Introduction: Several studies have reported regeneration of the pulp-dentin complex when treating noninfected root canal systems. However, current protocols applied to infected root canal systems are much less predictable for the formation of dentin. Converging lines of evidence implicate residual biofilm as an important factor for these variable histologic outcomes. Here we studied the effect of a residual polymicrobial biofilm on the release of transforming growth factor beta 1 (TGF-beta 1) from dentin. We hypothesized that the presence of bacterial biofilm attenuates the release of bioactive molecules from dentin. Methods: Using bacteria commonly found in infected immature teeth, we developed a multispecies biofilm in an organotypic root canal model. Root segments were then subjected to various irrigation or intracanal medicament protocols. Subsequently, the release of TGF-beta 1 from dentin was measured using the enzyme-linked immunosorbent assay. Results: Our data show that sterile root segments released greater amounts of TGF-beta 1 when conditioned with 17% EDTA alone (P < .001) or with the combination of 1.5% sodium hypochlorite and 17% EDTA (P < .05) compared with root segments infected with the multispecies biofilm. Similar results were also observed with the intracanal medicament protocol. Sterile root segments medicated with various concentrations of triple antibiotic paste and full-strength calcium hydroxide released greater amounts of TGF-beta 1 when compared with their infected counterparts. Conclusions: This is the first study to report the detrimental effects of a residual biofilm on dentin conditioning and, therefore, the release of growth factors critical for regenerative procedures.

BACKGROUND:RNA sequencing (RNA-seq) is a high throughput technology that profiles gene expression in a genome-wide manner. RNA-seq has been mainly used for testing differential expression (DE) of transcripts between two conditions and has recently been used for testing differential alternative polyadenylation (APA). In the past, many algorithms have been developed for detecting differentially expressed genes (DEGs) from RNA-seq experiments, including the one we developed, XBSeq, which paid special attention to the context-specific background noise that is ignored in conventional gene expression quantification and DE analysis of RNA-seq data.RESULTS:We present several major updates in XBSeq2, including alternative statistical testing and parameter estimation method for detecting DEGs, capacity to directly process alignment files and methods for testing differential APA usage. We evaluated the performance of XBSeq2 against several other methods by using simulated datasets in terms of area under the receiver operating characteristic (ROC) curve (AUC), number of false discoveries and statistical power. We also benchmarked different methods concerning execution time and computational memory consumed. Finally, we demonstrated the functionality of XBSeq2 by using a set of in-house generated clear cell renal carcinoma (ccRCC) samples.CONCLUSIONS:We present several major updates to XBSeq. By using simulated datasets, we demonstrated that, overall, XBSeq2 performs equally well as XBSeq in terms of several statistical metrics and both perform better than DESeq2 and edgeR. In addition, XBSeq2 is faster in speed and consumes much less computational memory compared to XBSeq, allowing users to evaluate differential expression and APA events in parallel. XBSeq2 is available from Bioconductor: http://bioconductor.org/packages/XBSeq/.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2017MP39-06 WHOLE GENOME TRANSCRIPTIONAL ANALYSIS OF CLEAR CELL RENAL CELL CARCINOMA WITH VENOUS TUMOR THROMBUS REVEALS INTRATUMORAL HETEROGENEITY AND GENES ASSOCIATED WITH POOR OUTCOME Dharam Kaushik, Wasim Chowdhury, Ping Wu, Teresa Johnson-Pais, Yidong Chen, Michael A. Liss, and Ronald Rodriguez Dharam KaushikDharam Kaushik More articles by this author , Wasim ChowdhuryWasim Chowdhury More articles by this author , Ping WuPing Wu More articles by this author , Teresa Johnson-PaisTeresa Johnson-Pais More articles by this author , Yidong ChenYidong Chen More articles by this author , Michael A. LissMichael A. Liss More articles by this author , and Ronald RodriguezRonald Rodriguez More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1180AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Overall, 4 to 10% of newly diagnosed renal cell carcinoma (RCC) patients have been found to have a venous tumor thrombus (VTT). Intratumoral heterogeneity may contribute to progression of the disease and metastases. Previous studies have focused on studying the heterogeneity of metastases, recurrence and tumor. There has been no study to date evaluating VTT and characterizing its transcriptional profile. We report results of the transcriptional analysis of primary tumor (PT), VTT and adjacent normal parenchyma (NP). METHODS We performed Whole Transcriptome Sequencing on fresh tissue specimens from 6 patients with clear cell RCC and VTT collected at the time of radical nephrectomy with tumor thrombectomy. We evaluated transcriptional alterations between PT/NP, VTT/NP and VTT/PT. We compared our data set with The Cancer Genome Atlas (TCGA) data. For multiple testing corrections, we utilized false discovery estimation and differential expression criterion. RESULTS 1. We surveyed 23,228 genes and identified differential expression in 1455, 1344 and 26 genes between tumor and normal parenchyma (PT/NP), tumor thrombus and normal parenchyma (VTT/NP) and tumor thrombus and primary tumor (VTT/PT) respectively. We identified altered key pathways (cytokine activity, regulation of apoptosis, cytoskeleton organization, immune response). 2. We compared these genes with TCGA data and identified 35 genes, which predicted poor outcome. 3. VTT demonstrated statistically significant differential expression of OSM (2.37 fold), INHBA (1.9 fold), CCL2 (2.3 fold), CCL20 (2.9 fold) and IL1B genes (4.5 fold) compared to primary tumor (TT/T), (Figure1). 4. We identified statistically significant higher expression of OSM, INHBA, CCL20, and IL1B genes in higher tumor thrombus level compared to lower tumor thrombus level. CONCLUSIONS RNA sequencing of RCC with VTT reveals significant genomic intratumoral heterogeneity. We identified key molecular pathways and differential expression of genes in VTT compared to primary tumor (VTT/PT). Furthermore, these genes were found to be upregulated in higher VTT level compared to lower VTT level. These results will require validation in a larger cohort. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e495 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Dharam Kaushik More articles by this author Wasim Chowdhury More articles by this author Ping Wu More articles by this author Teresa Johnson-Pais More articles by this author Yidong Chen More articles by this author Michael A. Liss More articles by this author Ronald Rodriguez More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

