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PDF file - 148KB, Exploratory Baseline Analytes.

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo.Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib–sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib–sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates.Results: From 2005–2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib–sulindac (omnibus comparison, two-sided Kruskal–Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib–sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib–sulindac > erlotinib > placebo (two-sided exact Jonckheere–Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R2 = 0.312, P = 0.024).Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR–COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target. Clin Cancer Res; 20(12); 3289–98. ©2014 AACR.

PDF file - 176KB, Assessing the Adequacy of Randomization.

PDF file - 61KB, Antibodies used to evaluate candidate biomarkers in the TMA.

Ovarian steroid cell tumor is a rare type of ovarian tumor, accounting for ~0.1% of all neoplasms of the ovary. Patients suffering from this type of tumor exhibit virilization due to high testosterone levels. The present study reported a case of an elderly female patient with high testosterone serum levels, resulting in hirsutism and deepening of the voice. Magnetic resonance imaging did not reveal any solid ovarian tumor. However, gray-scale ultrasound indicated suspicious solid nodules on the right ovary. A clear outline of the tumor, characterized by ring-shaped uniform enhancement, was revealed by contrast-enhanced ultrasound (CEUS) scanning. In addition, laparoscopic resection of both fallopian tubes and ovaries confirmed the right ovarian steroid cell tumor. After the operation, the patient's symptoms were completely relieved and testosterone levels returned to normal. In the present study, a case of ovarian steroid tumor diagnosed by CEUS was reported, supporting the significant role of CEUS in the detection of adnexal tumors.

Urbanization can induce environmental changes such as the urban heat island effect, which in turn influence the terrestrial ecosystem. However, the effect of urbanization on the phenology of subtropical vegetation remains relatively unexplored. This study analyzed the changing trend of vegetation photosynthetic phenology in Dongting Lake basin, China, and its response to urbanization using nighttime light and chlorophyll fluorescence datasets. Our results indicated the start of the growing season (SOS) of vegetation in the study area was significantly advanced by 0.70 days per year, whereas the end of the growing season (EOS) was delayed by 0.24 days per year during 2000–2017. We found that urbanization promoted the SOS advance and EOS delay. With increasing urbanization intensity, the sensitivity of SOS to urbanization firstly increased then decreased, while the sensitivity of EOS to urbanization decreased with urbanization intensity. The climate sensitivity of vegetation phenology varied with urbanization intensity; urbanization induced an earlier SOS by increasing preseason minimum temperatures and a later EOS by increasing preseason precipitation. These findings improve our understanding of the vegetation phenology response to urbanization in subtropical regions and highlight the need to integrate human activities into future vegetation phenology models.

Elevated expression of the DNA damage response proteins PARP1 and poly(ADP-ribose) glycohydrolase (PARG) in glioma stem cells (GSCs) suggests that glioma may be a unique target for PARG inhibitors (PARGi). While PARGi-induced cell death is achieved when combined with ionizing radiation, as a single agent PARG inhibitors appear to be mostly cytostatic. Supplementation with the NAD+ precursor dihydronicotinamide riboside (NRH) rapidly increased NAD+ levels in GSCs and glioma cells, inducing PARP1 activation and mild suppression of replication fork progression. Administration of NRH+PARGi triggers hyperaccumulation of poly(ADP-ribose) (PAR), intra S-phase arrest and apoptosis in GSCs but minimal PAR induction or cytotoxicity in normal astrocytes. PAR accumulation is regulated by select PARP1- and PAR-interacting proteins. The involvement of XRCC1 highlights the base excision repair pathway in responding to replication stress while enhanced interaction of PARP1 with PCNA, RPA and ORC2 upon PAR accumulation implicates replication associated PARP1 activation and assembly with pre-replication complex proteins upon initiation of replication arrest, the intra S-phase checkpoint and the onset of apoptosis.

The extensive crosstalk between the developing heart and lung is pivotal for their proper morphogenesis and maturation. However, there remains a lack of model systems for investigating the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human pluripotent stem cells (hPSCs) via temporal specific tuning of WNT and TGF-β signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (μTs), and observed expedited alveolar maturation in the presence of cardiac accompany. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage μT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect our multilineage differentiation model to offer an experimentally tractable system for investigating human cardio-pulmonary interplay and tissue boundary formation during embryogenesis.

