Side-chain dynamics of the α1B -adrenergic receptor determined by NMR via methyl relaxation.

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~µs-ms). On a faster timescale (~ps-ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized α1B -adrenergic receptor (α1B -AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However, ρ-TIA increases the flexibility of Ile1764×56 and possibly of Ile2145×49 , adjacent to Pro2155×50 of the highly conserved P5×50 I3×40 F6×44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side-chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α1B -AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.

Early Molecular Insights into Thanatin Analogues Binding to A. baumannii LptA.

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro.

Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae.

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth.

PURPOSE: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. METHODS: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. RESULTS: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. CONCLUSIONS: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

Peptides in BioNMR Research.

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ρ-TIA to the human G-protein coupled α1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics.

Analysis of Compositional Gradients in Cu(In,Ga)(S,Se)2 Solar Cell Absorbers Using Energy Dispersive X-ray Analysis with Different Acceleration Energies.

The efficiency of Cu(In,Ga)(S,Se)2 (CIGSSe) solar cell absorbers can be increased by the optimization of the Ga/In and S/Se gradients throughout the absorber. Analyzing such gradients is therefore an important method in tracking the effectiveness of process variations. To measure compositional gradients in CIGSSe, energy dispersive X-ray analysis (EDX) with different acceleration energies performed at both the front surface and the backside of delaminated absorbers was used. This procedure allows for the determination of compositional gradients at locations that are millimeters apart and distributed over the entire sample. The method is therefore representative for a large area and yields information about the lateral homogeneity in the millimeter range. The procedure is helpful if methods such as secondary ion-mass (SIMS), time-of-flight SIMS, or glow-discharge optical emission spectrometry (GDOES) are not available. Results of such EDX measurements are compared with GDOES, and they show good agreement. The procedure can also be used in a targeted manner to detect local changes of the gradients in inhomogeneities or points of interest in the µm range. As an example, a comparison between the compositional gradients in the regular absorber and above the laser cut separating the Mo back contact is shown.

Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy.

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Optimizing the α1B-adrenergic receptor for solution NMR studies.

Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes 13C, 15N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the α1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1-2 mg/L of D2O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the α1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [15N,1H]- as well as [13C,1H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [13C,1H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.

The Impact of Lidocaine on Adipose-Derived Stem Cells in Human Adipose Tissue Harvested by Liposuction and Used for Lipotransfer.

The local anesthetic lidocaine, which has been used extensively during liposuction, has been reported to have cytotoxic effects and therefore would be unsuitable for use in autologous lipotransfer. We evaluated the effect of lidocaine on the distribution, number, and viability of adipose-derived stem cells (ASCs), preadipocytes, mature adipocytes, and leukocytes in the fatty and fluid portion of the lipoaspirate using antibody staining and flow cytometry analyses. Adipose tissue was harvested from 11 female patients who underwent liposuction. Abdominal subcutaneous fat tissue was infiltrated with tumescent local anesthesia, containing lidocaine on the left and lacking lidocaine on the right side of the abdomen, and harvested subsequently. Lidocaine had no influence on the relative distribution, cell number, or viability of ASCs, preadipocytes, mature adipocytes, or leukocytes in the stromal-vascular fraction. Assessing the fatty and fluid portions of the lipoaspirate, the fatty portions contained significantly more ASCs (p < 0.05), stem cells expressing the preadipocyte marker Pref-1 (p < 0.01 w/lidocaine, p < 0.05 w/o lidocaine), and mature adipocytes (p < 0.05 w/lidocaine, p < 0.01 w/o lidocaine) than the fluid portions. Only the fatty portion should be used for transplantation. This study found no evidence that would contraindicate the use of lidocaine in lipotransfer. Limitations of the study include the small sample size and the inclusion of only female patients.

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cß, C') resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Triterpenes for Well-Balanced Scar Formation in Superficial Wounds.

Triterpenes are demonstrably effective for accelerating re-epithelialisation of wounds and known to improve scar formation for superficial lesions. Among the variety of triterpenes, betuline is of particular medical interest. Topical betuline gel (TBG) received drug approval in 2016 from the European Commission as the first topical therapeutic agent with the proven clinical benefit of accelerating wound healing. Two self-conducted randomized intra-individual comparison clinical studies with a total of 220 patients involved in TBG treatment of skin graft surgical wounds have been screened for data concerning the aesthetic aspect of wound healing. Three months after surgery wound treatment with TBG resulted in about 30% of cases with more discreet scars, and standard of care in about 10%. Patients themselves appreciate the results of TBG after 3 months even more (about 50%) compared to standard of care (about 10%). One year after surgery, the superiority of TBG counts for about 25% in comparison with about 10%, and from the patients' point of view, for 25% compared to 4% under standard of care. In the majority of wound treatment cases, there is no difference visible between TBG treatment and standard of care after 1 year of scar formation. However, in comparison, TBG still offers a better chance for discreet scars and therefore happens to be superior in good care of wounds.

Visible tumor surface response to physical plasma and apoptotic cell kill in head and neck cancer.

