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Sample records for fluorescent protein-expressing cancer

  1. TRPM4 protein expression in prostate cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Soldini, Davide; Jung, Maria

    2016-01-01

    BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level.......79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high...

  2. Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.

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    Kim, Hyojin; Yoo, Seol-Bong; Choe, Ji-Young; Paik, Jin Ho; Xu, Xianhua; Nitta, Hiroaki; Zhang, Wenjun; Grogan, Thomas M; Lee, Choon-Taek; Jheon, Sanghoon; Chung, Jin-Haeng

    2011-08-01

    Accurate determination of ALK rearrangement is important in lung cancer patients, especially in determining their eligibility for crizotinib therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting ALK rearrangement. However, FISH requires a fluorescence microscope, and the signals are labile and rapidly fade over time. This study evaluates the concordance between ALK gene rearrangement in non-small cell lung cancer assessed by ALK FISH and a newly developed ALK chromogenic in situ hybridization (CISH) and correlates the results with ALK protein expression assessed by immunohistochemistry. A total of 465 formalin-fixed, paraffin-embedded non-small cell lung cancer samples were analyzed by ALK FISH (PathVysion, Vysis, Abbott) and ALK CISH. For comparison, all specimens were stained by immunohistochemistry (clone 5A4, Novocastra) and interobserver reproducibility was assessed. We found that agreement between the pathologists on the CISH-determined ALK status was achieved in 449 patients (96.6%), and ALK rearrangement was identified in 18 patients (4.0%) in CISH method. Among these cases, 443 cases (95.3%) had results matching the corresponding FISH results: 17 rearranged, 425 wild types, and 1 discordant case. There was high concordance in the assessment of ALK gene rearrangement between FISH and CISH techniques (κ = 0.92) and between observers (κ = 0.97). In addition, there was high concordance in the ALK gene status and ALK protein expression between CISH and IHC tests (κ = 0.82). CISH is a highly reproducible and practical method to detect ALK gene rearrangement and correlated well with ALK protein expression. Here, we present a diagnostic algorithm (Chung's SNUBH ALK protocol) to detect lung cancer with ALK rearrangements using IHC, FISH and CISH. Because CISH allows a concurrent analysis of histological features of the tumors and gene rearrangement, it appears to be a useful method in determining ALK gene

  3. Kiss-1/GPR54 protein expression in breast cancer.

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    Papaoiconomou, Eleni; Lymperi, Maria; Petraki, Constantina; Philippou, Anastassios; Msaouel, Pavlos; Michalopoulou, Fani; Kafiri, Georgia; Vassilakos, George; Zografos, Georgios; Koutsilieris, Michael

    2014-03-01

    Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (pbreast cancer and fibrocystic disease specimens. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

  4. BAX protein expression and clinical outcome in epithelial ovarian cancer.

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    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

  5. HER 2/neu protein expression in colorectal cancer

    International Nuclear Information System (INIS)

    Schuell, B; Gruenberger, T; Scheithauer, W; Zielinski, Ch; Wrba, F

    2006-01-01

    Conflicting data exist about the prevalence of HER-2/neu overexpression in colorectal cancer ranging from 0 to 83 %. In our study we tried to clarify the extent of expression and its relationship to clinicopathological parameters. This study involved 77 specimens of malignant colorectal cancer lesions of surgically resected patients. HER-2/neu immunohistochemistry was performed using the Hercep-Test Kit. Out of 77 specimens, 56 were Her-2/neu negative (70%), 20 (26%) showed a barely immunostaining (1+), only 1 (1%) was moderately (2+) and 2 (3%) were strongly positive (3+). Her-2/neu staining (moderately and strongly positive) was only detected in primary tumours of patients with confirmed metastases. No relationship was found between membranous HER-2 expression and patients' gender or differentiation. The median survival time of patients with positive HER-2/neu immunostaining was 21 versus 39 months in patients without HER-2/neu expression (p = 0.088). The c-erbB protein expression was observed in colorectal cancer but rarely in the therapeutic range (2+ and 3+). There was no significant association with tumour grade, gender, localization of the primary tumour or survival. These data indicate that c-erbB-2 is unlikely to play a major role in the therapeutic management of colorectal cancer

