagulation system, and the pantothenate and CoA biosynthesis pathway  identified in a recent genome-wide association study of IgE [This is the first study to correlate genome-wide CD4+ T cell transcript abundance with IgE level in humans and is, to our knowledge, the largest gene-expression study of as asthma-related quantitative phenotype published to date. Our results indicate that IL17RB may be the only gene expressed in CD4+ T cells whose transcript measurement is correlated with the variation in IgE level in asthmatics. Given that IgE level is strongly influenced by environmental factors  it is surprising that 12% of the variation in IgE can be explained by IL17RB transcript measurement, a correlation that is substantially greater than that explained genetic determinants from three significant genomic regions  but has much higher binding avidity to interleukin 17 E  . While s2 = 19%), but not in females (r2 = 3%); evidence for a multivariate gene signature of total IgE levels in males, but not in females; and discrete, non-overlapping canonical gene pathway enrichment in each sex. Notably, we found limited statistical evidence to suggest that there is significant overlap between CD4+ T cell genes whose transcript measurements correlate with IgE level in both male and female asthmatics. Our work stands with a body of literature now implicating an important role for sex in the heritability, genome-wide linkage analyses, [this report) of IgE, and strongly support the notion that genetic and genomic studies of IgE should consider the role of sexual dimorphism.Another finding of our analyses is that the magnitude of the relationship between patterns of CD4+ lymphocyte gene expression and total serum IgE levels is substantially different between males and females. Several lines of evidence are provided, including: strong association of IL17RB transcript abundance with IgE levels in males  to detect the male-specific associations in our female sample, replication in larger sample sizes could help to determine if the lack of overlap we note between males and females is primarily an issue of the magnitude of the correlations or of true differences in the genomic regulation of IgE. In addition, our gene expression analyses include gene transcript measurements from unstimulated peripheral blood CD4+ lymphocytes in a population of adolescents and young adults recruited on the basis of childhood asthma. We urge caution in extrapolating our findings to other populations and cell types. Finally, while an assessment of IL17RB protein levels would provide additional supportive information, we do not have intact CD4+ lymphocytes enabling suitable cell surface protein quantification.TSLP polymorphisms on the regulation of specific, and total serum IgE in children with asthma [While the precise role that sex plays in the genomic regulation of IgE is unknown, sex differences in the immune environments experienced by CD4+ T cells are well described. In animals, females are more likely to develop a Th1 response after antigen challenge, except during pregnancy when a Th2 environment dominates . Although asthma . This ish asthma .-6), particularly in light of prior studies suggesting the importance of interferon-γ (INFG) in the female  allergic response [Though we have limited evidence that single genes, or genetic models strongly correlate with IgE level among females, it is intriguing that the canonical pathway most significantly correlated with IgE in females was the interferon signaling pathway  correlated with total serum IgE in all subjects.Click here for fileCanonical pathways (and genes) correlated with total serum IgE in males.Click here for fileCanonical pathways (and genes) correlated with total serum IgE in females.Click here for file


KRAS and EGFR somatic mutations in NSCLC may predict resistance and responsiveness to TKI, respectively. Nevertheless, most research to date has been conducted on samples from primary tumors. For many patients with advanced disease, their samples can only be obtained from metastases for test. The molecular characteristics of metastasized tumors may be different from those of primary tumors.Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) have been widely used for the treatment of non-small cell lung cancer (NSCLC). KRAS and EGFR between primary tumors and local lymph node metastases of 80 Chinese patients with NSCLC were analyzed by direct sequencing. Five of them were given gefitinib as neoadjunvant treatment after the EGFR-TKI sensitive mutations were detected in their biopsies of mediastinal lymph nodes metastases. McNemar's test was used to compare the EGFR and KRAS mutation status between primary tumors and corresponding local lymph node metastases. Data evaluation was carried out with SPSS_13.0 statistical software.Mutation status of KRAS mutations and 21 primary tumors and 26 metastases were found to have EGFR mutations. KRAS and EGFR mutation status was different between primary tumors and corresponding metastases in 6 (7.5%) and 7 (8.75%) patients, respectively. One patient with no TKI sensitive mutations detected in the primary tumor showed disease progression.Among the 160 samples, one primary tumor and seven metastases were identified with KRAS and EGFR mutation status between primary tumors and corresponding metastases. This observation may have important implication for the use of targeted TKI therapy in the treatment of NSCLC patients.Our results suggest that a considerable proportion of NSCLC in Chinese population showed discrepancy in  EGFR gene, whereas the presence of KRAS mutations seems to be correlated with primary resistance to these agents [KRAS and EGFR for selection of patients who are more likely to benefit from TKI. Although almost 70% of patients with NSCLC present with locally advanced or metastatic disease at the time of diagnosis [KRAS and EGFR mutation status is most commonly assessed only in the primary tumor tissue based on the assumption that primary and metastases are pathologically concordant. However, it has been known that lung cancers are often heterogeneous at the molecular level even within the same tumor and many key molecular alterations may occur during metastatic progression [KRAS and EGFR mutation status in primary tumors is reflected in their corresponding metastases in Chinese patients with NSCLC, although several recent relevant studies in western countries have been performed and published [Lung cancer is one of the leading causes of cancer-related mortality both in China and throughout the world ,2. Non-se agents -15. So iiagnosis ,17, KRASgression -20. It iublished -26.KRAS and EGFR mutation status using PCR-based sequencing analyses in 80 primary tumor samples and their corresponding local lymph node metastases from Chinese patients with NSCLC. The goal is to determine whether KRAS and EGFR mutation profile is stable during the metastatic progress and to investigate the clinical usefulness of mutational analyses in primary tumor versus in metastases for planning EGFR-targeted therapies for the treatment of patients with NSCLC.In the present study, we investigate Patients were selected from a pathological database of lung cancer cases undergoing curative resection for excision of primary tumor and the corresponding lymph nodes metastases at the Pathology Department of Tianjin Medical University Cancer Hospital from March 2009 to September 2009. Only patients with paraffin embedded tissues from surgically resected primary lung cancers and lung cancer-related local lymph node metastatic samples with histologically confirmed NSCLC were included. Patients who had been exposed to TKI before surgical treatment were excluded from this study. In each case, hematoxylin and eosin-stained sections of formalin-fixed paraffin-embedded tissue of primary tumor and corresponding synchronous lymph node metastases were reviewed by two pathologists to identify neoplastic areas and the amount of tumor cells in order to ensure that they contained more than 70% of tumor components for DNA extraction and mutation analysis. Tissue blocks were macro-dissected using a safety blade when samples were less than 70% of tumor cells. Primary tumor and lymph node specimens were obtained from all patients by surgical resection of primary tumors with lymph nodes dissection according to prevailing surgical standards. Consequently, 80 pairs of primary tumors and the corresponding lymph nodes metastases were analyzed. All samples were from patients of Chinese origin with NSCLC. The characteristics of the included patients were shown in Table The inclusive criteria for selecting patients to receive gefitinib as neoadjunvant therapy were as follows: (1) NSCLC verified by cytology or histology; (2) age 18 to 70 years; (3) NSCLC with stage ⅢA or ⅢB and the tumors were confined in homolateral thoracic cavity; (4) patients without metastases in contralateral mediastinal lymph node; (5) patients who have never received treatment; (6) patients who could tolerate the surgery; (7) patients who were willing to receive preoperative target therapy. The exclusive criteria were: (1) without definite diagnosis; (2) age ≥ 70 years; (3) NSCLC with N3 or distant metastases; (4) small cell lung cancer; (5) patients who have been treated before; (6) patients who were unable to tolerate radical surgery. The local ethics committee granted approval, and written informed consent was obtained from each patient.Thirty mg of frozen tissue was shredded by scissors. The E.Z.N.ATM Tissue DNA Kit (purchased by OMEGA) was used to extract genomic DNA. Quality and concentration of the DNA samples were examined by Nano Drop (Thermo™). Genomic DNA was then diluted to a working concentration of 5-10 ng/ul.KRAS (12 and 13) and two exons of EGFR (19 and 21) were amplified by PCR using the following forward and reverse primers: exon 1 of KRAS: 5'-AAAGGTACTGGTGGAGTATTTGATAGTG-3', 5 ' -TCATGAAAATGGTCAGAGAAACCT- 3 '; EGFR e x o n 1 9: 5 ' -AGCATGTGGCACCATCTCAC-3',5'-GCAGGGTCTAGAGCAGAGCAG-3'; E G F R e x o n 2 1: 5 ' - C T G A A T T C G G A T G C A G A G C T T - 3 ', 5 ' - C T A G T G G G A A G G C A G C C T G G T - 3. A total of 20 μl PCR reaction system included the following: 1x HotStarTaq buffer, 2.0 mM Mg2+, 0.2 mM dNTP, 0.2 μM of each primer, 1U HotStarTaq Polymerase (Qiagen), and 10ng DNA template. PCR reaction procedures were performed using 35 cycles of 15 sec at 94°C, 30 sec at 56°C, 1 min at 72°C and extension for 2 min at 72°C. Sequencing reactions were performed on an ABI3700 genetic analyzer after PCR products were purified. Sequence variations were determined using Seqscape software (Applied Biosystems) with the KRAS and EGFR reference sequence .The two codons of In order to avoid contamination during PCR steps, gloves and lab coats were worn at all times when PCR is performed. Pipette tips with aerosol filters were used to prevent microdroplets being injected into the PCR mixture. DNA sample preparation was done in a separate room from the area where PCR reaction mixes were prepared. Additionally negative control was also included during PCR procedure.Five patients received gefitinib as first-line treatment after being identified to harbor EGFR-TKI