The aim of the study was to learn, whether clinical application of cold atmospheric pressure plasma (CAP) is able to cause (i) visible tumor surface effects and (ii) apoptotic cell kill in squamous cell carcinoma and (iii) whether CAP-induced visible tumor surface response occurs as often as CAP-induced apoptotic cell kill. Twelve patients with advanced head and neck cancer and infected ulcerations received locally CAP followed by palliative treatment. Four of them revealed tumor surface response appearing 2 weeks after intervention. The tumor surface response expressed as a flat area with vascular stimulation (type 1) or a contraction of tumor ulceration rims forming recesses covered with scabs, in each case surrounded by tumor tissue in visible progress (type 2). In parallel, 9 patients with the same kind of cancer received CAP before radical tumor resection. Tissue specimens were analyzed for apoptotic cells. Apoptotic cells were detectable and occurred more frequently in tissue areas previously treated with CAP than in untreated areas. Bringing together both findings and placing side by side the frequency of clinical tumor surface response and the frequency of analytically proven apoptotic cell kill, detection of apoptotic cells is as common as clinical tumor surface response. There was no patient showing signs of an enhanced or stimulated tumor growth under influence of CAP. CAP was made applicable by a plasma jet, kINPen(®) MED (neoplas tools GmbH, Greifswald, Germany).

Hypericin-bearing magnetic iron oxide nanoparticles for selective drug delivery in photodynamic therapy.

Combining the concept of magnetic drug targeting and photodynamic therapy is a promising approach for the treatment of cancer. A high selectivity as well as significant fewer side effects can be achieved by this method, since the therapeutic treatment only takes place in the area where accumulation of the particles by an external electromagnet and radiation by a laser system overlap. In this article, a novel hypericin-bearing drug delivery system has been developed by synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) with a hypericin-linked functionalized dextran coating. For that, sterically stabilized dextran-coated SPIONs were produced by coprecipitation and crosslinking with epichlorohydrin to enhance stability. Carboxymethylation of the dextran shell provided a functionalized platform for linking hypericin via glutaraldehyde. Particle sizes obtained by dynamic light scattering were in a range of 55-85 nm, whereas investigation of single magnetite or maghemite particle diameter was performed by transmission electron microscopy and X-ray diffraction and resulted in approximately 4.5-5.0 nm. Surface chemistry of those particles was evaluated by Fourier transform infrared spectroscopy and ζ potential measurements, indicating successful functionalization and dispersal stabilization due to a mixture of steric and electrostatic repulsion. Flow cytometry revealed no toxicity of pure nanoparticles as well as hypericin without exposure to light on Jurkat T-cells, whereas the combination of hypericin, alone or loaded on particles, with light-induced cell death in a concentration and exposure time-dependent manner due to the generation of reactive oxygen species. In conclusion, the combination of SPIONs' targeting abilities with hypericin's phototoxic properties represents a promising approach for merging magnetic drug targeting with photodynamic therapy for the treatment of cancer.

A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation.

BACKGROUND: DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. RESULTS: This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. CONCLUSION: The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

Development of a diagnostic microarray assay to assess the risk of recurrence of prostate cancer based on PITX2 DNA methylation.

Prostate cancer is among the most common cancers. Although it has a relatively good prognosis, 15 to 30% of men with prostate cancer experience a relapse after radical prostatectomy. Identifying patients with an aggressive tumor will therefore help to improve prostate cancer management. DNA methylation of PITX2 has been established in several studies as a prognostic biomarker for breast and prostate cancer. These case control studies were conducted using research assay components; to facilitate its use in a diagnostic setting, the PITX2 biomarker was transferred to a validated diagnostic platform, the Affymetrix GeneChip System. A customized microarray (Epichip PITX2) was designed using features in high redundancy to ensure a robust determination of the methylation state of the PITX2 promoter. The developed method allowed for accurate assessment of prognosis in prostate cancer patients who underwent radical prostatectomy. Determination of PITX2 methylation in formalin-fixed and paraffin-embedded tissue samples from a cohort of 157 prostatectomy patients resulted in an excellent level of concordance of the clinical classification, as well as the measured output between the research assay and the Epichip PITX2. These analytical performance results describe the Epichip PITX2 as a robust and reliable diagnostic tool for assessing the methylation status of PITX2, enabling an improved outcome prediction in cancer patients following radical prostatectomy.

Quantitative DNA methylation profiling on a high-density oligonucleotide microarray.

Recently, the analysis and functional elucidation of CpG island methylation has become a focus area of genomic research. Deviations from the normal parental imprinting pattern have been shown to cause developmental defects associated with serious symptoms. Aberrant DNA methylation of tumor suppressor and other functional genes, especially when found in 5' untranslated regions and early exons, has been associated with tumorigenesis. In the context of applying DNA methylation analysis for the molecular characterization of cancer and other diseases, standardized protocols enabling parallel genome-wide methylation profiling of numerous samples are required. DNA methylation profiling is described using a CpG island microarray representing more than 50,000 CpG-rich DNA fragments. Fragments were selected to represent the vast majority of known 5'-untranslated regions as well as the first exons of thousands of genes. Measurement probes were designed to represent these fragments were displayed on an Affymetrix custom array. A modified procedure for differential methylation hybridization (DMH) is described for methylation enrichment. Application of a novel signal normalization concept enables accurate and reproducible measurements using a single fluorescence channel. The use of defined calibrator material allows quantification of DNA methylation patterns by DMH in a massively parallel fashion.

Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer.

BACKGROUND: The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case-control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage. METHODS: A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls. RESULTS: The SEPT9 assay workflow yielded 1.9 microg/L (CI 1.3-3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%-50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study. CONCLUSIONS: Circulating methylated SEPT9 DNA, as measured in the new (m)SEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

Analysis of DNA methylation of multiple genes in microdissected cells from formalin-fixed and paraffin-embedded tissues.

A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells.

DNA methylation biomarkers for blood-based colorectal cancer screening.

BACKGROUND: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. METHODS: We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. RESULTS: Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%). CONCLUSIONS: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.