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae. We also assessed mRNA concentrations and their synthesis rates in nonsense-mediated decay (NMD) targets in euploid strains. We found that the altered synthesis rates in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not counterbalanced. In addition, the stability of NMD targets was not specifically compensated by the changes in synthesis rate. We conclude that there is no genetic compensation of NMD mRNA targets in yeast, and total mRNA buffering uses mostly a global system rather than a gene-specific one., This work was funded with grants from the Spanish Ministry of Economy and Competitiveness, the European Union (FEDER) [BFU2016-77728-C3-1-P to S. C.], [BFU2016-77728-C3-3-P to J.E.P -O] and [RED2018-102467-T to J.E.P -O and S.C.], the Regional Valencian Government [AICO2019/088 to J.E.P-O] and the Junta de Andalucía [BIO-271 to S.C.].

CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 A resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

Functional loss of expression of breast cancer susceptibility gene 1(BRCA1) has been implicated in genomic instability and cancer progression. There is emerging evidence that BRCA1 gene product (BRCA1) also plays a role in cancer cell migration. We performed a quantitative proteomics study of EOC patient tumor tissues and identified changes in expression of several key regulators of actin cytoskeleton/cell adhesion and cell migration (CAPN1, 14-3-3, CAPG, PFN1, SPTBN1, CFN1) associated with loss of BRCA1 function. Gene expression analyses demonstrate that several of these proteomic hits are differentially expressed between early and advanced stage EOC thus suggesting clinical relevance of these proteins to disease progression. By immunohistochemistry of ovarian tumors with BRCA1(+/+) and BRCA1(null) status, we further verified our proteomic-based finding of elevated PFN1 expression associated with BRCA1 deficiency. Finally, we established a causal link between PFN1 and BRCA1-induced changes in cell migration thus uncovering a novel mechanistic basis for BRCA1-dependent regulation of ovarian cancer cell migration. Overall, findings of this study open up multiple avenues by which BRCA1 can potentially regulate migration and metastatic phenotype of EOC cells.

BACKGROUND Esophageal adenocarcinoma (EAC) is associated with a dismal prognosis. The identification of cancer biomarkers can advance the possibility for early detection and better monitoring of tumor progression and/or response to therapy. The authors present results from the development of a serum-based, 4-protein (biglycan, myeloperoxidase, annexin-A6, and protein S100-A9) biomarker panel for EAC. METHODS A vertically integrated, proteomics-based biomarker discovery approach was used to identify candidate serum biomarkers for the detection of EAC. Liquid chromatography-tandem mass spectrometry analysis was performed on formalin-fixed, paraffin-embedded tissue samples that were collected from across the Barrett esophagus (BE)-EAC disease spectrum. The mass spectrometry-based spectral count data were used to guide the selection of candidate serum biomarkers. Then, the serum enzyme-linked immunosorbent assay data were validated in an independent cohort and were used to develop a multiparametric risk-assessment model to predict the presence of disease. RESULTS With a minimum threshold of 10 spectral counts, 351 proteins were identified as differentially abundant along the spectrum of Barrett esophagus, high-grade dysplasia, and EAC (P

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo. Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib–sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib–sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates. Results: From 2005–2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib–sulindac (omnibus comparison, two-sided Kruskal–Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib–sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib–sulindac > erlotinib > placebo (two-sided exact Jonckheere–Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R2 = 0.312, P = 0.024). Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR–COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target. Clin Cancer Res; 20(12); 3289–98. ©2014 AACR.

Recent studies have implicated the neuronal calcium-sensing protein visinin-like 1 protein (Vilip-1) as a peripheral biomarker in Alzheimer disease (AD), but little is known about expression of Vilip-1 in the brains of patients with AD. We used targeted and quantitative mass spectrometry to measure Vilip-1 peptide levels in the entorhinal cortex (ERC) and the superior frontal gyrus (SF) from cases with early to moderate stage AD, frontotemporal lobar degeneration (FTLD), and cognitively and neuropathologically normal elderly controls. We found that Vilip-1 levels were significantly lower in the ERC, but not in SF, of AD subjects compared to normal controls. In FTLD cases, Vilip-1 levels in the SF were significantly lower than in normal controls. These findings suggest a unique role for cerebrospinal fluid Vilip-1 as a biomarker of ERC neuron loss in AD.

