Oncomicrobial Community Profiling Identifies Clinicomolecular and Prognostic Subtypes of Colorectal Cancer.

BACKGROUND & AIMS: Dysbiosis of gut microbiota is linked to the development of colorectal cancer (CRC). However, microbiota-based stratification of CRC tissue and how this relates to clinicomolecular characteristics and prognosis remains to be clarified. METHODS: Tumor and normal mucosa from 423 patients with stage I to IV CRC were profiled by bacterial 16S rRNA gene sequencing. Tumors were characterized for microsatellite instability (MSI), CpG island methylator phenotype (CIMP), APC, BRAF, KRAS, PIK3CA, FBXW7, SMAD4, and TP53 mutations, subsets for chromosome instability (CIN), mutation signatures, and consensus molecular subtypes (CMS). Microbial clusters were validated in an independent cohort of 293 stage II/III tumors. RESULTS: Tumors reproducibly stratified into 3 oncomicrobial community subtypes (OCSs) with distinguishing features: OCS1 (Fusobacterium/oral pathogens, proteolytic, 21%), right-sided, high-grade, MSI-high, CIMP-positive, CMS1, BRAF V600E, and FBXW7 mutated; OCS2 (Firmicutes/Bacteroidetes, saccharolytic, 44%), and OCS3 (Escherichia/Pseudescherichia/Shigella, fatty acid ß-oxidation, 35%) both left-sided and exhibiting CIN. OCS1 was associated with MSI-related mutation signatures (SBS15, SBS20, ID2, and ID7) and OCS2 and OCS3 with SBS18 related to damage by reactive oxygen species. Among stage II/III patients, OCS1 and OCS3 both had poorer overall survival compared with OCS2 for microsatellite stable tumors (multivariate hazard ratio [HR], 1.85; 95% confidence interval [CI], 1.15-2.99; P = .012; and HR, 1.52; 95% CI 1.01-2.29; P = .044, respectively) and left-sided tumors (multivariate HR, 2.66; 95% CI, 1.45-4.86; P = .002; and HR, 1.76; 95% CI, 1.03-3.02; P = .039, respectively). CONCLUSIONS: OCS classification stratified CRCs into 3 distinct subgroups with different clinicomolecular features and outcomes. Our findings provide a framework for a microbiota-based stratification of CRC to refine prognostication and to inform the development of microbiota-targeted interventions.

Tumour-specific phosphorylation of serine 419 drives alpha-enolase (ENO1) nuclear export in triple negative breast cancer progression.

BACKGROUND: The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer. RESULTS: Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins. CONCLUSIONS: The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase's role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

Colorectal carcinogenesis: a cellular response to sustained risk environment.

The current models for colorectal cancer (CRC) are essentially linear in nature with a sequential progression from adenoma through to carcinoma. However, these views of CRC development do not explain the full body of published knowledge and tend to discount environmental influences. This paper proposes that CRC is a cellular response to prolonged exposure to cytotoxic agents (e.g., free ammonia) as key events within a sustained high-risk colonic luminal environment. This environment is low in substrate for the colonocytes (short chain fatty acids, SCFA) and consequently of higher pH with higher levels of free ammonia and decreased mucosal oxygen supply as a result of lower visceral blood flow. All of these lead to greater and prolonged exposure of the colonic epithelium to a cytotoxic agent with diminished aerobic energy availability. Normal colonocytes faced with this unfavourable environment can transform into CRC cells for survival through epigenetic reprogramming to express genes which increase mobility to allow migration and proliferation. Recent data with high protein diets confirm that genetic damage can be increased, consistent with greater CRC risk. However, this damage can be reversed by increasing SCFA supply by feeding fermentable fibre as resistant starch or arabinoxylan. High protein, low carbohydrate diets have been shown to alter the colonic environment with lower butyrate levels and apparently greater mucosal exposure to ammonia, consistent with our hypothesis. Evidence is drawn from in vivo and in vitro genomic and biochemical studies to frame experiments to test this proposition.

Epigenetic liquid biopsies for minimal residual disease, what's around the corner?

Liquid biopsy assays for minimal residual disease (MRD) are used to monitor and inform oncological treatment and predict the risk of relapse in cancer patients. To-date, most MRD assay development has focused on targeting somatic mutations. However, epigenetic changes are more frequent and universal than genetic alterations in cancer and circulating tumor DNA (ctDNA) retains much of these changes. Here, we review the epigenetic signals that can be used to detect MRD, including DNA methylation alterations and fragmentation patterns that differentiate ctDNA from noncancerous circulating cell-free DNA (ccfDNA). We then summarize the current state of MRD monitoring; highlight the advantages of epigenetics over genetics-based approaches; and discuss the emerging paradigm of assaying both genetic and epigenetic targets to monitor treatment response, detect disease recurrence, and inform adjuvant therapy.

