Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer

Purpose : The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. Experimental design : We performed 2‐D gel electrophoresis and MALDI‐TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. Results : Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). Conclusion and clinical relevance : Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.     

Methods for classification of two thyroid follicular tumour classes

The goal of the present paper is to show that certain cytometric (morphological, photometric and textural) features of isolated cell nuclei can be useful for discrimination and classification of two thyroid tumour classes, namely, follicular adenoma and follicular carcinoma which cannot in general be visually discriminated in cytological smears. Several linear classifiers both at cell and specimen levels are proposed, in order to estimate the true error rate of these classifiers, a ‘hold one specimen out’ scheme is employed.     

Identification of distinctive protein expression patterns in colorectal adenoma

Purpose: As a pre‐malignant precursor, adenoma provides an ideal tissue for proteome profiling to investigate early colorectal cancer development and provide possible targets for preventive interventions. The aim of this study was to identify patterns of differential protein expression that distinguish colorectal adenoma from normal tissue. Experimental design: Twenty paired samples of adenoma and normal mucosa were analysed by 2‐DE and MALDI‐TOF/TOF MS to detect proteins with ≥2‐fold differential expression. Results: Four proteins were up‐regulated in adenoma (Annexin A3, S100A11, S100P and eIF5A‐1) and three were down‐regulated (Galectin‐1, S100A9 and FABPL). S100P, galectin‐1, S100A9 and FABPL expression was localised by immunohistochemistry. Conclusions and clinical relevance: Distinctive patterns of in vivo protein expression in colorectal adenoma were identified for the first time. These proteins have important functions in cell differentiation, proliferation and metabolism, and may play a crucial role in early colorectal carcinogenesis. The ability to recognise premalignant lesions may have important applications in cancer prevention.     

NanoProbeArrays for the analysis of ultra-low-volume protein samples using piezoelectric liquid dispensing technology

Antibody microarrays are gaining popularity as a high-throughput technology to investigate the proteome. However, protein extracts from most body fluid or biopsy samples are available in very small volumes and are often unsuitable for large-scale antibody microarray studies. To demonstrate the potential for protein analysis with as little as a few nanoliters of sample, we have developed a new technology called NanoProbeArrays based on piezoelectric liquid dispensing for non-contact printing and probing of antibody arrays. Instead of flooding the protein sample on the antibody microarray surface, as in conventional microarray screening, a piezoelectric inkjet printer is used to dispense nanoliters of fluorescently labeled proteins over the antibody spots on the array. The ability of NanoProbeArrays to precisely identify and reliably distinguish between test proteins from different sources, without any loss of sensitivity and specificity as compared with conventional antibody microarrays, is illustrated here. The utility of NanoProbeArrays for biomarker identification in a complex biological sample was tested by detecting the cytokine interleukin-4 in serum. The significant reduction in volume of sample during NanoProbeArray analysis, as compared with conventional antibody microarrays, offers new opportunities for basic and applied proteomic research.     

Identification of cancer-associated proteins by proteomics and downregulation of β-tropomyosin expression in colorectal adenoma and cancer

Elucidating the molecular mechanism underlying the development of adenoma, the major precursor lesion of colorectal cancer (CRC), would provide a basis for early detection, prevention as well as treatment of CRC. Using the highly sensitive 2-D DIGE method coupled with MS, we identified 24 differentially expressed proteins in adenoma tissues compared with matched normal colonic mucosa and CRC tissues. Fifteen proteins were downregulated and three proteins were upregulated in adenoma tissues when compared with individual-matched normal colonic mucosa. Five proteins were downregulated, while one protein was upregulated in adenoma tissues when compared with matched CRC tissues. A protein, β-tropomyosin (TM-β), recently suggested to be a biomarker of esophageal squamous carcinoma, was downregulated in both adenoma and CRC tissues. Additionally, the reduction in the level of TM-β in adenoma and CRC tissues was further validated by Western blotting (p<0.05) and RT-PCR (p<0.001). Our findings suggest that downregulation of TM-β is involved in the early development of CRC and that differentially expressed proteins might serve as potential biomarkers for detection of CRC.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

Identification of differentially expressed proteins in papillary thyroid carcinomas with V600E mutation of BRAF

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway. V600E mutation of BRAF protein is the most common genetic alteration occurring in papillary thyroid carcinomas and is prognostic of poor clinicopathological outcomes. Protein expression in the subclass of PTC bearing the BRAF(V600E) mutation was investigated by using 2-DE and MS/MS techniques and compared to that of matched normal thyroid tissues from seven patients. 2-D gel image analysis revealed that the expression of eight polypeptide spots, corresponding to five proteins, were significantly underexpressed in PTC bearing BRAF(V600E) mutation whereas 25 polypeptides, representing 19 distinct proteins, were significantly upregulated in tumour tissue, as compared to normal thyroid. Among the differentially expressed polypeptides, mitochondrial proteins, ROS-scavenger enzymes, apoptosis-related proteins as well as proteins involved in tumour cell proliferation were identified. Although dissimilarities between the present results and those previously reported can be ascribed to the use of different 2-DE techniques, the possibility that BRAF(V600E) mutation is responsible for changes in protein expression distinct from those induced by other oncogenes cannot be ruled out.     