sensitive mutations in mediastinal lymph nodes metastases obtained by mediastinoscope. One tablet of gefitinib (250 mg) was taken once daily at about the same time. Patients continued the course uninterrupted until disease progression, intolerable toxicity or withdrawal of consent. All drugs were supplied by AstroZeneca.Baseline evaluation included medical history and physical examination, electrocardiogram, chest radiography, thorax CT scan and ultrasonography of the upper abdomen. Laboratory investigations included complete blood counts, urinalysis, renal function and liver function tests. Performance status was evaluated according to the Eastern Cooperative Oncology Group (ECOG) criteria. Patients were re-evaluated, using the same method at the end of the first and third months of therapy, and then every 3 months. Objective tumor response and its duration were assessed according to the RECIST criteria , and allEGFR and KRAS mutation status between primary tumors and corresponding local lymph node metastases. Two-sided p values <0.05 were considered significant. Data evaluation was carried out with SPSS_13.0 statistical software.McNemar's test was used to compare the KRAS mutations were detected in one primary tumor and seven lymph node metastases . The clinicopathologic characteristics that were significantly associated with EGFR mutations were gender, smoke history and pathologic type. Woman, non-smoker and adenocarcinoma showed a higher percentage of EGFR mutations . Discordant cases included five cases with no EGFR mutation in the primary tumors  and 8.75% (7/80), respectively. To our knowledge, there have been several recent similar studies in western countries. For example, Kalikaki et al. reported that the discordance in KRAS and EGFR gene status between primary tumors and corresponding metastases was 24% and 28% in 25 patients with NSCLC, respectively [KRAS and EGFR gene status in primary tumors and lymph node metastases were discordant in 25 (26%) and 6 (6.25%) patients among 96 patients, respectively [KRAS status [KRAS gene status in primary tumors and their matched metastases. They found that the use of ME-PCR allowed a resolution of the discordance in 3 of the 6 cases by demonstrating the presence of low levels of mutant KRAS in lesions that were found negative by direct sequencing. Their data suggests that some gene discordance could be resolved by using techniques with increased sensitivity and that highly sensitive tools are required to identify biomarkers [KRAS mutation rate is high in NSCLC patients, especially in those with adenocarcinoma (30%-50%), but EGFR mutation rate is low (3%-8%). However, Asian patients with NSCLC harbor more EGFR mutations (30%-60%) and fewer KRAS mutation (4%-24%) than western patients [KRAS and EGFR genes in patients from Asia and western countries, it is very likely that changes of the mutation status during disease progression are different. Because relevant data about Chinese or Asian was not searched, further study should be performed to disclose the molecular mechanism.In this study we assessed nt in 25 % and 6 , consistent with those previous reports. This result also suggests that the KRAS mutations might play an important role during metastases of NSCLC, especially squamous cell carcinomas.Moreover, several previous studies assessing the Neoadjuvant or presurgical therapy is a novel therapeutic strategy that is now being investigated in the treatment of NSCLC. In part predicated on the success of this paradigm in other malignancies , presurgical therapy has the potential to provide real-time clinical feedback on the responsiveness of the patient's overall tumor burden to a given systemic therapy before committing the patient to what could be a highly morbid surgical procedure. Other potential benefits of this approach include local tumor down-staging, which may make subsequent surgical extirpation less morbid. In the case of locally advanced NSCLC, presurgical therapy may eliminate micrometastatic disease at its earliest stage, thus diminishing the risk of metastatic progression postoperatively. With the development and implementation of molecular targeted therapies that can meaningfully affect the biology of both primary tumors and metastases, the practice has largely been extended into the era of targeted therapy. In our study among five patients with EGFR TKI-sensitive mutations in mediastinal lymph node metastases, there were four patients who showed tumor regression in response to EGFR TKI and underwent surgery. These responses included dimension reductions in both primary tumors and mediastinal lymph nodes, suggesting tumor down-staging. Therefore, it is intriguing to consider the utilization of targeted therapies as an adjunct to make the "unresectable" become resectable. Neoadjuvant target therapy for NSCLC could potentially become a new treatment option for locally advanced and metastatic disease. On the other hand, we should not ignore the possibility that gene mutation status of primary tumors is different from that of their metastases when neoadjuvant target therapy is considered. If discordance between primary tumors and metastases is not evaluated before therapy, the patients may not benefit from the targeted therapies. Taken together, we propose that biopsies of both primary tumors and metastatic tumors of patients with advanced NSCLC, though difficult to obtain, should be pursued to ascertain the mutation status of key genes. This will allow clinicians to better understand gene mutation status and the biology of patient tumors, so that better treatment options can be selected based on tumor responsiveness to those available targeted therapies such as EGFR TKI.KRAS and EGFR mutation status between primary tumors and metastatic tumors may have therapeutic implications for EGFR-targeted therapy strategy. For NSCLC patients with metastases, determining the KRAS and EGFR mutation status in both primary and metastatic tumors may be critical for making meaningful decisions regarding the appropriate use of targeted therapies.In summary, the substantial discordance of The authors declare that they have no competing interests.ZZ, CW and BS designed the study; LS and QZ performed experiments; LS and HL analyzed data and prepared the Tables and Figures; LS and BS drafted the manuscript. All authors have read and approved the final manuscript.


The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems  Tumors develop a chaotic vascular network characterized by variable blood pressure and vascular permeability that inhibits effective drug delivery By transiently altering tumor blood vessel physiology during systemic anti-cancer treatment, tissue perfusion and drainage can be enhanced, thereby relieving interstitial hypertension in vivo.Normalizing blood flow promotes drug delivery by reducing the interstitial pressure that counteracts diffusion. However, normalizing agents can also reduce vascular permeability. Vascular permeability greatly influences the extravasation of drugs associated with carriers, including liposomes, micelles or other nanoparticles Classically, vascular permeability has been measured using the Miles Assay ex ovo chick model. The leakage of small versus large molecular weight dextrans from the vasculature in this model may provide a high-resolution measure of vascular permeability predictive of drug localization in vivo.Here, we present an integrated method to visualize and quantify the real-time dynamics of dextrans in a shell-less chick chorioallantoic (CAM) model. Regional and temporal differences in vessel permeability within the tumor microenvironment are captured at high resolution using an intravital imaging approach. The use of dextrans of different molecular weights allows for the concurrent evaluation of vascular permeability and vascular structural integrity. The dynamics of anti-cancer drugs as they move through the vasculature and into tumor tissues can be mimicked with dextrans ex ovo model, the CAM is directly accessible for experimental manipulation and imaging. Paired with a fluorescence microscopy platform, this model is well-suited for analyzing drug-induced changes in vascular permeability in tumor xenografts and their microenvironment.The CAM is a thin, respiratory tissue for the developing chick embryo characterized by a dense, highly organized network of blood vessels We demonstrate, using this intravital imaging approach, that vascular permeability can be manipulated to modulate the extravasation of small molecules into the local tumor microenvironment. Treatment with vascular endothelial growth factor (VEGF) or a permeability enhancing peptide (PEP) fragment of IL-2 121  or PEP  in 50 µL volumes. For local applications, reagents were applied to the CAM via a small hole in a sterilized glass coverslip (18 mm diameter). Embryos were then incubated for 2 hours at which time 100 µL of 0.5% Evan's Blue, 5% BSA in PBS was injected and embryos were further incubated for 60 minutes. After incubation, the embryos were perfused with saline. The tissue underlying the coverslip was removed after the treatment period and blotted dry, weighed, homogenized and incubated in 200 µL of 100% formamide to release the extravasated dye. Tissue samples were homogenized for 30 sec and then incubated for 48 hr at 38°C. The samples were centrifuged (14000 g for 10 minutes) and 175 µL of supernatant quantified spectrophotometrically against a formamide blank at 620 nm. Vascular permeability index was calculated as dye concentration in treated tissue sections/dye concentration in matched vehicle (PBS) treated samples. For rat studies, Evans Blue dye solution  in endotoxin-free PBS) was injected intravenously. Ten minutes after injection, each rat (n = 5) was injected intradermally with 25 µL of PBS into the left ear and 0.15nmoles of reagent (∼25 µL) into the right ear. Thirty minutes later, rats were anesthetized, their ears photographed, and then perfused with 100 mL PBS thru a ventricular infusion to remove free intravascular dye. The ears were removed, the area of extravasation cut out with a biopsy punch (8 mm wide), and then the tissue was weighed and subsequently placed into 1 mL of formamide for elution of the Evans Blue dye at 60°C over the course of 48 hours. The amount of extravasated dye was then determined spectrophotometrically as described above. The absolute amount of dye was determined using a standard curve.A modified Miles assay 6 tumor cells in serum free media applied directly to a section of the CAM surface that had been lightly abraded with a piece of filter paper. For embryos being prepared for intravital imaging, sterilized coverslips were applied on top of the tumor 24 hr post tumor cell application.Epidermoid carcinoma (HEp3) or breast cancer (MDA-MB435) cells expressing green fluorescent protein (GFP) were maintained as described previously Fertilized Dekalb White chicken eggs were received from Cox Bros Poultry Farm  and incubated in a humidified chamber at 38°C. At day 4, embryos were removed from their shells using a Dremel tool with a cutting wheel and maintained under shell-less conditions, in a covered dish in a humidified air incubator at 38°C and 60% humidity