Development and Optimization of a Subtraction-Normalized Immunocyte Profiling Signature for Prostate Cancer Active Surveillance Risk Stratification.

PURPOSE: Less invasive decision support tools are desperately needed to identify occult high-risk disease in men with prostate cancer (PCa) on active surveillance (AS). For a variety of reasons, many men on AS with low- or intermediate-risk disease forgo the necessary repeat surveillance biopsies needed to identify potentially higher-risk PCa. Here, we describe the development of a blood-based immunocyte transcriptomic signature to identify men harboring occult aggressive PCa. We then validate it on a biopsy-positive population with the goal of identifying men who should not be on AS and confirm those men with indolent disease who can safely remain on AS. This model uses subtraction-normalized immunocyte transcriptomic profiles to risk-stratify men with PCa who could be candidates for AS. MATERIALS AND METHODS: Men were eligible for enrollment in the study if they were determined by their physician to have a risk profile that warranted prostate biopsy. Both training (n = 1017) and validation cohort (n = 1198) populations had blood samples drawn coincident to their prostate biopsy. Purified CD2+ and CD14+ immune cells were obtained from peripheral blood mononuclear cells, and RNA was extracted and sequenced. To avoid overfitting and unnecessary complexity, a regularized regression model was built on the training cohort to predict PCa aggressiveness based on the National Comprehensive Cancer Network PCa guidelines. This model was then validated on an independent cohort of biopsy-positive men only, using National Comprehensive Cancer Network unfavorable intermediate risk and worse as an aggressiveness outcome, identifying patients who were not appropriate for AS. RESULTS: The best final model for the AS setting was obtained by combining an immunocyte transcriptomic profile based on 2 cell types with PSA density and age, reaching an AUC of 0.73 (95% CI: 0.69-0.77). The model significantly outperforms (P < .001) PSA density as a biomarker, which has an AUC of 0.69 (95% CI: 0.65-0.73). This model yields an individualized patient risk score with 90% negative predictive value and 50% positive predictive value. CONCLUSIONS: While further validation in an intended-use cohort is needed, the immunocyte transcriptomic model offers a promising tool for risk stratification of individual patients who are being considered for AS.

Evaluation of an RNAseq-Based Immunogenomic Liquid Biopsy Approach in Early-Stage Prostate Cancer.

The primary objective of this study is to detect biomarkers and develop models that enable the identification of clinically significant prostate cancer and to understand the biologic implications of the genes involved. Peripheral blood samples (1018 patients) were split chronologically into independent training (n = 713) and validation (n = 305) sets. Whole transcriptome RNA sequencing was performed on isolated phagocytic CD14+ and non-phagocytic CD2+ cells and their gene expression levels were used to develop predictive models that correlate to adverse pathologic features. The immune-transcriptomic model with the highest performance for predicting adverse pathology, based on a subtraction of the log-transformed expression signals of the two cell types, displayed an area under the curve (AUC) of the receiver operating characteristic of 0.70. The addition of biomarkers in combination with traditional clinical risk factors (age, serum prostate-specific antigen (PSA), PSA density, race, digital rectal examination (DRE), and family history) enhanced the AUC to 0.91 and 0.83 for the training and validation sets, respectively. The markers identified by this approach uncovered specific pathway associations relevant to (prostate) cancer biology. Increased phagocytic activity in conjunction with cancer-associated (mis-)regulation is also represented by these markers. Differential gene expression of circulating immune cells gives insight into the cellular immune response to early tumor development and immune surveillance.

In vitro radiosensitization by gold nanoparticles during continuous low-dose-rate gamma irradiation with I-125 brachytherapy seeds.