Proteomics-based identification of HSP60 as a tumor-associated antigen in colorectal cancer

Patients with cancer frequently develop autoantibodies. The identification of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteomics analysis (SERPA) to identify tumor proteins that elicit humoral response in colorectal cancer (CRC). The CRC cell line HCT116 was used as a source of proteins for 2-DE and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. An autoantibody against HSP60 identified by MS was detected in 13 out of 25 patients with CRC and 1 out of 15 healthy subjects. In addition, the HSP60 expressions in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry, and serum specimens from 100 patients with cancer and 30 healthy controls were screened for antibody titer to HSP60 by ELISA. The results showed that expressions of HSP60 in tumor tissue and serum antibody titer to HSP60 were significantly higher in patients with CRC than in healthy subjects. Thus, we conclude that the SERPA is an excellent assay for the identification of tumor-associated antigens and tumor markers. The detection of HSP60 may have clinical utility in CRC screening, diagnosis, and immunotherapy.     

Real time medical data monitoring and iodine 131 treatment of thyroid cancer nursing analysis based on embedded system

Iodine is a normal function of the thyroid gland, is essential in order to make the thyroid hormone. Thyroid, is equipped with a system known as "NA + / I- thyroid symporter pump" for moving iodine into the cell. Therefore either bear radioiodine I-131, RAI is the ability of the thyroid cells to such those as accurately iodine, have been used by the medical staff for the diagnosis and treatment of various thyroid disorders. Embedded medical devices also include some existing embedded medical devices. On fetal monitors, oximeters, defibrillators, etc., which are easier and more efficient in the medical field. RAI has not been concentrated in the thyroid gland, which is excreted from the body through sweat and urine. Beta radiation can be used at high doses in order to destroy the residual thyroid tissue after treatment of exclusion and differentiated thyroid carcinoma of thyroid nodules and surgical diseases, and gamma; however, unique I-131 is approximately 8 days have a beta and gamma radiation half-life performance radiation can be measured for imaging and uptake. Evaluate the effectiveness of lifestyle care instructions for patients undergoing Radioactive Iodine Therapy (RAI) for thyroid disease. Patients and Methods: conducted in the Department of Nuclear Medicine. Adults are useful for men and women with thyroid disease who are admitted to the Department of Nuclear Medicine for the treatment of RAI.     

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2‐DE and LC‐tandem mass chromatography to separate and identify differentially expressed proteins. Forty‐five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.     

A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue offers significant diagnostic utility but is complicated due to the high level of covalently crosslinked proteins arising from formalin fixation. To address these challenges, we developed a reliable protein extraction method for FFPE tissue, based on heat-induced antigen retrieval within a pressure cooker. The protein extraction yield from archival FFPE tissue section is approximately 90% of that recovered from frozen tissue. This method demonstrates preservation of immunoreactivity and recovery of full-length proteins by Western blotting. Additionally, we developed a well-based RP protein array platform utilizing an electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, remain stable over 1?month at room temperature. Theses data suggest that this new protein-profiling platform coupled with the protein extraction method can be used for molecular profiling analysis in FFPE tissue, and contribute to the validation and development of biomarkers in clinical studies.     

Proneurotensin/neuromedin N secreted from small cell lung carcinoma cell lines as a potential tumor marker

Proteins secreted from specific cancer cells have a high potential for use as tumor markers. We identified secreted proteins produced by 15 different carcinoma cell lines grown in serum-free medium using MS/MS. Proneurotensin/neuromedin N (proNT/NMN) was found in conditioned medium from four of seven small cell lung carcinoma cell lines but not from eight nonsmall cell lung carcinoma cell lines. These results indicate proNT/NMN has potential as a specific tumor marker of small cell lung carcinoma.     

Integrative analysis of cancer pathway progression and coherence

We analyzed the cancer pathways in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The database provides a collective of signaling pathway members involved in cancer progression. However, the KEGG cancer pathways, unlike signaling pathways, have not been analyzed extensively with gene expression and mutation data. We transformed the colorectal cancer pathway into discrete X and Y scales and analyzed the relative expression levels of adenoma and carcinoma samples as well as the distribution of mutation targets. The X scale corresponds to the downstream location in a pathway, whereas the Y scale corresponds to the stage of the tumor. The gene expression values of the early stage pathway members are significantly higher than of the rest of the pathway members in colorectal adenoma tissues. The colorectal cancer pathway shows some degree of coherence in the carcinoma samples. The correlated gene pairs responsible for the coherence of the colorectal cancer pathway in the carcinoma samples are supported, in part, by the literature and may suggest novel regulatory associations. Finally, there are more mutation targets in the nucleus as well as the late tumor stages of the KEGG colorectal cancer pathway.     