Identification of potential pathways involved in induction of apoptosis by butyrate and 4-benzoylbutyrate in HT29 colorectal cancer cells.

Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure-activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4-benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4-benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4-benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects.

A review of the potential mechanisms for the lowering of colorectal oncogenesis by butyrate.

Colorectal cancer (CRC) is a leading cause of preventable cancer deaths worldwide, with dietary factors being recognised as key risk modifiers. Foods containing dietary fibre are protective to a degree that the World Cancer Research Fund classifies the evidence supporting their consumption as 'convincing'. The mechanisms by which fibre components protect against CRC remain poorly understood, especially their interactions with the gut microbiome. Fibre is a composite of indigestible plant polysaccharides and it is emerging that fermentable fibres, including resistant starch (RS), are particularly important. RS fermentation induces SCFA production, in particular, relatively high butyrate levels, and in vitro studies have shown that this acid has strong anti-tumorigenic properties. Butyrate inhibits proliferation and induces apoptosis of CRC cell lines at physiological concentrations. These effects are attributed to butyrate's ability to alter gene transcription by inhibiting histone deacetylase activity. However, the more recent discovery of G-protein coupled receptors that bind butyrate and other SCFA and data obtained from proteomic and genomic experiments suggest that alternative pathways are involved. Here, we review the mechanisms involved in butyrate-induced apoptosis in CRC cells and, additionally, the potential role this SCFA may play in mediating key processes in tumorigenesis including genomic instability, inflammation and cell energy metabolism. This discussion may help to inform the development of strategies to lower CRC risk at the individual and population levels.

Early evolution of the venom system in lizards and snakes.

Among extant reptiles only two lineages are known to have evolved venom delivery systems, the advanced snakes and helodermatid lizards (Gila Monster and Beaded Lizard). Evolution of the venom system is thought to underlie the impressive radiation of the advanced snakes (2,500 of 3,000 snake species). In contrast, the lizard venom system is thought to be restricted to just two species and to have evolved independently from the snake venom system. Here we report the presence of venom toxins in two additional lizard lineages (Monitor Lizards and Iguania) and show that all lineages possessing toxin-secreting oral glands form a clade, demonstrating a single early origin of the venom system in lizards and snakes. Construction of gland complementary-DNA libraries and phylogenetic analysis of transcripts revealed that nine toxin types are shared between lizards and snakes. Toxinological analyses of venom components from the Lace Monitor Varanus varius showed potent effects on blood pressure and clotting ability, bioactivities associated with a rapid loss of consciousness and extensive bleeding in prey. The iguanian lizard Pogona barbata retains characteristics of the ancestral venom system, namely serial, lobular non-compound venom-secreting glands on both the upper and lower jaws, whereas the advanced snakes and anguimorph lizards (including Monitor Lizards, Gila Monster and Beaded Lizard) have more derived venom systems characterized by the loss of the mandibular (lower) or maxillary (upper) glands. Demonstration that the snakes, iguanians and anguimorphs form a single clade provides overwhelming support for a single, early origin of the venom system in lizards and snakes. These results provide new insights into the evolution of the venom system in squamate reptiles and open new avenues for biomedical research and drug design using hitherto unexplored venom proteins.

A combined free-flow electrophoresis and DIGE approach to identify proteins regulated by butyrate in HT29 cells.

Many biologically active agents exert a pleiotropic response in cells and tissues. This presents challenges in descriptive and comparative analysis of the proteome in response to these agents. Although free-flow electrophoresis has been applied in a number of proteomic studies as a protein separation technique, the combination of free-flow electrophoresis and DIGE has not yet been investigated for comparative proteomic analysis. In this study, we have compared the effects of butyrate on HT29 colorectal cancer cells with a particular focus on apoptosis and describe the utility of a novel approach combining free-flow electrophoresis with DIGE to identify differentially expressed proteins. We verify the results obtained by the combined free-flow electrophoresis and DIGE approach with Western blot analysis of selected proteins. We also report for the first time the regulation of a number of proteins by butyrate in HT29 colorectal cells including peptidyl-prolyl cis-trans isomerase A (cyclophilin A) and profilin-1.

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response.

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.

Comparative assessment of soil quality parameters after implementing afforestation programme in forest areas of Hong Kong.

The objective of this research is to investigate and compare the alteration of soil parameters with and without afforestation programme in a Hong Kong forest, analysed by Principal Component Analysis (PCA). One hundred soil samples were collected from the following sites: Pak Ngau Shek (PNS), Shing Mun (SM), Tai Po Kau (TPK), Tai Tong (TT) (forest with afforestation programme) and Lantau Peak (LP) (control site). A significant difference was found in only two out of 16 parameters: pH (8.34-8.87) and PAHs (4.35-6.32 µg/kg) by comparing the soils taken in the forest with and without an afforestation programme implemented. Three principle components are responsible for soil quality variations in the studied sites. The first, second and third components included pH (0.167) and EC (0.176), PAHs (0.331) and PAHs (0.207), respectively. This framework provides information on the least disturbance of soil properties for the afforestation programme. To conclude, a rigorous monitoring of soil quality is necessary to assess forest health after an afforestation programme. Besides, in the long term, an appropriate forest preservation programme should be implemented to achieve rural area sustainability.