This communication reports the first experimental evidence of gold nanoparticle (AuNP) radiosensitization during continuous low-dose-rate (LDR) gamma irradiation with low-energy brachytherapy sources. HeLa cell cultures incubated with and without AuNP were irradiated with an I-125 seed plaque designed to produce a relatively homogeneous dose distribution in the plane of the cell culture slide. Four sets of irradiation experiments were conducted at low-dose rates ranging from 2.1 to 4.5cGy/h. Residual γH2AX was measured 24h after irradiation and used to compare radiation damage to the cells with and without AuNP. The data demonstrate that the biological effect when irradiating in the presence of 0.2mg/ml concentration of AuNP is about 70%-130% greater than without AuNP. Meanwhile, without radiation, the AuNP showed minimal effect on the cancer cells. These findings provide in vitro evidence that AuNP may be employed as radiosensitizers during continuous LDR brachytherapy. FROM THE CLINICAL EDITOR: In this basic science paper, the application of gold nanoparticles as radiosensitizing agents for low dose rate gamma radiation therapy is discussed, demonstrating efficacy in cell culture models.

Bystander effect in tumor cells produced by Iodine-125 labeled human lymphocytes.

PURPOSE: To investigate the ability of human lymphocytes labeled with DNA-incorporated (125)I to exert an inhibitory (antiproliferative) bystander effect on co-cultured human colon adenocarcinoma LS174T cells in vitro. MATERIALS AND METHODS: Human peripheral blood lymphocytes were stimulated to synthesize DNA in the presence of phytohemagglutinin (PHA) and labeled with 5-[(125)I]iodo-2'-deoxyuridine. Human colon adenocarcinoma LS174T cells were co-cultured with the (125)I-labeled lymphocytes in various ratios for 5 days and the proliferation of the LS174T cells was assessed. Further, the supernatant media from these co-cultures were: (i) Transferred to LS174T cells and their proliferation measured after 5 days, (ii) used to assess the clonogenic survival of LS174T cells, and (iii) screened for factors that suppress growth. RESULTS: A significant reduction in the proliferation of LS174T cells was observed when co-cultured either with (125)I-labeled lymphocytes (56 ± 3.5%) or the supernatant media (52.5 ± 1.3%) obtained from these co-cultures. Clonogenic survival of LS174T cells grown in the supernatant media corroborated the decrease in tumor cell growth. CONCLUSION: The observed reduction in the proliferation of LS174T cells in presence of (125)I-labeled lymphocytes or media obtained from such co-cultures can be attributed to an inhibitory (antiproliferative) bystander effect, probably mediated by factor(s) released from the dying (125)I-labeled lymphocytes.

Effect of distance between decaying (125)I and DNA on Auger-electron induced double-strand break yield.

PURPOSE: To determine the possible effects of (125)I-to-DNA distance on the magnitude and mechanism of Auger-electron induced-double-strand break (DSB) production. MATERIALS AND METHODS: We have synthesized a series of (125)I-labeled Hoechst (H) derivatives ((125)IE-H, (125)IB-H, (125)I-C(8)-H and (125)I-C(12)-H). While all four molecules share a common DNA minor groove binding bis-benzimidazole motif, they are designed to position (125)I at varying distances from the DNA helix. Each Hoechst derivative was incubated at 4°C in phosphate buffered saline (PBS) together with supercoiled (SC) (3)H-pUC19 plasmid DNA (ratio 3:1) ± the •OH scavenger dimethyl sulfoxide (DMSO) (0.2 M). Aliquots were analyzed on agarose gels over time and DSB yields per decay of (125)I atom were determined. Docking of the iodinated compounds on a DNA molecule was carried out to determine the distance between the iodine atom and the central axis of DNA. RESULTS: In the absence of DMSO, the results show that the DSB yields decrease monotonically as the (125)I atom is distanced - by 10.5 Å to 13.9 Å - from the DNA helix ((125)IEH: 0.52 ± 0.01; (125)IB-H: 0.24 ± 0.03; (125)I-C(8)-H: 0.18 ± 0.02; (125)I-C(12)-H: 0.10 ± 0.00). In the presence of DMSO, DSB yields for (125)IEH (0.49 ± 0.02) and (125)IB-H (0.26 ± 0.04) remain largely unchanged indicating that DSB are entirely produced by direct effects. Strikingly, (125)I-C(8)-H or (125)I-C(12)-H, did not produce detectable DSB in the presence of DMSO under similar conditions suggesting when (125)I atom is positioned > 12 Å from the DNA, DSB are entirely produced by indirect effects. CONCLUSION: These results suggest that at a critical distance between the (125)I atom and the DNA helix, DSB production switches from an 'all' direct to an 'all' indirect mechanism, the latter situation being comparable to the decay of (125)I free in solution. These experimental findings were correlated with theoretical expectations based on microdosimetry.