  6. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    International Nuclear Information System (INIS)

    Tsutsui, Shinichi; Yasuda, Kazuhiro; Suzuki, Kosuke; Takeuchi, Hideya; Nishizaki, Takashi; Higashi, Hidefumi; Era, Shoichi

    2006-01-01

    Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

  7. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  8. Intrafocal heterogeneity of ERG protein expression and gene fusion pattern in prostate cancer.

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    Suh, Ja Hee; Park, Jeong Hwan; Lee, Cheol; Moon, Kyung Chul

    2017-10-01

    Prostate cancer is considered to be highly heterogeneous, with various morphologic features and biologic behaviors. The TMPRSS2-ERG gene fusion is the most frequently observed genetic aberration in prostate cancer. The aim of this study was to elucidate the intrafocal heterogeneity of ERG gene fusion status. ERG immunohistochemistry (IHC) was performed in samples from 168 prostate cancer patients who had undergone radical prostatectomy, and 40 cases showing ERG-positive IHC staining were selected for tissue microarray (TMA) construction. Two to six representative cores were selected from each tumor focus. In the cases with heterogeneous ERG IHC staining intensity, the areas showing different intensities were separately selected. Using the TMA blocks, IHC and fluorescence in situ hybridization (FISH) were conducted to evaluate the heterogeneity of ERG protein expression and ERG fusion gene patterns, respectively, in a single tumor focus. Heterogeneity of ERG IHC staining was defined as the simultaneous presence of negative and positive cores in the same tumor focus. Heterogeneity of ERG FISH was defined by the presence of cores with positive and negative FISH signals or cores with break-apart and interstitial deletion FISH signals in the same tumor focus. A total of 202 TMA cores were isolated from 40 ERG-positive cases. Of the 202 total cores, 19 were negative for ERG IHC staining, and 46 showed 1+, 52 showed 2+, and 85 showed 3+ ERG staining intensity. Eleven cores were negative for ERG FISH signal, 119 cores showed ERG break-apart FISH signals, and the remaining 72 cores revealed interstitial deletion. Intrafocal heterogeneity of ERG IHC staining was found in 20% (8/40) of cases, and intrafocal heterogeneity of ERG gene fusion pattern was found in 32.5% (13/40) of cases. In summary, this study showed significantly frequent intrafocal heterogeneity of ERG protein expression, gene fusion status and fusion pattern. This heterogeneity can be caused by the development

  9. Measuring ERCC1 protein expression in cancer specimens

    DEFF Research Database (Denmark)

    Smith, David Hersi; Fiehn, Anne-Marie Kanstrup; Fogh, Louise

    2014-01-01

    Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most......, the specificity of antibody 4F9 was tested by immunoblotting, immunohistochemistry and immunofluorescence. Scoring guidelines to aid in the evaluation of ERCC1 tumor expression were developed and evaluated in archival formalin-fixed paraffin embedded colorectal cancer specimens. Antibody 4F9 was found...... to be specific by all methods applied and it was possible to evaluate the ERCC1 expression in the majority (85%) of colorectal cancer tumor specimens....

  10. Protein expression based multimarker analysis of breast cancer samples

    International Nuclear Information System (INIS)

    Presson, Angela P; Horvath, Steve; Yoon, Nam K; Bagryanova, Lora; Mah, Vei; Alavi, Mohammad; Maresh, Erin L; Rajasekaran, Ayyappan K; Goodglick, Lee; Chia, David

    2011-01-01

    Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups. We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-β1, and TGF β receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis. We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach. We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes

  11. Methylation status and protein expression of RASSF1A in breast cancer patients.

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    Hagrass, Hoda A; Pasha, Heba F; Shaheen, Mohamed A; Abdel Bary, Eman H; Kassem, Rasha