as previously described Real-time imaging of vascular leak was performed using an upright epifluorescence microscope with a motorized Z stage  controlled by Volocity software . A four dimensional image series was collected by capturing a mosaic of 3D image stacks at distinct time-points from regions of interest within the CAM. Specifically, a 150 nm image stack mosaic was captured with a 15 nm step size every 15 min for 3–6 hours (13–25 frames). From this raw data at each time point, the image stack was cropped to a 100 nm stack containing the in-focus images of the tumor. This was then flattened into a maximum intensity projection for the majority of the analyses using Volocity software .http://bigwww.epfl.ch/) of ImageJ . To generate the time-dependent changes in fluorescence localization in the CAM, the time 0 image stack was subtracted from the subsequent time-points using the Image Calculator function within Image J, hence time 0 intensity was set at 0. For the generation of surface plots, end point images were processed using the Interactive 3D Surface Plot plug-in for ImageJ . Relative intensities in the surface plots were qualified using a spectrum LUT normalized from standard 255 levels to 100 levels for ease of interpretation. The pseudo colored spectrum LUT is based on 255 shades of grey in which a Value 0 = black and a Value 255 = white.Time 0 was defined as the time of the first image capture, 5 minutes after injection of the fluorescent dextran mixture. The captured images were corrected for drift and rotation using the Stackreg plugin , which are largely retained by structurally intact vasculature The tumor vasculature is heterogeneous, consisting of irregularly formed vessels that are both leaky and often fenestrated Given that the vascular permeability in tumors is elevated, and that the vasoactive agents VEGF and PEP enhance vascular permeability, we hypothesized that the delivery of chemotherapeutic drugs to tumor sites could be improved using these agents. To test this, we measured the delivery of chemotherapeutic agents to tumor sites in real time using intravital imaging in the presence and absence of VEGF . DoxorubHere, we present a novel approach to visualize and quantitate hemodynamics and vascular leak in tumors. In contrast to traditional, endpoint analysis methods, real-time intravital imaging is sensitive to changes in both permeability and vessel integrity, and can effectively track rapid and dynamic changes in vascular permeability. We demonstrate that vascular permeability is increased in xenograft tumors compared to distal normal tissues, and that it can be further enhanced by VEGF and the PEP fragment of IL-2. Utilizing standard epifluorescence microscopy, we could also monitor localization of the chemotherapeutic, doxorubicin, which is a naturally fluorescent DNA intercalating agent, using this approach. We show that the uptake of systemically administered doxorubicin in xenograft tumors is enhanced by co-administered VEGF, suggesting that transiently increasing the vascular leak in tumors using adjuvant therapies can improve the uptake of chemotherapy at the tumor site.As an alternative to the model we present here, vascular dynamics can be visualized in rodent models with the use of surgically placed skin flaps. Skin flaps have been used to estimate the vascular leakage of florescent-labeled particles under various conditions in tumors in rats ex ovo chicken embryo model The chick CAM responds predictably to permeabilization factors and supports the growth of human tumor xenografts. While increased vascular permeability induced by VEGF and PEP occurred within minutes, imaging time courses of up to 72 hours, can be accommodated in the Selective modification of the tumor vasculature is emerging as a powerful means to enhance drug delivery and ultimately efficacy. Common vasoactive agents used in oncology follow two principle approaches; perturbation of the tumor vasculature by vascular disrupting agents (VDAs) or normalization of the tumor vasculature by anti-angiogenic agents. For example, the tubulin-binding agent, combretastatin-serine (AVE8062) is a small molecular weight VDA that causes a rapid and extensive shutdown of established tumor vasculature. Prior dosing with AVE8062 can therapeutically synergize with docetaxel, oxaliplatin or cisplatin Increasing the uptake of co-administered chemotherapies by overcoming the high interstitial fluid pressures in the tumor microenvironment has previously been accomplished through inhibition of the PDGF receptor with imatinib in vivo and to screen the most appropriate strategies for improving drug uptake.Given the dynamic interplay of vascular signaling factors and their individual roles in the modulation of vascular permeability, it is difficult to predict the impact of adjuvant permeability enhancement agents on drug uptake at the tumor site. The approach and the model presented here offer a powerful tool to investigate mechanisms of vasopermeability Figure S1Comparison to rodent ear model of vascular permeability. Injection of PEP (0.15 nmoles) into the ears of rats (n = 5) induces significant levels of vascular permeability similar to CAM data in (TIF)Click here for additional data file.Video S1Intravital imaging assesses real-time changes in vascular permeability. Representative intravital imaging experiments representing the extravasation of 158 kDa dextran are shown for PBS, VEGF and PEP-treated embryos over 3 hours. Top panels represent the raw imaging data; bottom panels are normalized to the first time point to denote leaked dextran.(AVI)Click here for additional data file.Video S2Intravital imaging of vascular permeability in HEp3 tumor. Intravital imaging experiments of 158 kDa (red) and 2000 kDa (green) dextran extravasation over time are shown for human epidermoid carcinoma (HEp3) tumors established in the CAM. Raw and normalized data are shown.(AVI)Click here for additional data file.Video S3Intravital imaging of vascular permeability in MDA-MB435 tumor. Intravital imaging experiments of 158 kDa (red) and 2000 kDa (green) dextran extravasation over time are shown for human breast carcinoma (MDA-MB435) tumors established in the CAM. Raw and normalized data are shown.(AVI)Click here for additional data file.


Canis lupus) show population genetic structure in the absence of geographic barriers, and across relatively short distances for this highly mobile species. Additional information on the location of and divergence between population clusters is required, particularly because wolves are currently recolonizing parts of Europe. We evaluated genetic structure in 177 wolves from 11 countries using over 67K single nucleotide polymorphism (SNP) loci. The results supported previous findings of an isolated Italian population with lower genetic diversity than that observed across other areas of Europe. Wolves from the remaining countries were primarily structured in a north-south axis, with Croatia, Bulgaria, and Greece  differentiated from northcentral wolves that included individuals from Finland, Latvia, Belarus, Poland and Russia. Carpathian Mountain wolves in central Europe had genotypes intermediate between those identified in northcentral Europe and the Dinaric-Balkan cluster. Overall, individual genotypes from northcentral Europe suggested high levels of admixture. We observed high diversity within Belarus, with wolves from western and northern Belarus representing the two most differentiated groups within northcentral Europe. Our results support the presence of at least three major clusters  in southern and central Europe. Individuals from Croatia also appeared differentiated from wolves in Greece and Bulgaria. Expansion from glacial refugia, adaptation to local environments, and human-related factors such as landscape fragmentation and frequent killing of wolves in some areas may have contributed to the observed patterns. Our findings can help inform conservation management of these apex predators and the ecosystems of which they are part.European wolves ( Lynx canadensis) Canis latrans) C. lupus) Population genetic structure can occur across relatively short distances in the absence of geographic barriers in highly mobile species, such as lynx  with the Polish Ministry of Science and Higher Education in compliance with all requirements. No ethics permit was required as the project did not involve collection of samples from live animals. Samples from Finland, Latvia, Russia, Belarus, Ukraine, Slovakia, Croatia, Bulgaria, and Greece were obtained from collaborators and used with their permission.We extracted genomic DNA from tissues of n = 272 European canids sampled 1995–2010, using a DNeasy Tissue Kit (Qiagen) according to the manufacturer’s protocol. We performed DNA quantity and purity control using the spectrophotometer NanoDrop ND-1000  and examined DNA quality using electrophoresis with a 1% agarose gel. Samples were genotyped at AROS Applied Biotechnology A/S in Aarhus, Denmark, for 170 000 loci using the CanineHD BeadChip microarray from Illumina®  according to their Infinium HD Assay Ultra assay protocol. Samples included n = 20 Italian dogs and three known first-generation captive wolf-dog hybrids to help identify and remove individuals suspected to have dog ancestry. The dogs were of unknown breed/ancestry sampled in villages close to wolf distributions. Four wolf samples  were processed and genotyped in duplicates to verify genotyping reliability and showed consistent individual profiles.We used GenomeStudio™ and accompanying guidelines from Illumina 2)>0.8 within a window of 50 SNPs) using the adegenet-package We performed principal component analyses (PCA) on a subset of markers pruned for linkage disequilibrium in PLINK . Six Italian individuals showed dog ancestry qdog≥0.10 (range 0.10–0.35). Three other European outliers that had been removed prior to PCA and STRUCTURE analyses showed apparent dog ancestry. These were from northern Poland (qdog = 0.22), western Ukraine (qdog = 0.38), and Greece (qdog = 0.76). None of the wolves from Belarus showed dog ancestry.We performed additional analyses in STRUCTURE to evaluate whether outlier individuals had profiles similar to that of dogs and known wolf-dog hybrids from Italy (n = 16), including three first-generation wolf-dog hybrids. The results (data not presented) based on 21K SNPs indicated that the four outliers from southern Ukraine  had dog Our results indicate clear genetic divergence between Italian wolves and individuals from other European countries. We found differentiation between profiles from northern and southern Europe, with individuals from the Carpathian Mountains in central Europe displaying intermediate genotypes. Our results also reveal high genetic diversity within Belarus that exceeded the variation observed in neighbouring countries.The presence of a distinct Italian population with comparatively low values of heterozygosity accords with earlier reports of long-term isolation and relatively low genetic diversity et al.Our results support the presence of distinct wolf populations in Europe et al.et al.ST values should be interpreted with caution considering that some clusters are based