Computational and biological evaluation of quinazolinone prodrug for targeting pancreatic cancer.

Our concept of enzyme-mediated cancer imaging and therapy aims to use radiolabeled compounds to target hydrolases over-expressed on the extracellular surface of solid tumors. A data mining approach identified extracellular sulfatase 1 (SULF1) as an enzyme expressed on the surface of pancreatic cancer cells. We designed, synthesized, and characterized 2-(2'-sulfooxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-S)) as well as its radioiodinated form ((125) IQ(2-S)) as a prodrug with potential for hydrolysis by SULF1. IQ(2-S) was successfully docked in silico into three enzymes - homolog of SULF1, alkaline phosphatase, and prostatic acid phosphatase. The incubation of (125) IQ(2-S) and (125) IQ(2-P) with the three enzymes in solution confirms the docking results and enzyme selectivity for the analogs. The hydrolysis of both radioactive compounds produces the water-insoluble, fluorescent product 2-(2'-hydroxyphenyl)-6-[(125) I]iodo-4-(3H)-quinazolinone ((125) IQ(2-OH)). The in vitro incubation of (127) IQ(2-S) and (127) IQ(2-P) with pancreatic, ovarian, and prostate cancer cells expressing studied hydrolases also results in their hydrolysis and the precipitation of (127) IQ(2-OH) fluorescent crystals on the cell surface. To our knowledge, these findings are the first to report the targeting of a radioactive substrate to SULF1 and that this prodrug may be potentially useful in the imaging ((123) I/(124) I/(131) I) and radiotherapy ((131) I) of pancreatic cancer.

Target discovery from data mining approaches.

Data mining of available biomedical data and information has greatly boosted target discovery in the 'omics' era. Target discovery is the key step in the biomarker and drug discovery pipeline to diagnose and fight human diseases. In biomedical science, the 'target' is a broad concept ranging from molecular entities (such as genes, proteins and miRNAs) to biological phenomena (such as molecular functions, pathways and phenotypes). Within the context of biomedical science, data mining refers to a bioinformatics approach that combines biological concepts with computer tools or statistical methods that are mainly used to discover, select and prioritize targets. In response to the huge demand of data mining for target discovery in the 'omics' era, this review explicates various data mining approaches and their applications to target discovery with emphasis on text and microarray data analysis. Two emerging data mining approaches, chemogenomic data mining and proteomic data mining, are briefly introduced. Also discussed are the limitations of various data mining approaches found in the level of database integration, the quality of data annotation, sample heterogeneity and the performance of analytical and mining tools. Tentative strategies of integrating different data sources for target discovery, such as integrated text mining with high-throughput data analysis and integrated mining with pathway databases, are introduced.

Human placental alkaline phosphatase-mediated hydrolysis correlates tightly with the electrostatic contribution from tail group.

Human placental alkaline phosphatase has been identified as a hydrolase that is significantly overexpressed on the surface of various solid tumor cells, and is therefore a suitable prodrug design target for non-invasive cancer imaging and therapy. Structure-based prediction of enzymatic activities is essential for rational prodrug design. We have been probing the catalytic proficiency--(k(cat) /K(M) )/k(w)--of placental alkaline phosphatase toward several widely diverse substrate structures experimentally and correlating these results to in silico predictions that are based on the free energy estimates obtained from docking of each substrate structure with placental alkaline phosphatase. We have found that electrostatic contribution from the tail group is the most crucial factor to determine the catalytic efficiencies of the substrates. The electrostatic contribution and the total binding energy of the tail group are well correlated with catalytic efficiencies (R² = 0.79 and 0.89, respectively). However, hydrophobic contribution from the tail group does not correlate with the catalytic efficiencies (negative correlation, R² = 0.27). This supports the prior hypothesis stating that alkaline phosphatase-mediated differential hydrolysis of its substrates is attributable to the differential interactions with the tail group, determined by the electrostatic contributions from the non-bridging oxygen atoms. Calculation of the electrostatic potentials within the active site of human placental alkaline phosphatase also suggests that the local positive electrostatic environment may account for its capability to distinguish various substrates. Our study is likely to have immediate implications in the design of prodrugs against human placental alkaline phosphatase and other esterases overexpressed by human tumor cells.