To monitor eukaryotic mRNA metabolism, we developed comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. cDTA uses nonperturbing metabolic labeling that supersedes conventional methods for mRNA turnover analysis. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are fivefold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4–Not complex causes decreased decay rates as expected, but also decreased synthesis rates. Extended kinetic modeling reveals mutual feedback between mRNA synthesis and degradation that may be achieved by a factor that inhibits synthesis and enhances degradation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endoimetrial cancers and normal post-menopausal endometrium controls (Q

Introduction:Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer and the relatively favorable survival associated with early-stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit.Methods:We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry in a set of pooled non-small cell lung cancer case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by liquid chromatography-tandem mass spectrometry. The tandem mass spectrum data were searched against the human proteome database, and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting-derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring mass spectrometry assays.Results:A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (p < 0.01). Functional analysis with Ingenuity Pathway Analysis tools showed significant enrichment of inflammatory response proteins, key molecules in cell-cell signaling and interaction network, and differential physiological responses for the two common non-small cell lung cancer subtypes.Conclusions:We identified a set of candidate serum biomarkers with statistically significant differential abundance across the lung cancer case/control pools, which, when validated, could improve lung cancer early detection.

The heterogeneity of breast cancer requires the discovery of more incisive molecular tools that better define disease progression and prognosis. Proteomic analysis of homogeneous tumor cell populations derived by laser microdissection from formalin-fixed, paraffin-embedded (FFPE) tissues has proven to be a robust strategy for conducting retrospective cancer biomarker investigations. We describe an MS-based analysis of laser microdissected cancerous epithelial cells derived from twenty-five breast cancer patients at defined clinical disease stages with the goal of identifying protein abundance characteristics indicative of disease progression and recurrence. Comparative analysis of stage 0 and stage III patients revealed 113 proteins that significantly differentiated these groups and included known factors associated with disease pathogenesis, such as CDH1 and CTNNB1, as well as those previously implicated in breast cancer, such as TSP-1. Similar analyses of patients presenting with stage II disease that did or did not exhibit recurrence two years postdiagnosis revealed 42 proteins that significantly differentiated these subgroups and included IRS-1 and PARK7. These data provide evidence supporting the utility of FFPE tissues for functional proteomic analyses and protein biomarker discovery and yielded protein candidates indicative of disease stage and recurrence in breast cancer that warrant further investigation for diagnostic utility and biological relevance.

Psychological stress is recognized as a factor that contributes to increased susceptibility to a number of diseases, including cancer. Psychological stress, via release of chemical mediators, can induce long-term changes in the organism resulting in an altered responsiveness of the organism to external carcinogens. The present investigation sought to evaluate the impact of stress on polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. We utilized a repetitive restraint stress mouse model and the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). Restraint stress was applied three times a week for 6 weeks and DMBA was administered intra-gastrically once a week for 6 weeks and formation of skin, mammary and ovarian tumours was examined at 26 weeks by pathological analyses. The results indicate that stress accelerates the onset of tumour formation produced in response to DMBA and significantly influences the type and location of tumours. Taken together, these results demonstrate that stress enhances the carcinogenicity of DMBA. These results clearly indicate the need for further characterization of the impact of stress on carcinogen metabolism in oncology. Copyright © 2010 John Wiley & Sons, Ltd.

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment.

Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens implicated to underlie development of several types of cancers. Cytochrome P450 (CYP) enzymes play key roles in the conversion of PAHs to highly potent carcinogens, namely diol epoxides. 7,12-Dimethylbenz(a)anthracene (DMBA), a PAH, is highly carcinogenic, where in mouse models it is known to be responsible for initiating tumor formation in many organs including mammary tissues, ovaries, and skin. Psychological stress, via release of biochemical mediators, can greatly impact carcinogenesis. The present investigation examined the hypothesis that psychological stress modulates metabolism and carcinogenicity of DMBA through alteration of key drug metabolizing enzyme abundance levels in the liver utilizing mass spectrometry-based proteomics. To test this hypothesis, four groups of mice were treated as follows: nonstressed, stressed, nonstressed/DMBA-exposed, and stressed/DMBA-exposed, where the stressor was a well-accepted model of restraint. Liver proteins were extracted, resolved by one-dimensional gel electrophoresis, digested in-gel with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. This investigation resulted in the unique identification of 59 isoforms of CYP enzymes. Changes in protein abundances derived from spectral counting indicates that stress alone results in increases in the abundance of proteins responsive to oxidative stress, along with Phase I and II metabolizing enzymes, such as CYP2J5 and UDP glucoronytransferases. The proteomic results further indicate that exposure to DMBA induces increases in the abundance of CYP1A2 and serine protease inhibitors and decreases the abundance of CYP4 V3. Finally, significant changes in the abundance of proteins such as CYP1A2, CYP3A11, and Topoisomerase-2 were found between nonstressed and stressed/DMBA-treated mice. These data support the hypothesis that psychological stress modulates DMBA-induced regulation of drug metabolizing proteins in the liver.