on <20 samples.Carpathian individuals were distinct from Dinaric-Balkan wolves, which in our sample comprised the most isolated group outside Italy according to PCA and Structure results. Importantly, however, we did not have samples from Romania, and an important research priority will be to determine whether a gradient in wolf profiles might be present and extend from the Carpathian Mountains into the Dinaric-Balkan population. Carpathian individuals were more similar to wolves from the east (i.e. the Ukrainian Steppe) than they were to wolves from northcentral Europe, although wolves in northern Poland  are nevertheless geographically closer to the Carpathian Mountains than to the Ukrainian Steppe. Factors other than geographic distance appear therefore to be important in shaping population structure. The Carpathian Mountains are a meeting point for different wolf haplogroups and subpopulations based on mtDNA analyses et al. Geffen Alces alces) and also wild forest reindeer (Rangifer tarandus fennicus) are important wolf prey in areas of northern Europe Although wolves might have had an extensive distribution in northern Eurasia during the late Pleistocene C. aureus) Wolves in Belarus exhibited unexpected diversity and structure, and western and northern Belarus wolves showed the most divergent genotypes within northcentral Europe. The country is located near the centre of our sampling area and there are no major landscape barriers to dispersal whereas wolf harvest is high et al. Wolves in Finland appeared well-connected to populations in Russia and southward, despite the geographic distance. Jansson We identified certain canids with apparent dog ancestry, including four individuals from southern Ukraine where multiple instances of wolf-dog hybridization have been reported Within the Dinaric-Balkan cluster, we observed divergence between wolves from Croatia and individuals from Greece and Bulgaria. A recent evaluation of the European wolf distribution suggest relatively good landscape connectivity from the Carpathians and southward , althougThe definition of management units from population genetic data should consider the extent to which populations are demographically independent Figure S1Principal component analysis of European wolves (n = 127) using 67K single nucleotide polymorphism (SNP) markers. Genetic diversity is represented by distance and colour; individuals further away and with more different colours have more divergent genotypes. The first axis represents 3.6% of the variation, the second axis 2.4%.(DOC)Click here for additional data file.Figure S2Principal component analysis of Italian wolves (n = 50) using 67K single nucleotide polymorphism (SNP) markers. Genetic diversity is represented by distance and colour; individuals further away and with more different colours have more divergent genotypes. The first axis represents 5.7% of the variation, the second axis 4.3%.(DOC)Click here for additional data file.Table S1Quality control of single nucleotide polymorphism (SNP) data from n = 272 canids  for evaluation of wolf population structure. The resulting data set had n = 177 samples .(DOC)Click here for additional data file.Table S2Quality control of 79 462 single nucleotide polymorphism (SNP) loci in European wolf samples, resulting in a data set of 67 784 SNP loci.(DOC)Click here for additional data file.Table S3Summary of STRUCTURE results for Europe minus Italy and outliers  for 3 repetitions of each K-value. These suggest highest Delta K support for K2, then K4. .(DOC)Click here for additional data file.Table S4Summary of STRUCTURE results for Italian wolves  for 3 repetitions of each K-value. The results suggest highest Delta K support for K2.(DOC)Click here for additional data file.


Near-infrared diffuse correlation spectroscopy (DCS) is an emerging technology for monitoring blood flow in various tissues. This article reviews the recent progress of DCS for the assessment of skeletal muscle blood flow, including the developments in technology allowing use during dynamic exercise and muscular electrical stimulation, the utilization for diagnosis of muscle vascular diseases, and the applications for evaluating treatment effects. The limitations of current DCS studies and future perspective are finally discussed. Skeletal muscles comprise approximately 40% of total body mass, facilitating posture and locomotion and utilizing up to 85% total cardiac output . Many vaBlood flow in skeletal muscle has been quantified by a number of noninvasive methods. Conventional venous occlusion plethysmography has been employed in muscle perfusion investigations for many years ,6. This 2) has been quantified by a variety of techniques. Spirometry measures whole-body V̇O2, but cannot provide information about local muscle tissues  and [Hb]), total hemoglobin concentration (THC), and blood oxygen saturation (StO2), were acquired using a commercial NIRS oximeter . Baseline blood flow was derived as the average rate of initial increase in THC during the first 5 seconds of three venous occlusions. Baseline V̇O2 was calculated as the linear regression of the oxygen desaturation rate  during the first 60 seconds of arterial occlusion. The absolute values of blood flow and V̇O2 throughout  exercise were calculated by multiplying the rBF and rV̇O2 and their absolute baseline values measured by the occlusion protocols, respectively. Here rV̇O2 represents the relative change of V̇O2 that was calculated from relative changes of blood flow and oxygenation data measured by the DCS/NIRS devices [2  rather than its absolute value  study to facilitate blood flow and oxygenation measurements during ES on quadriceps muscle . AccordiAs an advanced application, DCS was used in another ES study to extract the strain rate of contracting or relaxing muscle fibers through fitting the correlation curve to a unique physical model . Fast-anDCS/NIRS has been used to assess muscle circulatory and metabolic function in diseased skeletal muscles ,4,39. A DCS/NIRS was also used to quantify muscle hemodynamic and metabolic responses in older women with fibromyalgia following isometric pedal exercise . CompareDCS technology was first applied in clinical surgery by monitoring muscle blood flow in patients with PAD during femoral arterial revascularization . DCS demIn another application, DCS was used to evaluate the effect of massage on muscle microcirculation . ImmediaB) in DCS technology is mostly calculated using the semi-infinite analytical solution [Currently, the blood flow index (αDsolution ,59,60, wsolution ,28. Realsolution , thus ti


The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples. Epithelial ovarian cancer (EOC) is the fifth-leading cause of cancer-related deaths in women, with a higher fatality-to-case ratio than any other gynecologic malignancy in the United States. Although proteomic technologies have improved dramatically, discovering novel blood biomarkers for cancers remains formidable due to the vast complexity of the plasma proteome and the likelihood that any tumor-specific proteins will be present at very low abundance. In addition, comparison of patient and control serum or plasma to discover biomarkers is complicated by the fact that cancers and other conditions induce inflammatory responses involving changes in abundance of multiple blood proteins, and these changes are not very specific to a single disease. The primary experimental systems where migration into the blood is assured are mouse models. Hence, one approach is to identity quantitative differences in plasma or serum of genetically engineered mice bearing murine ovarian tumors compared with appropriate controls. We recently identified 106 human proteins shed by a human endometrial ovarian cancer cell line (TOV-112D) into the blood of SCID mice using an in-depth 4D proteome analysis of this xenografted mouse serum. Specifically, serum was depleted of the three most abundant serum proteins followed by microscale solution isoelectrofocusing (MicroSol IEF), SDS-PAGE, and LC-MS/MS. The current study was designed to improve ovarian cancer biomarker discovery using the xenograft mouse model system, improve the efficiency of MRM assay development and preliminary verification of biomarker candidates, and identify candidates that may include biomarkers with some degree of specificity for the serous ovarian cancer subtype. High grade serous ovarian cancers are very aggressive and account for most deaths from EOC, although they only constitute approximately half of EOC cases. Plasma from SCID mice bearing OVCAR-3 serous ovarian tumors was analyzed using an optimized 4-D plasma proteome analysis method, which resulted in identification of 268 human proteins based on two or more peptides. Parallel analysis of supernatants from the serous tumor excised from the SCID mice and incubated briefly in cell culture media increased the sequence coverage for many of the human proteins, thereby both confirming these proteins were human and providing more proteotypic peptides for MRM assay development. Selected high-priority candidate biomarkers were then compared to a dataset from a label-free quantitative comparison of serum pools from cancer patients with advanced-stage EOC and benign controls. Finally, candidate biomarkers observed to be elevated in pooled sera from advanced EOC cancer patients in the label-free analysis were selected for multiplexed assay development using targeted mass spectrometry analysis with the multiple reaction monitoring (MRM) method. MRM analysis is an alternative mass spectrometry approach utilizing the discriminating power of triple quadropole mass spectrometers, or their equivalent, to select and quantify a series of specific analytes and associated fragment ions. MRM enables rapid, quantitative measures of proteins in complex mixtures such as plasma and serum, without a dependency on the generation of antibodies or immunoassays. N,N-dimethylacrylamide (DMA), ammonium bicarbonate; and iodoacetamide were purchased from Sigma-Aldrich . Sodium dodecyl sulfate (SDS), 2-mercaptoethanol, and Tris were purchased from Bio-Rad . ZOOM focusing buffers and thiourea were obtained from Invitrogen . PlusOne reagents dithiothreitol (DTT), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and urea were purchased from GE Healthcare . HPLC-grade acetonitrile was purchased from Thomas Scientific . Tris(2-carboxyethyl)phosphine (TCEP) was obtained from Pierce , and sequencing-grade modified trypsin was purchased from Promega .Molecular-biology-grade ethanol (200 proof); LC-MS-grade formic acid; sodium phosphate monobasic; 2–95% air atmosphere in RPMI-1640 medium (ATCC) supplemented with 10% fetal calf serum.The human EOC serous cell line OVCAR-3 was obtained from the American Type Culture Collection . The cells were maintained in a 37°C incubator with a 5% COThis study was carried out in accordance with the recommendations in the Guide for the Care and use of Laboratory Animals of the National Institutes of Health under protocol #111959 using severe combined immunodeficiency (SCID) mice. The study protocol was reviewed and approved by The Wistar Institute's Institutional Animal Care and Use Committee (IACUC). All