Novel method for quantifying radiation-induced single-strand-break yields in plasmid DNA highlights 10-fold discrepancy.

The widely used agarose gel electrophoresis method for assessing radiation-induced single-strand-break (SSB) yield in plasmid DNA involves measurement of the fraction of relaxed-circular (C) form that migrates independently from the intact supercoiled (SC) form. We rationalized that this method may underestimate the SSB yield since the position of the relaxed-circular form is not altered when the number of SSB per DNA molecule is >1. To overcome this limitation, we have developed a novel method that directly probes and quantifies SSBs. Supercoiled (3)H-pUC19 plasmid samples were irradiated with γ-rays, alkali-denatured, dephosphorylated, and kinated with γ-[(32)P]ATP, and the DNA-incorporated (32)P activities were used to quantify the SSB yields per DNA molecule, employing a standard curve generated using DNA molecules containing a known number of SSBs. The same irradiated samples were analyzed by agarose gel and SSB yields were determined by conventional methods. Comparison of the data demonstrated that the mean SSB yield per plasmid DNA molecule of [21.2±0.59]×10(-2)Gy(-1) as measured by direct probing is ~10-fold higher than that obtained from conventional gel-based methods. These findings imply that the SSB yields inferred from agarose gels need reevaluation, especially when they were utilized in the determination of radiation risk.

Integrated bioinformatics analysis for cancer target identification.

The exponential growth of high-throughput Omics data has provided an unprecedented opportunity for new target identification to fuel the dried-up drug discovery pipeline. However, the bioinformatics analysis of large amount and heterogeneous Omics data has posed a great deal of technical challenges for experimentalists who lack statistical skills. Moreover, due to the complexity of human diseases, it is essential to analyze the Omics data in the context of molecular networks to detect meaningful biological targets and understand disease processes. Here, we describe an integrated bioinformatics analysis strategy and provide a running example to identify suitable targets for our in-house Enzyme-Mediated Cancer Imaging and Therapy (EMCIT) technology. In addition, we go through a few key concepts in the process, including corrected false discovery rate (FDR), Gene Ontology (GO), pathway analysis, and tissue specificity. We also describe popular programs and databases which allow the convenient annotation and network analysis of Omics data. We provide a practical guideline for researchers to quickly follow the protocol described and identify those targets that are pertinent to their work.

General approach to identifying potential targets for cancer imaging by integrated bioinformatics analysis of publicly available genomic profiles.

Molecular imaging has moved to the forefront of drug development and biomedical research. The identification of appropriate imaging targets has become the touchstone for the accurate diagnosis and prognosis of human cancer. Particularly, cell surface- or membrane-bound proteins are attractive imaging targets for their aberrant expression, easily accessible location, and unique biochemical functions in tumor cells. Previously, we published a literature mining of potential targets for our in-house enzyme-mediated cancer imaging and therapy technology. Here we present a simple and integrated bioinformatics analysis approach that assembles a public cancer microarray database with a pathway knowledge base for ascertaining and prioritizing upregulated genes encoding cell surface- or membrane-bound proteins, which could serve imaging targets. As examples, we obtained lists of potential hits for six common and lethal human tumors in the prostate, breast, lung, colon, ovary, and pancreas. As control tests, a number of well-known cancer imaging targets were detected and confirmed by our study. Further, by consulting gene-disease and protein-disease databases, we suggest a number of significantly upregulated genes as promising imaging targets, including cell surface-associated mucin-1, prostate-specific membrane antigen, hepsin, urokinase plasminogen activator receptor, and folate receptors. By integrating pathway analysis, we are able to organize and map "focused" interaction networks derived from significantly dysregulated entity pairs to reflect important cellular functions in disease processes. We provide herein an example of identifying a tumor cell growth and proliferation subnetwork for prostate cancer. This systematic mining approach can be broadly applied to identify imaging or therapeutic targets for other human diseases.

Molecular and cellular radiobiological effects of Auger emitting radionuclides.