BACKGROUNDCirculating tumor cells (CTCs) hold great promise as biomarkers and are a direct source of tumor cells through a simple blood draw. However, CTCs are rare and their detection requires sensitive and specific methods to overcome the overwhelming hematocyte population. Therefore, CTC detection remains technically challenging.METHODSAn assay was developed for detecting viable and tissue‐specific CTCs using a tropism‐enhanced and conditionally replicating reporter adenovirus (CTC‐RV). Adenoviral replication was made prostate‐specific by placing the E1A gene under the control of the probasin promoter and prostate‐specific antigen enhancer (PSE‐PBN). Viral tropism was expanded through capsid‐displayed integrin targeting peptides. A secreted reporter, humanized Metridia Luciferase (hMLuc), was engineered for expression during the major late phase of viral replication. The assay involves red blood cell lysis, cell collection, viral infection, and subsequent quantification of reporter activity from cellular media. Assay and reporter stability, cell specificity and sensitivity were evaluated in cell dilution models in human blood.RESULTSA conditionally replicating prostate‐selective adenovirus reporter and CTC assay system were generated. The secreted reporter, MLuc, was found to be stable for at least 3 days under assay conditions. CTC detection, modeled by cell dilution in blood, was selective for androgen receptor positive prostate cancer (PCa) cells. Serial dilution demonstrated assay linearity and sensitivity to as few as three cells. Prostate cancer cell viability declined after several hours in anticoagulated blood at ambient temperatures.CONCLUSIONSConditionally replicative adenoviral vectors and secreted reporters offer a functional method to detect viable CTCs with cell specificity and high sensitivity. Prostate 74: 1286–1296, 2014. © 2014 Wiley Periodicals, Inc.