Don't Terminate Me! DNA transcription progresses through three phases—initiation, elongation, and termination—of messenger RNA chains. The transcribing enzyme, RNA polymerase (Pol) II, recruits factors that assist in each of these phases. Mayer et al. (p. 1723 ) now show that the C-terminal domain (CTD) of actively elongating Pol II is phosphorylated at conserved tyrosine residues. This modification impairs recruitment of termination factors. Factor exchange on the transcribing polymerase enzyme may be explained by an extended CTD code that is based on differential phosphorylation of the tyrosines and two well-characterized serine residues in the CTD.

Esophageal adenocarcinoma (EAC) is associated with a dismal prognosis. The identification of cancer biomarkers can advance the possibility for early detection and better monitoring of tumor progression and/or response to therapy. The authors present results from the development of a serum-based, 4-protein (biglycan, myeloperoxidase, annexin-A6, and protein S100-A9) biomarker panel for EAC.A vertically integrated, proteomics-based biomarker discovery approach was used to identify candidate serum biomarkers for the detection of EAC. Liquid chromatography-tandem mass spectrometry analysis was performed on formalin-fixed, paraffin-embedded tissue samples that were collected from across the Barrett esophagus (BE)-EAC disease spectrum. The mass spectrometry-based spectral count data were used to guide the selection of candidate serum biomarkers. Then, the serum enzyme-linked immunosorbent assay data were validated in an independent cohort and were used to develop a multiparametric risk-assessment model to predict the presence of disease.With a minimum threshold of 10 spectral counts, 351 proteins were identified as differentially abundant along the spectrum of Barrett esophagus, high-grade dysplasia, and EAC (P.05). Eleven proteins from this data set were then tested using enzyme-linked immunosorbent assays in serum samples, of which 5 proteins were significantly elevated in abundance among patients who had EAC compared with normal controls, which mirrored trends across the disease spectrum present in the tissue data. By using serum data, a Bayesian rule-learning predictive model with 4 biomarkers was developed to accurately classify disease class; the cross-validation results for the merged data set yielded accuracy of 87% and an area under the receiver operating characteristic curve of 93%.Serum biomarkers hold significant promise for the early, noninvasive detection of EAC.

Objective: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. Results: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. Conclusion: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute.

Recently, sudden natural disasters strike worldwide frequently, seriously prevent the economic development and increasing living standard of people in affected countries. Emergency management is gradually being seen as the most important task by all the governments, the academia has also conducted in-depth exploration and rational thinking. Based on USA and Japan the two developed countries¡¯ emergency practices, summaries and analyses the successfully experiences build up during the emergency management mechanism process, and gives some proposal to help our country¡¯s emergency management practitioners.

The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative dynamic transcriptome analysis (cDTA) that uses nonperturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1- dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network.

Osteosarcomas rarely occur in older adults. Proteomics has not been reported to date in osteosarcoma occurring in the older adult population. This proteomic investigation was conducted to identify differentially expressed proteins in osteosarcoma occurring in various backgrounds from older adults. Desmoid tumors, known to recur locally but not metastasize, were also analyzed. Protein digests isolated from formalin-fixed, paraffin-embedded tumor tissue specimen representing 14 primary osteosarcomas of soft tissue and bone and 18 desmoid tumors were analyzed by high-resolution liquid chromatography-tandem mass spectrometry for protein identification and relative quantification by spectral counting. Elevated abundance levels of several proteins including heat shock protein 90 (HSP90), elastin microfibril interface-located protein 1, and clusterin were identified in osteosarcoma with slight differences in proteomic profiles. Desmoids had an abundance of collagen II and periostin only. The findings were confirmed by immunohistochemical staining for HSP90 and clusterin in the experimental samples and additionally in 16 posttherapy conventional osteosarcomas in tissue microarrays constructed from heterogeneous sarcomas and benign lesions. All osteosarcomas were positive for HSP90 and clusterin to a variable extent. One case of well-differentiated parosteal osteosarcoma was negative. Thirty of 75 other high-grade sarcomas including cases of chondrosarcoma were positive for HSP90. Low-grade and benign lesions and scars and 18 desmoid tumors had little or no expression of these proteins. HSP90 and clusterin represent candidate markers of aggressiveness in osteosarcoma occurring in older adults and may be indicative of drug resistance.