Although the general radiobiologic principles underlying external beam therapy and radionuclide therapy are similar, significant differences in the biophysical and radiobiologic effects from the two types of radiation continue to accumulate. Here, I will address the unique features that distinguish the molecular and cellular radiobiological effects of Auger electron-emitting radionuclides consequent to (1) the physical characteristics of the decaying atom and its subcellular localisation, (2) DNA topology and (3) the bystander effect. Based on these experimental findings, I postulate that the ability of track structural simulations as primary tools in modelling DNA damage and cellular survival at the molecular level would be greatly enhanced when these contributions are factored in.

Solid-tumor radionuclide therapy dosimetry: new paradigms in view of tumor microenvironment and angiogenesis.

PURPOSE: The objective of this study is to evaluate requirements for radionuclide-based solid tumor therapy by assessing the radial dose distribution of beta-particle-emitting and alpha-particle-emitting molecules localized either solely within endothelial cells of tumor vasculature or diffusing from the vasculature throughout the adjacent viable tumor cells. METHODS: Tumor blood vessels were modeled as a group of microcylindrical layers comprising endothelial cells (one-cell thick, 10 microm diameter), viable tumor cells (25-cell thick, 250 microm radius), and necrotic tumor region (> 250 microm from any blood vessel). Sources of radioactivity were assumed to distribute uniformly in either endothelial cells or in concentric cylindrical 10 microm shells within the viable tumor-cell region. The EGSnrc Monte Carlo simulation code system was used for beta particle dosimetry and a dose-point kernel method for alpha particle dosimetry. The radioactive decays required to deposit cytocidal doses (> or = 100 Gy) in the vascular endothelial cells (endothelial cell mean dose) or, alternatively, at the tumor edge [tumor-edge mean dose (TEMD)] of adjacent viable tumor cells were then determined for six beta (32P, 33P, 67Cu, 90Y 131I, and 1188Re) and two alpha (211At and 213Bi) particle emitters. RESULTS: Contrary to previous modeling in targeted radionuclide therapy dosimetry of solid tumors, the present work restricts the region of tumor viability to 250 microm around tumor blood vessels for consistency with biological observations. For delivering > or = 100 Gy at the viable tumor edge (TEMD) rather than throughout a solid tumor, energetic beta emitters 90Y, 32P, and 188Re can be effective even when the radionuclide is confined to the blood vessel (i.e., no diffusion into the tumor). Furthermore, the increase in tumor-edge dose consequent to beta emitter diffusion is dependent on the energy of the emitted beta particles, being much greater for lower-energy emitters 131I, 67Cu, and 33P relative to higher-energy emitters 90Y, 32P, and 188Re. Compared to alpha particle emitters, a approximately 150-400 times higher number of beta-particle-emitting radioactive atoms is required to deposit the same dose in tumor neovasculature. However, for the alpha particle emitters 211At and 213Bi to be effective in irradiating viable tumor-cell regions in addition to the vasculature the carrier molecules must diffuse substantially from the vasculature into the viable tumor. CONCLUSION: The presented data enable comparison of radionuclides used for antiangiogenic therapy on the basis of their radioactive decay properties, tumor neovasculature geometry, and tumor-cell viability. For alpha particle emitters or low-energy beta particle emitters, the targeting carrier molecule should be chosen to permit the radiopharmaceutical to diffuse from the endothelial wall of the blood vessel, while for long-range energetic beta particle emitters that target neovasculature, a radiopharmaceutical that binds to newly formed endothelial cells and does not diffuse is preferable. The work is a first approximation to modeling of tumor neovasculature that ignores factors such as pharmacokinetics and targeting capability of carrier molecules. The calculations quantify the interplay between irradiation of neovasculature, the surrounding viable tumor cells, and the physical properties of commonly used radionuclides and can be used to assist estimation of radioactivity to be administered for neovasculature-targeted tumor therapy.

Putative molecular signatures for the imaging of prostate cancer.