Changes in proteolytic activity are associated with several diseases, including cancer. Proteases are potential drug targets and targeting of proteases is used for treatment of various conditions/diseases, like high blood pressure and HIV. We present here detailed protocols for basic evaluation of the effects of peptides on the activity of proteases, using kallikrein-related peptidases KLK2 and KLK3 (also known as hK2 and PSA), and trypsin as examples. KLK2 and KLK3 are major prostatic proteases, and they are potential targets for prostate cancer treatment. KLK2 has trypsin-like activity and KLK3 chymotrypsin-like activity. By phage display technology, we have developed peptides that specifically stimulate KLK3-activity and other peptides that inhibit KLK2 or trypsin. The effect of the peptides on the proteolytic activity of proteases can be studied using substrates, the cleavage of which generates detectable signal, allowing rapid evaluation of protease activity. The cleavage of protein substrates can be detected by SDS-PAGE, followed by staining of the proteins. We also describe graphical analysis of the IC50-value, the effect of a peptide on Michaelis-Menten constant (K(m)) and the maximal reaction rate (V(max)).

Abstract Circulating tumor cells (CTCs) offer a new strategy to access cancer tissue through a simple blood sample. CTCs levels can be predictive of overall survival in patients with metastatic cancers and changes in CTC levels following therapy can be indicative of therapeutic response. Moreover, CTCs may provide a means to select patients for specific therapeutic approaches by identifying active or susceptible pathways. However, most of the current methods for detecting CTCs have limitations including the need for specialized equipment, the reliance on cell capture through cell surface markers such as EpCAM, limited sensitivity, the inability to distinguish cancer from normal, tissue type, active pathways, or cell viability. Here we present a novel CTC detection approach which applies tissue-selective replicating adenoviruses and highly sensitive secreted reporters. These Circulating Tumor Cell Reporter Viruses (CTC-RVs) were generated by replacing the early viral E1A gene promoter with the prostate specific probasin promoter and prostate specific antigen enhancer (PSE-PBN). The secreted reporter gene, humanized metridia luciferase (hMLuc), was incorporated into the viral major late transcriptional unit, at the fiber gene locus, to limit reporter expression to only those cells in which the virus has replicated. Cells from total blood, including mononuclear cells and CTCs, are placed in tissue culture media and infected with the CTC-RV. After three days, viral replication dramatically amplifies reporter levels within infected prostate cancer cells with active Androgen Receptor pathways leading to the secretion of the bioluminescent reporter. Non-prostatic cells will not amplify the virus or express the reporter gene. LNCaP cellular dilution study results show that as few as five CTCs per million lymphocytes cells could be readily detected without any cell isolation. LNCaP cells diluted in healthy volunteer blood samples identified the optimal time and dosing conditions. Initial studies in clinical samples support the detection of viable CTCs with this assay. This new approach offers a means to quantify live CTC levels in a tissue-specific manner, without the need for cell capture, through the use of a secreted signal which can be detected separate from the large blood cell population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2392. doi:1538-7445.AM2012-2392

Abstract The convergence of phage-displayed peptide libraries and recombinant viral vectors launched a promising new direction in targeted viral gene therapeutics, but the translation of targeting peptides to functional cancer therapeutic agents has been challenging. Here, we report progress in developing a successful strategy to optimize targeted viral infection through adenovirus-displayed, semirandom peptide libraries. A phage-derived peptide targeting the prostate-specific membrane antigen (PSMA) was genetically incorporated into the adenoviral capsid Fiber protein and flanked by random peptide cassettes. The resulting adenovirus library was biopanned against PSMA-expressing cells and tumors to identify a PSMA-retargeted adenovirus. While the initial peptide alone could not target viral infection, the selected virus preferentially infects PSMA-expressing cells through the targeting peptide and infects LNCaP tumors after intravenous injection. Our results indicate that virus-displayed, semirandom peptide libraries can be used to optimize targeting infection. This approach represents a novel principle for developing targeted agents in a variety of disease models. Cancer Res; 70(23); 9549–53. ©2010 AACR.