efforts were made to minimize suffering.6) mixed 1∶1 with 50 µL Matrigel . Tumors were allowed to grow until final tumor size was estimated to be at least 1 cm3 using calipers, but less than 10% of body weight. Blood was collected, mice were euthanized, and tumors were removed at 12 weeks post-injection. Necrosis of tumor tissue was assessed by microscopic inspection of hematoxylin and eosin (H&E) stained, paraffin-embedded sections (5 µm).Nine SCID mice were injected subcutaneously in the flank with 50 µl of OVCAR-3 cells . The tubes were centrifuged for 3 min at room temperature, and aliquots of plasma from individual mice were snap-frozen and stored at −80°C. Plasma from the four mice that contained the largest, minimally neurotic tumors were subsequently thawed and pooled to average variations among individual mice. The pooled plasma was re-aliquoted, snap-frozen, and stored at −80°C until analysis using the 4-D fractionation method (immunoaffinity depletion/MicroSol IEF/SDS gel and trypsin digestion/LC-MS/MS). Total protein concentration of the pooled plasma was measured using a BCA Protein Assay (Pierce).Blood was collected from SCID mice containing visible OVCAR-3 tumors (see above section) by cardiac puncture under anesthesia into Microtainer 0.5 mL K3), placed in the upper chamber of a 5 µm PVDF microcentrifuge filter , and washed three times with 400 µL of PBS for 1 min each. The tissue sections were then transferred to the upper chamber of a 0.22 µm PVDF microcentrifuge filter and incubated in 400 µL of serum-free RPMI-1640 medium for 2 h in 5% CO2, 95% air at 37°C. After incubation, the supernatant (conditioned media) was recovered by centrifugation, then frozen and stored at −80°C until needed. For tumor supernatant analysis, 500 µL aliquots of conditioned media from tumors from four mice were thawed, pooled, and concentrated to ∼30 µL by ultrafiltration using a 10 K MWCO concentration unit. Membrane rinses using 1% SDS, 50 mM Tris, pH 8.5 were combined with the concentrated sample to maximize protein recovery.Immediately after removing tumors from eight of the nine SCID mice bearing the largest OVCAR-3 tumors, a section of fresh ovarian tumor tissue from each mouse was cut into small pieces  approved protocols, #2109171, #EX2110012, and #2602221 and in accordance with Health Insurance Portability and Accountability Act (HIPAA) requirements. All human samples were derived from subjects with informed written consent. Data were analyzed anonymously.Human sera from normal subjects and patients with benign ovarian tumors, early-stage ovarian cancer, and late-stage ovarian cancer were collected before clinical treatment at approximately the time of diagnosis and prior to surgery as previously described. The pooled mouse plasma was depleted using a 4.6×100 mm MARS Mouse-3 HPLC column , essentially as previously described, Human serum samples  were depleted of the 20-most-abundant serum proteins using a ProteoPrep20 Immunodepletion Column (Sigma), as described previously. Immunodepleted and concentrated mouse plasma (2.2 mg) was fractionated by MicroSol IEF as previously described, After evaluation of the MicroSol IEF separation on analytical SDS gels, concentrated samples were loaded onto multiple lanes of pre-cast 12% Bis-Tris NuPAGE gels (Invitrogen) and separated for discrete distances (1 or 4 cm). The concentrated tumor supernatants from both pools were loaded onto multiple lanes of pre-cast 12% Bis-Tris NuPAGE gels (Invitrogen) and separated for 6 cm. Gels were stained with Colloidal Blue (Invitrogen), each gel lane was sliced into uniform 1 mm slices, and corresponding slices from triplicate lanes were combined in a single well of a 96-well pierced digestion plate  and digested overnight with 0.02 µg/µL of modified trypsin, as previously described. m/z from 400 to 2000. The full MS scan was collected at 60,000 resolution in the Orbitrap in profile mode followed by data-dependant MS/MS scans on the six most abundant ions exceeding a minimum threshold of 1000 collected in the linear trap. Monoisotopic precursor selection was enabled and charge-state screening was enabled to reject z = 1 ions. Ions subjected to MS/MS were excluded from repeated analysis for 60 s.Tryptic digests were analyzed using an LTQ-Orbitrap XL mass spectrometer  interfaced with a Nano-ACQUITY UPLC system , as described previously. MS/MS spectra were extracted and searched using the SEQUEST algorithm  in Bioworks  against a combined human and mouse UniRef100 protein sequence database  to which commonly observed “contaminants”  were added. A decoy database was produced by reversing the protein sequence of each database entry, and the entire reversed database was appended in front of the forward human and mouse databases, respectively. Spectra were searched with a partial tryptic constraint of up to two missed cleavages, 100 ppm precursor mass tolerance, 1 Da fragment ion mass tolerance, static modification of cys , and variable modification of methionine (+15.9949). The use of a partial tryptic constraint and 100 ppm precursor tolerance for the database search had recently been shown to enhance depth of analysis for serum and plasma proteomes. Proteins identified in the database search were sorted into “human,” “mouse,” or “indistinguishable” based upon their species-specific sequences, as previously described. Table S2. Pools were immunodepleted and separated on a 1 D SDS gel, each serum proteome was separated into 40 fractions, and each slice was digested with trypsin and analyzed by LC-MS/MS using a 4 h gradient at 200 nL/min consisting of 5–28% B over 168 min, 28–50% B over 51.5 min, 50–80% B over 5 min, and 80% B for 4.5 min, before returning to 5% B over 0.5 min. A short blank gradient was run in between samples to minimize carryover. Full-MS and LC-MS/MS data from fractions 17–32, which encompassed the 8–50 kDa region of the gel, were analyzed using Rosetta Elucidator software  to compare peptide signal intensities in full MS scans. Based on peptide elution profiles and ion signal density, data for this label-free comparison was trimmed to 16–200 min. Retention time (RT) alignment, feature identification , feature extraction, and protein identifications were performed by the Elucidator system as previously described. To determine whether candidate biomarkers could be detected in ovarian cancer patient sera, in-depth discovery mode analyses of patient serum pools were conducted followed by global label-free quantitative comparisons. One pool of serum from benign patients and three pools of advanced ovarian cancer patient serum samples were made as described in MRM experiments were performed on a 5500 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer  interfaced with a Nano-ACQUITY UPLC system with the column heater maintained at 45°C. Tryptic digests were injected using the partial loop injection mode onto a UPLC Symmetry trap column (180 µm i.d. × 2 cm packed with 5 µm C18 resin), and then separated by RP-HPLC on a BEH C18 nanocapillary analytical column . Chromatography was performed with solvent A, consisting of Milli-Q water with 0.1% formic acid, and solvent B, as acetonitrile with 0.1% formic acid. Peptides and transitions used for quantitation were selected from discovery results  and further verified by MRM-initiated detection and sequencing (MIDAS) using the 5500 QTRAP mass spectrometer. MIDAS experiments were performed at 200 nL/min with a 77-min gradient consisting of 5–28% B over 42 min, 28–50% B over 25.5 min, 50–80% B over 5 min, and 80% B for 4.5 min, before returning to 5% B over 0.5 min. To increase throughput, after optimal peptides and transitions were established, label-free MRM assays were performed with a 41-min gradient, in which peptides were eluted at 400 nL/min for 5–35% B over 38 min and 35% B for 3 min, before returning to 5% B over 0.5 min.MRM data were acquired with a spray voltage of 3300 V, curtain gas of 20 p.s.i., nebulizer gas of 10 p.s.i., interface heater temperature of 150°C, and a pause time of 3 ms. Multiple MRM transitions were monitored per peptide at unit resolution in both Q1 and Q3 quadrupoles to maximize specificity. Scheduled MRM was used to reduce the number of concurrent transitions and maximize the dwell time for each transition. The detection window was set at 2 min, and the target scan time was set at 1.8 s. Data analysis was performed using Skyline v.1.2. Figure S1 B and C, respectively. Human proteins identified in the plasma by at least two peptides, at least one of which was uniquely human, were prioritized and verified as illustrated and described in further detail below. Although this biomarker candidate discovery study used a cell line representative of a late stage tumor, our working hypothesis is that the best cancer biomarkers will be shed by the tumor into the blood and will correlate with tumor size. These biomarkers will ideally be detectable in serum or plasma at higher levels than in control subjects, even when the tumors are small, and the levels of these biomarkers will increase as the tumor grows. By utilizing the xenograft mouse model and identifying human proteins, we are assured that the candidate biomarkers are derived from the tumor and shed into the blood, at least in this model system.The strategies used to improve ovarian cancer biomarker discovery using the xenograft mouse model system, select high priority candidate biomarkers, and improve the efficiency of MRM assay development and biomarker verification are outlined in 3 was pooled and analyzed using the 4D method described above. Fractionation using MicroSol IEF and 1D SDS gels yielded 180 fractions and subsequent analysis of these fractions by LC-MS/MS produced more than 1.1 million spectra, which were searched against a combined human and mouse database. A total of 3647 non-redundant human and mouse proteins were initially identified by 22,890 peptides at a peptide FDR of 5.7% for all proteins, and a 0.5% FDR for protein identifications with two or more peptides. After species classification, 268 human proteins were identified by two or more peptides and an additional 550 by a single peptide , and each sample was digested with trypsin followed by LC-MS/MS analysis resulting in 487,076 MS/MS spectra, which were searched against a combined mouse and human database. A total of 6066 unique proteins were identified from 46,111 peptides at a peptide FDR of 0.9%. Eliminating single peptide proteins resulted in 4619 unique protein entries, with a peptide FDR of 0.03%. This list of high-confidence proteins (≥2 peptides) identified from the combined human and mouse dataset is listed in Table S1. This complete dataset was divided into “human” and “mouse” based upon the presence of at least one peptide unique to that species, while “indistinguishable” proteins contained only peptides common to both species. A total of 2843 human proteins were identified by two or more peptides, and an additional 727 human proteins were identified by single peptides  present in the xenograft plasma samples by confirming the original peptide and protein identifications and, in most cases, providing more extensive peptide coverage.Examples of using the tumor supernatant