Prostate cancer is the most common cancer among men worldwide and is the second leading cause of death among those over 50 years of age in the USA. However, many men who develop a prostate tumor never exhibit symptoms in the early stage of the disease or even before it spreads to other parts of the body, such as bones and lymph nodes. Therefore, the successful prevention and treatment of prostate cancer relies on the early and accurate detection of the disease. Although prostate-specific antigen has been extensively used as a serum biomarker to detect prostate tumors in the past 20 years, this screening method has suffered from a lack of specificities and sensitivities, despite its wide use. Furthermore, fluorine-18-labeled fluorodeoxyglucose, a radiopharmaceutical useful in the detection (using PET) of various solid tumors, is not accurate for imaging cancer of the prostate. Therefore, there is an underlying necessity to discover upregulated tumor-specific markers that may serve as molecular targets for the imaging of prostate cancer. This review summarizes the most recent advances made in the discovery of tumor-specific signatures that could be useful for imaging and accurate detection of prostate cancer, using the tools of bioinformatics, genomics, proteomics and metabolomics approaches. Also introduced is the recent development of a few promising techniques, such as functional MRI, to facilitate the detection of prostate tumor signatures.

Target discovery from data mining approaches.

Data mining of available biomedical data and information has greatly boosted target discovery in the 'omics' era. Target discovery is the key step in the biomarker and drug discovery pipeline to diagnose and fight human diseases. In biomedical science, the 'target' is a broad concept ranging from molecular entities (such as genes, proteins and miRNAs) to biological phenomena (such as molecular functions, pathways and phenotypes). Within the context of biomedical science, data mining refers to a bioinformatics approach that combines biological concepts with computer tools or statistical methods that are mainly used to discover, select and prioritize targets. In response to the huge demand of data mining for target discovery in the 'omics' era, this review explicates various data mining approaches and their applications to target discovery with emphasis on text and microarray data analysis. Two emerging data mining approaches, chemogenomic data mining and proteomic data mining, are briefly introduced. Also discussed are the limitations of various data mining approaches found in the level of database integration, the quality of data annotation, sample heterogeneity and the performance of analytical and mining tools. Tentative strategies of integrating different data sources for target discovery, such as integrated text mining with high-throughput data analysis and integrated mining with pathway databases, are introduced.

DNA double-strand breaks induced by decay of (123)I-labeled Hoechst 33342: role of DNA topology.

PURPOSE: To determine double-strand-break (DSB) yields produced by decay of minor-groove-bound (123)I-labeled Hoechst 33342 ((123)IEH) in supercoiled (SC) and linear (L) forms of pUC19 DNA, to compare strand-break efficiency of (123)IEH with that of (125)IEH, and to examine the role of DNA topology in DSB induction by these Auger electron emitters. MATERIALS AND METHODS: Tritium-labeled SC and L pUC19 DNA were incubated with (123)IEH (0-10.9 MBq) at 4 degrees C. After (123)I had completely decayed (10 days), samples were analyzed on agarose gel, and single-strand-break (SSB) and DSB yields were measured. RESULTS: Each (123)I decay in SC DNA produces a DSB yield of 0.18 +/- 0.01. On the basis of DSB yields for (125)IEH (0.52 +/- 0.02 for SC and 1.62 +/- 0.07 for L, reported previously) and dosimetric expectations, a DSB yield of approximately 0.5 (3 x 0.18) per (123)I decay is expected for L DNA. However, no DSB are observed for the L form, even after approximately 2 x 10(11) decays of (123)I per microg DNA, whereas a similar number of (125)I decays produces DSB in approximately 40% of L DNA. CONCLUSION: (123)IEH-induced DSB yield for SC but not L DNA is consistent with the dosimetric expectations for Auger electron emitters. These studies highlight the role of DNA topology in DSB production by Auger emitters and underscore the failure of current theoretical dosimetric methods per se to predict the magnitude of DSB.

Synthesis and application of molecular probe for detection of hydroxyl radicals produced by Na(125)I and gamma-rays in aqueous solution.