Summary: Standard transcriptomics measures total cellular RNA levels. Our understanding of gene regulation would be greatly improved if we could measure RNA synthesis and decay rates on a genome-wide level. To that end, the Dynamic Transcriptome Analysis (DTA) method has been developed. DTA combines metabolic RNA labeling with standard transcriptomics to measure RNA synthesis and decay rates in a precise and non-perturbing manner. Here, we present the open source R/Bioconductor software package DTA. It implements all required bioinformatics steps that allow the accurate absolute quantification and comparison of RNA turnover. Availability: DTA is part of R/Bioconductor. To download and install DTA refer to http://bioconductor.org/packages/2.10/bioc/html/DTA.html Contact: schwalb@lmb.uni-muenchen.de

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.

Expressed prostatic secretion (EPS) is a proximal fluid directly derived from the prostate and, in the case of prostate cancer (PCa), is hypothesized to contain a repertoire of cancer-relevant proteins. Quantitative analysis of the EPS proteome may enable identification of proteins with utility for PCa diagnosis and prognosis. The present investigation demonstrates selective quantitation of proteins in EPS samples from PCa patients using a stable isotope labeled proteome standard (SILAP) generated through the selective harvest of the "secretome" from the PC3 prostate cancer cell line grown in stable isotope labeled cell culture medium. This stable isotope labeled secretome was digested with trypsin and equivalently added to each EPS digest, after which the resultant mixtures were analyzed by liquid chromatography-tandem mass spectrometry for peptide identification and quantification. Relative quantification of endogenous EPS peptides was accomplished by comparison of reconstructed mass chromatograms to those of the chemically identical SILAP peptides. A total of 86 proteins were quantified from 263 peptides in all of the EPS samples, 38 of which were found to be relevant to PCa. This work demonstrates the feasibility of using a SILAP secretome standard to simultaneously quantify many PCa-relevant proteins in EPS samples.

Background: Epithelial ovarian cancer (EOC) is the deadliest gynecologic malignancy in the United States. Unfortunately, a validated protein biomarker-screening test to detect early stage disease from peripheral blood has not yet been developed. The present investigation assesses the ability to identify tumor relevant proteins from ovarian cancer proximal fluids, including tissue interstitial fluid (TIF) and corresponding ascites, from patients with papillary serous EOC and translates these findings to targeted blood-based immunoassays. Methodology/Principal Findings: Paired TIF and ascites collected from four papillary serous EOC patients at the time of surgery underwent immunodepletion, resolution by 1D gel electrophoresis and in-gel digestion for analysis by liquid chromatography-tandem mass spectrometry, which resulted in an aggregate identification of 569 and 171 proteins from TIF and ascites, respectively. Of these, peroxiredoxin I (PRDX1) was selected for validation in serum by ELISA and demonstrated to be present and significantly elevated (p=0.0188) in 20 EOC patients with a mean level of 26.0 ng/mL (69.27 SEM) as compared to 4.19 ng/mL (62.58 SEM) from 16 patients with normal/benign ovarian pathology. Conclusions/Significance: We have utilized a workflow for harvesting EOC-relevant proximal biofluids, including TIF and ascites, for proteomic analysis. Among the differentially abundant proteins identified from these proximal fluids, PRDX1 was demonstrated to be present in serum and shown by ELISA to be elevated by nearly 6-fold in papillary serous EOC patients relative to normal/benign patients. Our findings demonstrate the facile ability to discover potential EOC-relevant proteins in proximal fluids and confirm their presence in peripheral blood serum. In addition, our finding of elevated levels of PRDX1 in the serum of EOC patients versus normal/benign patients warrants further evaluation as a tumor specific biomarker for EOC.