A Combined Free-Flow Electrophoresis and DIGE Approach to Compare Proteins in Complex Biological Samples.

Free-flow electrophoresis has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility and wide applicability to protein classes. As a stand-alone platform, however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with free-flow electrophoresis to simultaneously separate and identify differentially expressed proteins in a model cell system.

DNA Methylation Cancer Biomarkers: Translation to the Clinic.

Carcinogenesis is accompanied by widespread DNA methylation changes within the cell. These changes are characterized by a globally hypomethylated genome with focal hypermethylation of numerous 5'-cytosine-phosphate-guanine-3' (CpG) islands, often spanning gene promoters and first exons. Many of these epigenetic changes occur early in tumorigenesis and are highly pervasive across a tumor type. This allows DNA methylation cancer biomarkers to be suitable for early detection and also to have utility across a range of areas relevant to cancer detection and treatment. Such tests are also simple in construction, as only one or a few loci need to be targeted for good test coverage. These properties make cancer-associated DNA methylation changes very attractive for development of cancer biomarker tests with substantive clinical utility. Across the patient journey from initial detection, to treatment and then monitoring, there are several points where DNA methylation assays can inform clinical practice. Assays on surgically removed tumor tissue are useful to determine indicators of treatment resistance, prognostication of outcome, or to molecularly characterize, classify, and determine the tissue of origin of a tumor. Cancer-associated DNA methylation changes can also be detected with accuracy in the cell-free DNA present in blood, stool, urine, and other biosamples. Such tests hold great promise for the development of simple, economical, and highly specific cancer detection tests suitable for population-wide screening, with several successfully translated examples already. The ability of circulating tumor DNA liquid biopsy assays to monitor cancer in situ also allows for the ability to monitor response to therapy, to detect minimal residual disease and as an early biomarker for cancer recurrence. This review will summarize existing DNA methylation cancer biomarkers used in clinical practice across the application domains above, discuss what makes a suitable DNA methylation cancer biomarker, and identify barriers to translation. We discuss technical factors such as the analytical performance and product-market fit, factors that contribute to successful downstream investment, including geography, and how this impacts intellectual property, regulatory hurdles, and the future of the marketplace and healthcare system.

Characterisation of the protein composition of peripheral blood mononuclear cell microsomes by SDS-PAGE and mass spectrometry.

Approximately 340 leucocyte plasma membrane proteins have been characterised by the eight Human Leucocyte Differentiation Antigen workshops held between 1982 and 2004, based primarily on their reactivity with monoclonal antibodies. The human genome is predicted to encode approximately 34,000 cDNA transcripts, of which between 15% and 20% are predicted to contain one or more transmembrane helices. We have used SDS-PAGE separation coupled with mass spectrometry-based peptide mass tag identification to identify novel plasma membrane proteins in microsome preparations prepared from mononuclear cells obtained from human peripheral blood. A total of 361 distinct proteins were identified in a single preparation, including 37 known leucocyte plasma membrane proteins, 27 potential novel plasma membrane proteins whose expression on PBMC is poorly characterised, and 51 other proteins for which the subcellular location could not be determined. Expression analysis using cDNA panels indicates that several of these novel plasma membrane proteins are differentially expressed in lymphocyte subsets. These results show that previously unidentified lymphocyte plasma membrane proteins can be identified using this approach.

Blood-based protein biomarker panel for the detection of colorectal cancer.

BACKGROUND: The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test. PRINCIPAL FINDINGS: In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2). CONCLUSIONS: Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.

Activation of erythropoietin receptor through a novel extracellular binding site.

Activation of erythropoietin (EPO) receptor (EPOR) by a small peptide (ERP) was reported previously. ERP binds to a different receptor site than EPO, and binding of ERP does not change the dissociation constant and maximal binding for EPO binding to the receptor. The extracellular binding site for ERP is now characterized. The site is located in the membrane proximal, extracellular part of the receptor. ERP binds to a region on the EPOR that contains the same sequence as ERP. It is speculated that ERP binds to its identical sequence on EPOR, as ERP self-interacts. ERP is specific for EPOR and associates noncovalently with EPOR in a ratio 1:1. Peptide binding to the receptor results in receptor-mediated cellular proliferation, intracellular signaling, and erythroid colony-forming unit formation in bone marrow cells. The activity is comparable to that of EPO. Recognition of such receptor sites represents a new and important concept in receptor function.