PURPOSE: To synthesize N-(3-(3-aminopropylamino)propyl)-2-oxo-2H-chromene-3-carboxamide (7), a novel DNA-binding, coumarin-based, fluorescent hydroxylradical ((*)OH) indicator and to assess its quantum efficiency compared with that of coumarin-3-carboxylic acid (1) and N1,N12-bis[2-oxo-2H-chromene-3-carbonyl]- 1,12-diamine-4,9-diazadodecane (9). MATERIALS AND METHODS: Using computer-generated molecular modeling, 7 and 9 and their respective 7-hydroxylated derivatives 8 and 10 were docked onto DNA dodecamer d(CGCGAATTCGCG)2, the ligand-DNA complexes were energy minimized, and binding free energies and inhibition constants were calculated. Compound 7 was judged an appropriate target molecule and was synthesized. Compounds 1, 7, and 9 were incubated with Na(125)I or irradiated with (137)Cs gamma-rays, and the influence of pH, dose, type of radiation, and the concentration of indicator on fluorescence yield were determined. RESULTS: Non-fluorescent 7 and 9 are converted to fluorescent, 7-hydroxylated derivatives 8 and 10 after interaction with (*)OH in aqueous solution. For 1, 7, and 9, hydroxylation yield increases linearly with both Na(125)I dose (0-700 x 10(6) decays) and (137)Cs dose (0-11.0 Gy). Fluorescence induction is significantly reduced at acidic pH and the fluorescent quantum yield of 8 is approximately 3 times that of 2 or 10 at pH 7.0. With Na(125)I incubation and gamma-ray irradiation, the fluorescence signal of 7 increases linearly with concentration and saturates at approximately 50 microM. CONCLUSION: Compound 7 quantifies lower concentrations of (*)OH than do 1 and 9. This detector is therefore likely to be a good reporter of (*)OH produced within a few nanometers of DNA.

Integrative genomic data mining for discovery of potential blood-borne biomarkers for early diagnosis of cancer.

BACKGROUND: With the arrival of the postgenomic era, there is increasing interest in the discovery of biomarkers for the accurate diagnosis, prognosis, and early detection of cancer. Blood-borne cancer markers are favored by clinicians, because blood samples can be obtained and analyzed with relative ease. We have used a combined mining strategy based on an integrated cancer microarray platform, Oncomine, and the biomarker module of the Ingenuity Pathways Analysis (IPA) program to identify potential blood-based markers for six common human cancer types. METHODOLOGY/PRINCIPAL FINDINGS: In the Oncomine platform, the genes overexpressed in cancer tissues relative to their corresponding normal tissues were filtered by Gene Ontology keywords, with the extracellular environment stipulated and a corrected Q value (false discovery rate) cut-off implemented. The identified genes were imported to the IPA biomarker module to separate out those genes encoding putative secreted or cell-surface proteins as blood-borne (blood/serum/plasma) cancer markers. The filtered potential indicators were ranked and prioritized according to normalized absolute Student t values. The retrieval of numerous marker genes that are already clinically useful or under active investigation confirmed the effectiveness of our mining strategy. To identify the biomarkers that are unique for each cancer type, the upregulated marker genes that are in common between each two tumor types across the six human tumors were also analyzed by the IPA biomarker comparison function. CONCLUSION/SIGNIFICANCE: The upregulated marker genes shared among the six cancer types may serve as a molecular tool to complement histopathologic examination, and the combination of the commonly upregulated and unique biomarkers may serve as differentiating markers for a specific cancer. This approach will be increasingly useful to discover diagnostic signatures as the mass of microarray data continues to grow in the 'omics' era.

Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA.

Preparations of circular plasmid DNA in either supercoiled or nicked circular form often are contaminated with undesired linear DNA fragments arising from shearing/degradation of chromosomal DNA or linearization of plasmid DNA itself. We report a simple enzymatic method, using a combination of lambda exonuclease and RecJ(f), for the selective removal of linear DNA from such mixtures. lambda exonuclease digests one strand of linear duplex DNA in the 5' to 3' direction, whereas RecJ(f), a single-strand-specific exonuclease, digests the remaining complementary single strand into mononucleotides. This combination of exonucleases can remove linear DNA from a mixture of linear and supercoiled DNA, leaving the supercoiled form intact. Furthermore, the inability of lambda exonuclease to initiate digestion at nicks or gaps enables the removal of undesired linear DNA when nicked circular DNA has been enzymatically prepared from supercoiled DNA. This method can be useful in the preparation of homogeneous circular plasmid DNA required for therapeutic applications and biophysical studies.