Human trypsin isoenzymes share extensive sequence similarity, but certain differences in their activity and susceptibility to inhibitors have been observed. Using phage display technology, we identified seven different peptides that bind to and inhibit the activity of trypsin-3, a minor trypsin isoform expressed in pancreas and brain. All of the peptides contain at least two of the amino acids tryptophan, alanine and arginine, whereas proline was found closer to the N-terminus in all but one peptide. All peptides contain two or more cysteines, suggesting a cyclic structure. However, we were able to make synthetic linear variants of these peptides without losing bioactivity. Alanine replacement experiments for one of the peptides suggest that the IPXXWFR motif is important for activity. By molecular modeling the same amino acids were found to interact with trypsin-3. The peptides also inhibit trypsin-1, but only weakly, if at all, trypsin-2 and -C. As trypsin is a highly active enzyme which can activate protease-activated receptors and enzymes that participate in proteolytic cascades involved in tumor invasion and metastasis, these peptides might be useful lead molecules for the development of drugs for diseases associated with increased trypsin activity.

BACKGROUND. Prostate-specific antigen (PSA or KLK3) has been shown to inhibit angiogenesis, but it might also have tumor promoting activities. Thus, it may be possible to modulate prostate cancer growth by stimulating or inhibiting the activity of PSA. To this end we have previously identified peptides that stimulate the activity of PSA. As peptides have several limitations as drug molecules, we screened a chemical library to find drug-like compounds that could be used to modulate the function(s) of PSA.METHODS. Almost 50,000 compounds were analyzed for their ability to modulate PSA activity towards a fluorescent PSA-substrate. The ability of the most active compounds to affect the anti-angiogenic activity of PSA was analyzed by human umbilical vein endothelial cell (HUVEC) tube formation assay.RESULTS. In the initial screening we identified two compounds that inhibited PSA activity. Based on these, similar compounds were selected and tested for activity to define structure-activity relationships. Several compounds with micromolar IC50-values were found, but they were not entirely specific towards PSA, e.g., they inhibited chymotrypsin, which has similar substrate specificity as PSA. However, it was possibly to improve the selectivity of the compounds towards PSA by small structural changes. These compounds inhibited the anti-angiogenic activity of PSA in the HUVEC model, proving that the proteolytic activity of PSA is essential for inhibition of angiogenesis.CONCLUSIONS. We found several PSA inhibitors that could be useful tools for studying the role of PSA in cancer models and in normal physiology as showed in angiogenesis model.

Capsid-displayed adenoviral peptide libraries have been a significant, yet unfeasible goal in biotechnology. Three barriers have made this difficult: the large size of the viral genome, the low efficiency of converting plasmid-based genomes into packaged adenovirus and the fact that library amplification is hampered by the ability of two (or more) virus to co-infect one cell. Here, we present a novel vector system, pFex, which is capable of overcoming all three barriers. With pFex, modified fiber genes are recombined into the natural genetic locus of adenovirus through unidirectional Crelox recombination. Modified-fiber genes can be directly shuttled into replicating viral genomes in mammalian cells. The acceptor vector does not contain the fiber gene, and therefore does not propagate until it has received a donor fiber gene. Therefore, This methodology overcomes the low efficiency of transfecting large viral genomes and bypasses the need for transition to functional virus. Thus, with a fiber-shuttle library, one can generate and evaluate large numbers of fiber-modified adenovirus simultaneously. Finally, successful fiber genes can be rescued from virus and recombined back into shuttle plasmids, avoiding the need to propagate mixed viral pools. For proof of principal, we use this new system to screen a capsid-displayed peptide library for retargeted viral infection.

Prostate cancer (PCa) is the most common cancer in men and it is a major health problem in industrialized countries. Prostate-specific antigen (PSA) is a sensitive diagnostic marker for PCa, and measurement of serum PSA is widely used for early detection, screening and monitoring of patients with PCa. However, the usefulness of PSA testing is limited because of the high rate of false-positive results. Another problem with the use of PSA for early detection and screening is overdiagnosis, i.e. detection of slowly growing prostate cancers that do not need to be treated clinically. These can be reduced by assaying the proportion of free PSA in relation to total PSA. However, further development of more cancer-specific diagnostic methods facilitating identification of aggressive cancers is desirable. This chapter summarizes existing and new approaches for detection of various forms of PSA. PSA-based multivariate algorithms are also discussed.