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.

Stress triggers complex response mechanisms designed to recognize and adapt to perturbations in homeostasis. The immune system is highly responsive to stress, although the complete mechanisms linking stress and immune mediators including T lymphocytes, are not fully understood. Stress exerts its effects on immune effectors through two primary pathways: the sympathetic-adrenal-medullary pathway, and the hypothalamic-pituitary-adrenal pathway which modulate adaptive immunity and lymphocyte migration. In this report we show that stress via release of stress hormones induces early T cell activation and greatly impacts the cytoskeleton by modulating numerous actin-regulating proteins. In particular, proteomic profiling revealed significant decreases in numerous key actin-binding proteins including moesin. Although confocal microscopy showed that moesin and actin were uniformly distributed on the surface of resting T cells, a remarkable polarization and redistribution of moesin and actin was observed following treatment with stress hormones with moesin localizing at the distal pole complex. In addition, the alteration in moesin localization and eventual decrease in expression were accompanied by a loss of CD43; a receptor involved in negatively regulating T cell activation. In conclusion, we have defined a novel molecular mechanism whereby stress hormones negatively impact T cell activation and migration through regulation of key cytoskeletal and plasma membrane factors.

Targeted glycoproteomics represents an attractive approach for conducting peripheral blood based cancer biomarker discovery due to the well-known altered pattern of protein glycosylation in cancer and the reduced complexity of the resultant glycoproteome. Here we report its application to a set of pooled non-small cell lung cancer (NSCLC) case sera (9 adenocarcinoma and 6 squamous cell carcinoma pools from 54 patients) and matched controls pools, including 8 clinical control pools with computed tomography detected nodules but being non-malignant as determined by biopsy from 54 patients, and 8 matched healthy control pools from 106 cancer-free subjects. The goal of the study is to discover biomarkers which may enable improved early detection and diagnosis of lung cancer. Immunoaffinity subtraction was used to first deplete the top most abundant serum proteins; the remaining serum proteins were then subjected to hydrazide chemistry based glycoprotein capture and enrichment. Hydrazide resin in situ trypsin digestion was used to release non-glycosylated peptides. Formerly N-linked glycosylated peptides were released by peptide-N-glycosidase F (PNGase F) treatment and were subsequently analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A MATLAB® based in-house tool was developed to facilitate retention time alignment across different LC-MS/MS runs, determination of precursor ion m/z values and elution profiles, and the integration of mass chromatograms based on determined parameters for identified peptides. A total of 38 glycopeptides from 22 different proteins were significantly differentially abundant across the case/control pools (P

Spt6 is an essential transcription elongation factor and histone chaperone that binds the C-terminal repeat domain (CTD) of RNA polymerase II. We show here that Spt6 contains a tandem SH2 domain with a novel structure and CTD-binding mode. The tandem SH2 domain binds to a serine 2-phosphorylated CTD peptide in vitro, whereas its N-terminal SH2 subdomain, which we previously characterized, does not. CTD binding requires a positively charged crevice in the C-terminal SH2 subdomain, which lacks the canonical phospho-binding pocket of SH2 domains and had previously escaped detection. The tandem SH2 domain is apparently required for transcription elongation in vivo as its deletion in cells is lethal in the presence of 6-azauracil.

The pathogenesis of ovarian, fallopian tube, and peritoneal cancers has been difficult to elucidate despite intense effort. Recently, though, the care of women felt to be at high risk due to a strong family history of breast and/or ovarian cancer or a known germline BRCA1 or BRCA2 mutation has provided potential insight into the development of these malignancies. Risk-reducing surgical removal of the fallopian tubes and ovaries, called risk-reducing bilateral salpingo-oopherectomy (RRBSO), is commonly performed as a laparoscopic procedure to minimize recovery time. We describe here an optimized surgical sampling workflow for analyzing the proteomes of peritoneal, fallopian tube, and ovarian surface epithelial (OSE) specimens collected at the time of laparoscopic RRBSO, a technique which has not been described previously. This methodology presents a unique opportunity for closer examination of the proteomic alterations in the tissues at risk for malignant transformation in women with an inherited susceptibility to ovarian, fallopian tube, and peritoneal cancer development.