data to expand the utility of data from the xenograft plasma are shown in et al. that identified candidate biomarkers by comparing a genetically engineered mouse model and secretomes of ovarian cancer cells. et al., we identified eight proteins  in our tumor supernatant analysis, and three proteins  in the high-confidence xenograft plasma dataset.The tumor supernatant and plasma datasets were compared to a study by Pitteri We also compared the results from the current study using OVCAR-3 cells, a serous EOC cell line to an earlier xenograft mouse study using an endometrioid EOC cell line (TOV-112D) where we identified three new biomarkers of ovarian cancer that could distinguish cancer patients from normal individuals. Overall, these comparisons show that different biomarker discovery strategies result in detection of overlapping, but non-identical sets of biomarkers. These data also demonstrate that analysis of the tumor supernatant in parallel with xenograft mouse plasma is useful for confirming candidate biomarkers detected in xenograft mouse plasma.Efficient methods for selecting the best candidate biomarkers and economically verifying them in serum or plasma of EOC patients are needed because, some, but not all proteins shed by EOC tumors into blood are expected to be good biomarkers of the disease. Furthermore, some proteins detected in the xenograft mouse model may not be detectable in human blood using current methods either because the concentration in human blood is below detection limits of available assays or because in some cases the shedding may be unique to the mouse model. As noted above, when we evaluated a panel of candidate biomarkers from the TOV-112D xenograft mice tumors, the overall success in setting up MRM assays and demonstrating elevated levels of the targeted biomarker in EOC patient sera was only about 20%. Hence, an important challenge is to develop appropriate methods for more efficient triaging of candidate biomarkers and evaluating them in serum of EOC patients.The xenograft plasma proteome was prioritized starting with the 268 human proteins identified by two or more peptides Figure 2Table S2. Acquisition of full MS and data-dependent MS/MS scans were identical to those described for the xenograft proteome analyses, with the exception that ions subjected to MS/MS were excluded from repeated analysis for 180 s. Xenograft plasma candidate biomarkers that could be detected in these human serum pools were quantitatively compared across pools using peptide ion signal intensities from the Rosetta Elucidator System's peptide report results. Peptides were grouped into consensus proteins by protein description and peptide intensities were summed for each protein. The criteria for selecting candidates for further validation were proteins that showed increases in all three cancer pools compared with the benign serum, and where the average intensity of the three cancer pools was at least 1.7 times that of the benign serum pool (Table S2).Candidate biomarkers in the 15–50 kDa region of the gel were selected for further prioritization and verification because this was the region of the gel that contained the largest density of human proteins in the xenograft plasma analysis. By focusing on a discrete region of the gel we could increase the subsequent throughput of MRM assays by minimizing the number of fractions that need to be analyzed to quantitate the targeted group of candidate biomarkers. Candidates in this region that were identified with at least the same number of peptides in the tumor supernatant were considered further Table 1.To evaluate whether gene expression in ovarian tumor tissues could be a useful indicator of whether a protein is promising serum biomarker, we queried our candidate biomarkers from Overall, these comparisons suggest that gene expression levels are not reliable indicators of blood levels of a given protein, and use of gene expression levels to predict blood biomarkers is likely to be of limited value. This is not surprising because: 1) gene expression levels do not always correlate with protein abundance within cells; 2) shedding of proteins into the extracellular space and, more specifically, into the vascular system, does not necessarily depend upon the tissue levels of that protein; and 3) changes in proteolytic processing, PTM levels, or other processing of proteins that might affect their blood concentration may differ between normal and cancer states.Table S2. The peptides and transitions used in the integrated multiplex MRM assay, as well as the resulting relative quantitative data for the four pools, are shown in Table S3. Resulting relative protein quantities for the four pools are summarized in We subsequently attempted to set up MRM assays for the 11 novel and three known biomarkers shown in These results confirm that quantitative MRM assays were established for all 11 targeted proteins and that all targeted proteins showed elevated levels in initial analysis of sera from advanced EOC. This 100% success using these two criteria is dramatically better than the 20% success rate achieved for setting up MRM assays for TOV-112D derived candidate biomarkers showing elevated levels in initial screens of sera from advanced EOC. This more efficient selection of candidate biomarkers was achieved because the current strategy utilized additional criteria prior to attempting to set up MRM assays. The key advantages of the current approach include analysis of the tumor supernatant to extend sequence coverage for putative human plasma proteins in the xenograft mouse plasma and comparison of remaining high priority candidate biomarkers to an in-depth discovery mode quantitative comparison of serum pools from benign and advanced EOC patients. This latter analysis identified those proteins detectable in patient serum as well as those proteins exhibiting elevated levels in EOC patient sera. Interestingly, approximately two-thirds of the high priority candidates from the xenograft mouse plasma both expected to be in the 15–50 kDa region and verified in the tumor supernatant, were detected in the patient pools and half of these met the criteria used above for elevated levels in EOC serum. This 20% success rate is very similar to that obtained with the TOV-112D candidate biomarkers. The major difference in the current study is that time and expenses were not invested in attempting to set up MRM assays for the 75–80% of biomarkers that would ultimately fail to be detected in patient plasma or that would not show elevated levels in advanced EOC patient serum. Although substantial mass spectrometer and analysis time was invested in conducting the in-depth discovery mode quantitative comparison of serum pools from benign and advanced EOC patients, these analyses do not need to be repeated as the same dataset can be used to screen future candidate biomarkers. Additionally, this study includes pools of mixed histotypes that approximate the mixtures of cancer subtypes typically seen clinically because the numbers of available samples and the assay throughput were too low to distinguish potential subtype specific biomarkers. One goal of future studies using higher throughput assays such as sandwich ELISA will be to carefully evaluate potential relationships between EOC subtypes and these biomarker candidates.In the current study, improved strategies for both discovery and triaging novel blood biomarkers for EOC have been developed. The utility of analyzing xenograft mouse plasma and the corresponding tumor supernatant in parallel was demonstrated. The presence of human proteins in the plasma demonstrated these proteins were produced by the tumor and shed into the blood, but many of these assignments were based upon only a few peptides. Analysis of the tumor supernatant produced far more extensive sequence coverage for human proteins and confirmed many of the proteins identified in the plasma as human. In most cases an increased sequence coverage provided additional peptide candidates for potential MRM assays. A second key step in the prioritization and verification strategy was to compare candidate plasma biomarkers to an in-depth, label-free comparison of benign disease and advanced cancer patient serum pools to prescreen candidate biomarkers prior to setting up MRM assays. By extending reverse-phase gradients for the discovery mode analysis of these samples to four hours, the detection sensitivity is similar to that of MRM assays using shorter gradients. That is, if a protein cannot be detected in this dataset, it will probably not be feasible to set up an MRM assay and, therefore, effort is not wasted in assay development. Furthermore, by comparing candidate biomarker levels in the benign and advanced cancer patient pools, only those proteins showing elevated levels in the cancer sera advance to MRM assay development. This new approach reduces the effort invested in setting up MRM assays by about four-fold relative to the biomarkers detected in advanced EOC patient sera at elevated levels. Based upon initial screening of large pools of normal, benign, early, and advanced ovarian cancer sera, all of the biomarkers selected for MRM assay development in the current study should move forward and be further evaluated using serum or plasma from individual patients and controls. Although the fold changes observed in the pooled samples for most of these candidate biomarkers are not as large as HE4, the ranges of values for these biomarkers in individual EOC and control sera need to be determined in order to compare their diagnostic capacities to HE4 and CA125. While it is unlikely that most individual biomarkers will prove to be superior to HE4 or CA125, it is more likely that combinations with each other or with CA125 and HE4 could outperform the use CA125 or HE4 alone. Finally, the strategies developed in this study demonstrate that in-depth analysis of xenograft mouse plasma with efficient pilot verification using multiplexed assays can efficiently identify multiple promising candidate EOC biomarkers. This approach can be readily applied to further in-depth analysis of the OVCAR-3 cell line, as well as other EOC cell lines to identify additional EOC plasma biomarkers.Figure S1Analysis of OVCAR-3 xenograft plasma and tumor supernatant. (A) SDS-PAGE of concentrated media from the OVCAR-3 tumor supernatant. The sample was separated for 6 cm, the gel lane was sliced into 60 uniform fractions, digested with trypsin and analyzed by LC-MS/MS. (B) Analytical SDS-PAGE of unfractionated, depleted mouse plasma (DP), and MicroSol IEF fractions (F1–F4) and membrane extractions (M1–M5). (C) Representative preparative SDS-PAGE of the samples shown in panel B. Distances samples were separated are indicated and gel lanes were cut into 1 mm slices for trypsin digestion and subsequent LC-MS/MS analysis.(PDF)Click here for additional data file.Table S1Proteins identified in the OVCAR-3 tumor supernatant by two or more unique peptides.(PDF)Click here for additional data file.Table S2Sample classification for ovarian cancer patient sera.(PDF)Click here for additional data file.Table S3Peptide transitions monitored by MRM.(PDF)Click here for additional data file.