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from nonmalignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay-for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 mu g/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immuno-peptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.

OBJECTIVE:To explore the expressions of EGFR and Ki67 in non-small cell lung cancer and the relationships between above parameters and the occurrence, progression and prognosis of NSCLC. METHODS: The expressions of EGFR and Ki67 were detected in 60 NSCLC samples, 20 lung tissues adjacent to cancer and 5 normal lung tissues as control by S-P immunohistochemical method. RESULTS: The EGFR and Ki67 positive rates were 65% and 81.67% in 60 NSCLC,respectively. The brown granules of EGFR positive cell were mainly distributed in the cytoplasm, but the brown granules of Ki67 positive cell were in the nuclei. Positive cells were not found in 20 lung tissues adjacent to cancer and 5 normal lung tissues, otherwise , there were notable difference between cancer tissues and controls,P0.01.The EGFR and Ki67 positive expressions were not correlated to age,sex,smoking history and the pathologic types of tumor cell,P0.05, but were significantly correlated with tumor size,the sub-grade of tumor cell,lymph node metastasis and TNM stage,P0.05. The 3-year survival rate of EGFR and Ki67 positive patients(19.86% and 31.33%) was lower than that of EGFR and Ki67 negative patients (70.11% and 90.91%),P0.05. The EGFR and Ki67 positive patients who had received adjuvant chemotherapy had a higher metastasis rate (70.37% and 62.50%) than EGFR and Ki67 negative patients(11.11% and 0),P0.05. CONCLUSIONS: The aberrant expressions of EGFR and Ki67 in non-small cell lung cancer were highly correlated with tumor occurrence and development,and the expression of both presented co-ordination role. So the immunohistochemical expression of EGFR and Ki67 seems to be a useful tumor marker for patients with non-small cell lung cancer and can be used to evaluate the tumor biological behavior, sensitivity to the treatment and prognosis of the disease.

You have accessJournal of UrologyDiscussed Poster, Sunday, May 9, 2004, 1:00 - 5:00 pm1 Apr 2004583: Development of a Novel Immunopeptidometric Assay for Cancer-Associated form of Prostate-Specific Antigen Ping Wu, Lei Zhu, Jari Leinonen, and Ulf-Håkan Stenman Ping WuPing Wu More articles by this author , Lei ZhuLei Zhu More articles by this author , Jari LeinonenJari Leinonen More articles by this author , and Ulf-Håkan StenmanUlf-Håkan Stenman More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)37845-5AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "583: Development of a Novel Immunopeptidometric Assay for Cancer-Associated form of Prostate-Specific Antigen." The Journal of Urology, 171(4S), p. 155 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 171Issue 4SApril 2004Page: 155 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Ping Wu More articles by this author Lei Zhu More articles by this author Jari Leinonen More articles by this author Ulf-Håkan Stenman More articles by this author Expand All Advertisement PDF downloadLoading ...

Prostate‐specific antigen (PSA), a member of the kallikrein sub‐group of the trypsin serine protease family, is a widely used marker for prostate cancer. Several sequences with specific binding to PSA have been identified by using phage display peptide libraries. The GST‐fusion proteins of the characterized sequences have been shown to increase the enzyme activity of PSA to a synthetic substrate. The corresponding three cyclic synthetic analogues CVFTSNYAFC (A‐1), CVFAHNYNYLVC (B‐2) and CVAYCIEHHCWTC (C‐4) have similar PSA promoting activity. Despite differences in the amino acid sequences, all three peptides bind to the same region of PSA. The conformation of the peptides was investigated by proton NMR spectroscopy. In addition, alanine replacement was used to characterize the prerequisites for binding. It is proposed that interactions with PSA are based on the aromatic and hydrophobic features of the amino acid side chains. Furthermore, it is suggested that peptides form β–turn structures forced by cysteine bridges directing important aromatic side chains to the same side of the turn‐structure. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.