Practical quantitative biomedical applications of MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) is used to obtain fast and accurate determinations of molecular mass, but quantitative determinations are generally made by other techniques. In this study we illustrate the practical utility of automated MALDI-TOFMS as a tool for quantifying a diverse array of biomolecules covering an extensive molecular weight range, and present in biological extracts and fluids. Growth hormone was measured in rat pituitary tissue; insulin in human pancreatic tissue; homovanillic acid in human urine; and LVV-hemorphin-7, epinephrine and norepinephrine in human adrenal and pheochromocytoma tissues. Internal standards including compounds of similar molecular weight, structural analogs or isotopomers were incorporated into each analysis. We report on the current practical limitations of quantitative MALDI-TOFMS and highlight some of the potential benefits of this technique as a quantitative tool.

Colorectal cancer biomarkers: to be or not to be? Cautionary tales from a road well travelled.

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system. The time frame for development from premalignant to malignant disease typically spans 10-15 years, and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes. Currently, early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for population-based screening. New blood-based protein biomarkers for early detection of CRC are therefore urgently required. The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental, analytical, and biological factors that can interfere with the robust interpretation of results. In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies. Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.

Claudin-1 Expression Is Elevated in Colorectal Cancer Precursor Lesions Harboring the BRAF V600E Mutation.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/P) are now recognised precursors of colorectal cancer (CRC) including cancers harbouring somatic BRAF (V600E) mutations. While the morphological diagnostic criteria of SSA/P have been established, distinguishing between small/early SSA/P and microvesicular hyperplastic polyps (MVHP) is challenging and may not be possible in routine practice. METHODS: Gene expression profiling of MVHP (n=5, all BRAF V600E wild-type) and SSA/P (n=5, all BRAF V600E mutant) samples was performed. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical analysis was performed to verify the expression of claudin 1 (CLDN1) in MVHP and SSA/P. RESULTS: Gene expression profiling studies conducted between MVHP and SSA/P identified CLDN1 as the most statistically significant differentially expressed gene (p<0.05). Validation with qRT-PCR confirmed an up-regulation of CLDN1 in BRAF V600E mutant polyps regardless of polyp type (p<0.0005). Immunohistochemical analysis of CLDN1 expression in BRAF V600E mutant SSA/Ps (n=53) and MVHPs (n=111) and BRAF wild-type MVHPs (n=58), demonstrated a strong correlation between CLDN1 expression and the BRAF V600E mutation in both SSA/P and MVHP samples when compared to wild-type polyps (p<0.0001). CONCLUSION: This study demonstrates an up regulation of CLDN1 protein in serrated colorectal polyps including MVHP harbouring the BRAF V600E mutation. Our results demonstrated an apparent heterogeneity on the molecular level within the MVHP group and suggest that MVHP with somatic BRAF V600E mutation and up-regulated expression of CLDN1 are closely related to SSA/P and may in fact represent a continuous spectrum of the same neoplastic process within the serrated pathway of colorectal carcinogenesis.

Performance of serum lipocalin 2 as a diagnostic marker for colorectal cancer.

BACKGROUND: Lipocalin 2 has been implicated in colorectal tumorigenesis but its usefulness as a diagnostic marker for the disease has previously never been determined. METHODS: We have used ELISA immunoassay to measure the level of serum lipocalin 2 in a cohort consisting of colorectal cancer patients (n=196) and age/gender matched controls (n=99). RESULTS: The median concentration of lipocalin 2 was found to be significantly higher (p< 0.0001) in the patient group (105.9 ng/mL, range 10.8-444.7 ng/mL) when compared to the control subjects (86.4 ng/mL, range 17.1-190.0 ng/mL). Additionally, no significant difference was observed between disease stage (Dukes' or T stage) in the patient cohort. Receiver operating characteristic analysis was performed to determine its performance as a diagnostic marker. The area under the curve was found to be 0.641 (95% confidence interval 0.576-0.706). Furthermore, the sensitivity of lipocalin 2 was found to be 24% at 90% specificity. CONCLUSIONS: Our study indicates that lipocalin 2 is not a suitable serum biomarker for the diagnosis of CRC.

A combined free flow electrophoresis and DIGE approach to compare proteins in complex biological samples.

Free flow electrophoresis (FFE) has been applied in numerous studies as a protein separation technique due to its multiple advantages such as fast and efficient sample recovery, high resolving power, high reproducibility, and wide applicability to protein classes. As a stand-alone platform however, its utility in comparative proteomic analysis is limited as protein samples must be run sequentially rather than simultaneously which introduces inherent variability when attempting to perform quantitative analysis. Here we describe an approach combining fluorescent CyDye technology (DIGE) with FFE to simultaneously separate and identify differentially expressed proteins in a model cell system.