Our data provide insight into the mechanisms behind the observed negative prognostic impact of modest elevations in BNP and suggest that in an asymptomatic hypertensive cohort a peripheral BNP measurement may be a useful marker of an early, sub-clinical pathological process characterized by cardiac remodeling, inflammation and ECM alterations.In asymptomatic subjects B-type natriuretic peptide (BNP) is associated with adverse cardiovascular outcomes even at levels well below contemporary thresholds used for the diagnosis of heart failure. The mechanisms behind these observations are unclear. We examined the hypothesis that in an asymptomatic hypertensive population BNP would be associated with sub-clinical evidence of cardiac remodeling, inflammation and extracellular matrix (ECM) alterations. We performed transthoracic echocardiography and sampled coronary sinus (CS) and peripheral serum from patients with low (n = 14) and high BNP (n = 27). Peripheral BNP was closely associated with CS levels . CS BNP correlated significantly with CS levels of markers of collagen type I and III turnover including: PINP , CITP  and PIIINP , and with CS levels of inflammatory cytokines including: TNF-α , IL-6 , and IL-8 . The high BNP group had greater CS expression of fibro-inflammatory biomarkers including: CITP , TNF-α , IL-6  and hsCRP , and greater left ventricular mass index (97±20 versus 118±26 g/m Coronary sinus serum was sampled as this localizes events to the myocardium and may be more sensitive to detect subtle changes in biomarker expression.th percentile described in the Framingham Offspring Study (BNP 20 pg/ml for men and 23.3 pg/ml for women) We studied 41 Caucasian subjects with a history of hypertension who volunteered for the study. Patients were recruited prior to routine elective coronary catheterisation for suspected ischaemic chest pain. Patients had normal self-reported exercise capacity. At the time of recruitment patients were examined for signs of congestive cardiac failure/fluid overload which included assessment for: elevation in jugular venous pressure, basal pulmonary crepitations, a gallop rhythm (S3) or peripheral pitting oedema. Exclusion criteria included any findings suggestive of fluid overload, renal insufficiency (serum creatinine >1.5 mg/dl or estimated creatinine clearance (eCrCl) <45 ml/min as calculated using the Cockcroft-Gault formula), evidence of infection, conditions known to alter collagen turnover , conditions known to alter BNP levels , and recent surgery or physical trauma (<6 months). were categorised into high and low BNP groups according to values above and below the 80Office blood pressure monitoring was performed in the sitting position using a standard sphygmomanometer. Subjects underwent transthoracic echocardiography (TTE) and right heart catheterization for coronary sinus blood sampling within one week of each other. All subjects gave written informed consent to participate in the study. The Ethics Committee at St Vincent’s University Hospital approved the study protocol which conformed to the principles of the Helsinki Declaration.Following puncture of the right femoral vein, a 6F sheath was introduced using a Seldinger technique. A 5F catheter was positioned under fluoroscopic guidance to the os of the CS as confirmed by contrast injection. Peripheral blood samples were taken from the side arm of the femoral sheath and further samples from the CS os. Samples were collected into vacutainer tubes using ethylenediaminetetraacetic acid tubes for BNP and gel separation and clot activator tubes for biomarker determination. Samples were immediately stored at 4°C. Within 1 hour the serum tubes were centrifuged at 1500 rpm for 5 minutes at 4°C to separate off the serum. Serum was then aliquoted out into small volume microcentrifuge tubes and stored in a −80°C freezer until use.TTE was performed using a Philips IE33 by a single experienced echocardiographer. Measurements were performed by a single reader blinded to BNP results. The following measurements were performed as per ASE guidelines BNP was quantified using a Triage meter BNP assay (assay sensitivity <5 ng/ml) (Biosite Inc.).Serum procollagen type I amino-terminal (assay sensitivity 13.0 ng/ml) (PINP), carboxy-terminal (assay sensitivity 2.0 ng/ml) (PICP), carboxy telopeptide of type I collagen (assay sensitivity 0.5 ng/ml) (CITP) and procollagen type III amino-terminal (assay sensitivity 1.9 ng/ml) (PIIINP) were quantified using radioimmunoassay (Orion Diagnostica). A custom made ultrasensitive Meso Scale Discovery (MSD) 4-Plex ELISA enabled simultaneous detection of interleukin (IL)-6, IL-8 and tumour necrosis factor alpha (TNFα) using an MSD Sector Imager 2400 reader. For each of these inﬂammatory markers, the assay sensitivity of was ≤0.7 pg/ml. Duplicate measurements were performed in all the above tests. High sensitivity CRP (hsCRP) serum concentrations were also determined using ELISA (MSD). All assays were carried out according to manufacturer’s guidelines or as described previously th percentiles. Categorical variables are presented as frequencies and percentages (in parenthesis). Comparisons between the low and high BNP groups were made using independent t-test, Welsh test, or chi square test where appropriate. The relationships between BNP, markers of collagen turnover and markers of inflammation were assessed using Pearson’s correlation coefficient for variables that were approximately normally distributed. In addition, Pearson’s correlation coefficient was used with log-transformation of variables with non-normal distribution. A p value of <0.05 was considered statistically significant.For continuous variables, summary statistics are presented as the mean ± standard deviation (SD) or median and 25–75Baseline characteristics of the participants are presented in 2, p = 0.03) and LAVI  were significantly higher in the high BNP group when compared to the low BNP group. There were no other significant differences between the two groups.In samples collected from the periphery, only hsCRP was significantly different between the two groups  . CS sampPeripheral BNP was very closely associated with CS levels .CS BNP correlated significantly with CS levels of markers of collagen type I and III turnover including: PINP , CITP  and PIIINP . Furthermore, peripheral BNP also correlated significantly with CS PINP , CITP  and PIIINP  .Both CS and peripheral BNP correlated significantly with CS levels of inflammatory cytokines including: TNF-α , IL-6 , and IL-8  .In this population CS BNP was significantly associated with LAVI  and LVMI .The present cross-sectional study demonstrated that in an asymptomatic hypertensive population a modest elevation in BNP was associated with increased expression of fibro-inflammatory biomarkers in serum sampled from the coronary sinus. A modest elevation in BNP is associated with increased LVMI and LAVI. Furthermore, significant correlations exist between markers of inflammation, collagen turnover and BNP.It is estimated that between 25 and 30% of the adult population in the USA have hypertension 2, p = 0.008). It is possible that the observed elevation in CITP is as a result of this early LA remodeling.In the high BNP group we observed a significant increase in CITP expression when compared to the low BNP group suggesting a predominance of collagen type I catabolism. These data may appear counterintuitive in the context of increased LVMI observed in this group. However, previous studies have demonstrated an increase in CITP in HHD and have suggested that this may be an early indication of degradation in the ECM scaffolding with resultant progression to chamber dilatation et. al., where an increased risk of HF in subjects with LAVI measurements of 20–30 ml/m2 versus <20 ml/m2 was demonstrated. Consistent with the negative prognostic attributes of both BNP and LAVI, CITP has also been associated with adverse cardiac events It is interesting to note that the BNP cut-off identified previously and used in this study divides our population into LAVI groups similar to those used by Ristow et. al. recently demonstrated a significant increase in myocardial collagen volume fraction, ventricular stiffness and concentric LVH in rats infused with IL-6 in vitroIncreasing evidence suggests that inflammation may play a key role in the pathophysiology of hypertension The precise clinical implications of these observations are uncertain and it is not yet clear how best to manage asymptomatic hypertensive patients with a modestly elevated BNP. The prognostic evidence of elevated cardiovascular risk associated with such patients at a minimum underline the need for close attention to risk factor management. The observation that elevated BNP in hypertensive patients is associated with abnormal nocturnal blood pressure patterns underlines the need to completely assess blood pressure control In interpreting these data certain limitations need to be considered. First, we have used serum markers of collagen turnover taken from the CS as a surrogate of myocardial interstitial disease without endomyocardial biopsy evidence of fibrosis. Second, although BNP is a robust predictor of cardiovascular outcome in an unselected patient population Risk stratification allows the physician to identify those patients who will benefit most from more aggressive surveillance and management. For asymptomatic hypertensive patients a means of risk stratification which is rapid, readily available, relatively inexpensive and capable of providing information beyond a history and physical examination is of particular importance. In this regard, BNP is a useful marker of adverse outcomes in asymptomatic patients even at levels well below the contemporary thresholds used in the diagnosis of HF. Our data provide insight into the mechanisms behind these observations and suggest that in an asymptomatic hypertensive cohort a peripheral BNP measurement may be a useful marker of an early, sub-clinical pathological process characterized by cardiac remodeling, inflammation and extracellular matrix turnover.


Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo. At this stage of development three distinct cell types have emerged: the epiblast, trophectoderm and primitive endoderm, which give rise to the fetus, placenta and extraembryonic endoderm respectively Quantitative reverse transcription-polymerase chain reaction (qPCR) has emerged as a powerful technique to rapidly assess transcriptional differences between cell types and differing experimental conditions. However, accurate quantitative analysis is dependent upon proper, empirical selection of a suitable reference. Using published microarray data, and a novel statistical algorithm, (geNORM) Vandesompele and colleagues demonstrated that the geometric mean of three reference genes provided the most accurate and reliable means of normalizing qPCR expression data in vitro differentiated progeny. In order to help identify candidate genes we set two main criteria that the mRNAs would have to fulfill: 1) the transcripts needed to be expressed above background and easily detectable, and 2) candidate mRNAs needed to be stably expressed between each of the three stem cell lineages under investigation. To this end we surveyed the recent literature and compiled a short list of fourteen candidate genes, including Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sK, Sdha, Tbp and YwhazIn this study we sought to identify a list of genes most suitable for use as normalization controls in qPCR-based comparisons between ES, TS and XEN stem cells or their Pgk1, Sdha and Tbp transcripts as the most stably expressed reference genes between each of these stem cell types. To determine which of these candidates was most suitable for use during in vitro differentiation studies, we cultured ES and TS cells in the absence of crucial growth factors LIF and FGF4 respectively. Using three independent RNA samples isolated on Day 0 and Day 8, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of ES and TS cells. Our results suggest that normalization of qPCR data using the geometric means of the transcripts listed above will yield the most accurate quantification of gene expression between these three unique stem cell types.To evaluate the stability of our candidate genes we isolated RNA from three independent lines of varying genotypes for each of the three stem cell types. This RNA was quantified and seeded into five independent qPCR reactions measuring each of the candidate genes. Using the geNORM, NormFinder and BestKeeper algorithms, we identify the After a survey of the recent literature we curated a short list of fourteen commonly used reference genes and either designed new primers or pulled existing ones from references cited in the Mus musculus castaneus x mus musculus (C57Black6) F1 embryos. Polymorphisms between these genetic strains allow the examination of mono-allelic patterns of epigenetic marks and gene expression within loci regulated by genomic imprinting Previous studies in our laboratory have utilized stem cell lines derived from Rn7sk demonstrated the most robust expression averaging expression levels 125 fold higher than the remaining candidates; which were all readily detectable in each of the cell lines tested. To measure the relative stability of each of the candidate genes between the ES, TS and XEN lines, the CT values for the measured transcripts were compiled and run through the NormFinder, GENorm, and BestKeeper software packages Pgk1, Sdha, Tbp and H2afz as the most consistently stable reference genes between ES, TS and XEN stem cells  and Gapdh all demonstrate a significant down-regulation and therefore are not suitable reference genes for this experimental time course. Similar to results reported by Willems et al., examining ES cell differentiation induced by both DMSO and Retinoic acid, we also identify B2m and Hprt as among the most unstable transcripts Ywhaz, Pgk1 and Hk2 transcripts as the most stable during TS cell differentiation  and Day 8 and RNA seeded into five independent qPCR reactions measuring each of the fourteen candidate genes. Using a similar experimental design as described above, we identify Sdha, Tbp and Ywhaz as the three most stable transcripts . To examntiation . After and Rn7sk . PreviouPgk1, Sdha and Tbp as the most suitable reference genes and further find compelling evidence to suggest that both Actb and Gapdh are not suitable normalization controls for these experiments.Analysis of gene expression using qPCR has become the corner stone to nearly every facet of the biological sciences. However, despite numerous studies demonstrating the importance of careful selection and validation of appropriate reference genes, several studies continue to emerge utilizing inappropriate methods of qPCR normalization Pgk1 - phosphoglycerate kinase 1 is the seventh step of glycolysis and Sdha - Succinate dehydrogenase or succinate-coenzyme Q reductase is an enzyme complex that binds to the inner mitochondrial membrane and is an essential component of both the citric acid cycle and electron transport chain Pgk1 and Sdha are components of distinct pathways, they are both components of cellular respiration leaving the possibility that an experimental condition that impacts metabolic processes would significantly alter these normalization controls. The third and fourth candidates to emerge from our analysis were Tbp and H2afz respectively. Tbp is a central component of the RNA polymerase II pre-initiation complex and H2afz is an essential component of chromatin structure which is hypothesized to play a role in chromosome organization and stability Of the top three candidates to emerge from our analysis two are components of pathways controlling cellular respiration. Pgk1, Sdha and Tbp as the most stable reference genes with which to normalize qPCR data. We believe these three genes will serve as excellent reference controls examining the basis for the differing developmental and epigenetic properties unique to embryonic, trophectoderm and extraembryonic endoderm stem cells.The first differentiation event during mammalian embryogenesis is the formation of the epiblast, trophectoderm and primitive endoderm which go on to give rise to the three founding embryonic lineages. Stem cells derived from each of these lineages represent an excellent model system to study mammalian development and understand crucial aspects of stem cell biology necessary in developing regenerative therapies. Analysis of gene expression using qPCR will undoubtedly play a pivotal role in deciphering the cellular and molecular properties that define these different cell types. In these analysis, the identification of stable reference genes is an essential prerequisite to accurately interpreting experimental data. Using three independent, highly referenced and validated statistical methods, our analysis of fourteen potential candidate reference genes identify 1 embryos Primary ES cells, TS cells, and XEN cells were derived from either 129 strain . Cells were spun down, washed once in cold PBS, then RNA isolated using Trizol  according to the manufacturer's protocol. One µg of purified total RNA was treated with amplification grade DNaseI (Invitrogen) according to the manufacturer's protocol, and 250 ng RNA seeded into a reverse transcription reaction using the SuperScriptII system (Invitrogen) by combining 1 µl random hexamer oligonucleotides (Invitrogen), 1 µl 10 mM dNTP (Invitrogen), 11 µl RNA plus water. This mixture was brought to 70°C for 5 minutes then cooled to room temperature. SuperScriptII reaction buffer, DTT (Invitrogen) and SuperScriptII were then added according to manufacturer's protocol and the mixture was brought to 25°C for 5 minutes, 42°C for 50 minutes, 45°C for 20 minutes, 50°C for 15 minutes then 70°C for five minutes.Real-time PCR analysis of mRNA levels was carried out using the DyNAmo Flash SYBR Green qPCR Mastermix  following the manufacturer's instructions. Reactions were performed on a StepOnePlus Real Time PCR system . DNA primer information is available in Cycle Threshold) values for each sample were complied and the stability of each of the fourteen reference genes analyzed using the GENorm, NORMFinder and BESTKeeper software tools; which have been described in detail elsewhere The measured CT (