Impedance-controlled cell entrapment using microhole-array chips allows the isolation and identification of single, highly productive cells

Highly productive cells are required for the manufacture of biopharmaceutical proteins, but the best-performing cells can be difficult to isolate from a large and heterogeneous background. Although this problem has been addressed by gently positioning individual cells on microhole arrays, such a procedure is typically monitored by microscope which limits the size of the array to the field view of the microscope and also requires the use of direct illumination, which bleaches the sensitive fluorescent reagents needed to identify the most productive cells. We have circumvented these problems by developing an impedance-based method for cell positioning, and by defining a relationship between impedance and occupancy we are also able to monitor the progress of the cell positioning process. We identified cells secreting large amounts of a model antibody by mixing the cells with semi-solid medium and a light-sensitive antibody detection reagent, and then using impedance controlled positioning to capture cells on the microholes in absence of direct light. Following a 1-day incubation, antibody secretion could be determined qualitatively using fluorescence imaging and a simple image processing algorithm. For the first time, we have shown that antibody secretion from individual cells on a microhole array can be classified and used to identify the most productive cells. Because this procedure does not rely on optical analysis it could easily be scaled up to allow the screening of millions of cells at a time.     

Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.     

SMART: Student modeling approach for responsive tutoring

This paper describes a new student modeling paradigm called SMART. The premise is that a single, principled approach to student modeling, involving both theoretical and empirical methods, can render automated instruction more efficacious across a broad array of instructional domains. After defining key terms and discussing limitations to previous student modeling paradigms, I describe the SMART approach, as embedded within a statistics tutor called Stat Lady (Shute and Gluck, 1994). SMART works in conjunction with a tutor design where low-level knowledge and skills (i.e., curricular elements) are identified and separated into three main outcome types. Throughout the tutor, curricular elements with values below a pre-set mastery criterion are instructed, evaluated, and remediated, if necessary. The diagnostic part of the student model is driven by a series of regression equations based on the level of assistance the computer gives each person, per curriculum element. Remediation on a given element occurs when a subject fails to achieve mastery during assessment, which follows instruction. Remediation is precise because each element knows its location within the tutor where it is instructed and assessed. I end with a summary of results from two controlled evaluations of SMART examining the following research issues: (a) diagnostic validity, (b) individual differences in learning from Stat Lady, (c) affective perceptions of the tutorial experience, and (d) contributions of mastery and remediation to learning outcome and efficiency. Comments about related and future research with this paradigm are offered.     

Analysis of the differential proteome of human erythroblasts during in vitro erythropoiesis by 2-D DIGE

In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification.     

Hospital IoT system design and the efficacy of Iodine 131 in the treatment of thyroid cancer

Thyroid cancer of radioactive iodine (RAI) in IoT devices marker sensor for tracking a medical device's real-time location, such as oxygen pumps, wheelchairs, defibrillators, nebulizers, and have other monitoring devices. The hospital's current hospital information system is based on the fixed information solutions such as the IoT (IOT). The radiation can destroy the thyroid and occupied iodine thyroid cells, accumulate in; it does not have little effect on the other thyroid cells in the rest of your body. Retrospective analysis of differentiated thyroid carcinoma (DTC) cases was treated separately in the RAI-131 thyroid. Exclusion criteria are as follows. Malignancies existing or co no thyroidal, familial syndromes associated with multiple tumors, exposure to ionizing radiation or new second tumor with histopathology. For the survival of DTC, it was not related to developing the cumulative dose of RAI-131 Treatment and I-131 in a significantly long-term follow-up. Observation of a sudden event of the tendency of patients treated with RAI-131, the interaction of other risk factors, suggests that to predict the carcinogenic.     

Characterisation of tumoral markers correlated with ErbB2 (HER2/Neu) overexpression and metastasis in breast cancer

The receptor tyrosine kinase ErbB2 (HER2/neu) is overexpressed in ?30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis. Clinical treatments such as trastuzumab are effective in less than 35% of women diagnosed as ErbB2‐positive, highlighting the necessity of searching for novel targets and alternative therapies. Herein, a proteomic screening strategy combining quantitative‐based gel electrophoresis and MS was used to compare the protein expression of 48 normal human breast and tumour tissues differing in ErbB2 expression and lymph node status. The aim was to identify proteins associated with the aggressive phenotype of ErbB2‐positive breast cancer which could be potential biomarkers of the disease as well as therapy targets. In total, 177 protein isoforms (107 gene products) differentially expressed between tissue groups were identified. Immunohistochemical staining of a tissue‐microarray was used for validation of selected protein candidates. We found that expression of HSP90α, laminin and GSTP1 significantly correlated with ErbB2 expression, while others such as AGR2, NM23H1 and Annexin 2 were overexpressed in greater than 40% of tumours. Finally, knocking‐down the expression by RNA interference of three candidates, AGR2, Transgelin2 and NM23H1 resulted in an enhanced invasive capacity of MDA‐MB435 cells. These data support the involvement of these targets in tumour progression and identify them as novel biomarkers of the disease.