    2014-01-01

    Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

  12. YKL-40 protein expression is not a prognostic marker in patients with primary breast cancer

    DEFF Research Database (Denmark)

    Roslind, Anne; Knoop, Ann; Jensen, Maj-Britt

    2007-01-01

    in tumor tissue was assessed by immunohistochemistry in a cohort of 630 high-risk breast cancer patients with a median estimated potential follow-up time of 10 and 13 years for disease-free (DFS) and overall survival (OS), respectively. YKL-40 protein expression was found in malignant tumor cells......YKL-40 is a new biomarker in serum with a prognostic value in several localized and metastatic malignancies. The current knowledge regarding the biological functions of YKL-40 in cancer links YKL-40 to increased aggressiveness of the tumor. Utilizing tissue microarrays, YKL-40 protein expression...... and in inflammatory cells. High expression was associated with positive estrogen and progesterone receptor status and high tumor differentiation. Contrary to studies on serum YKL-40 as a prognostic biomarker, a high YKL-40 expression in tumor cells was not significantly associated with DSF and OS in univariate...

  13. A Breast Tissue Protein Expression Profile Contributing to Early Parity-Induced Protection Against Breast Cancer

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    Christina Marie Gutierrez

    2015-11-01

    Full Text Available Background/Aims: Early parity reduces breast cancer risk, whereas, late parity and nulliparity increase breast cancer risk. Despite substantial efforts to understand the protective effects of early parity, the precise molecular circuitry responsible for these changes is not yet fully defined. Methods: Here, we have conducted the first study assessing protein expression profiles in normal breast tissue of healthy early parous, late parous, and nulliparous women. Breast tissue biopsies were obtained from 132 healthy parous and nulliparous volunteers. These samples were subjected to global protein expression profiling and immunohistochemistry. GeneSpring and MetaCore bioinformatics analysis software were used to identify protein expression profiles associated with early parity (low risk versus late/nulliparity (high risk. Results: Early parity reduces expression of key proteins involved in mitogenic signaling pathways in breast tissue through down regulation of EGFR1/3, ESR1, AKT1, ATF, Fos, and SRC. Early parity is also characterized by greater genomic stability and reduced tissue inflammation based on differential expression of aurora kinases, p53, RAD52, BRCA1, MAPKAPK-2, ATF-1, ICAM1, and NF-kappaB compared to late and nulli parity. Conclusions: Early parity reduces basal cell proliferation in breast tissue, which translates to enhanced genomic stability, reduced cellular stress/inflammation, and thus reduced breast cancer risk.

  14. High SRPX2 protein expression predicts unfavorable clinical outcome in patients with prostate cancer

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    Zhang, Meng; Li, Xiaoli; Fan, Zhirui; Zhao, Jing; Liu, Shuzheng; Zhang, Mingzhi; Li, Huixiang; Goscinski, Mariusz Adam; Fan, Huijie; Suo, Zhenhe

    2018-01-01

    Background Sushi repeat-containing protein X-linked 2 (SRPX2) is overexpressed in a variety of different tumor tissues and correlated with poor prognosis in patients. Little research focuses on the role of SRPX2 expression in prostate cancer (PCa), and the clinicopathological significance of the protein expression in this tumor is relatively unknown. However, our previous transcriptome data from those cancer stem-like cells indicated the role of SRPX2 in PCa. Materials and methods In this study, RT-PCR and Western blotting were firstly used to examine the SRPX2 expression in three PCa cell lines including LNCaP, DU145, and PC3, and then SRPX2 protein expression was immunohistochemically investigated and statistically analyzed in a series of 106 paraffin-embedded PCa tissue specimens. Results Significantly lower levels of SRPX2 expression were verified in the LNCaP cells, compared with the expression in the aggressive DU145 and PC3 cells, in both mRNA and protein levels. Immunohistochemically, there were variable SRPX2 protein expressions in the clinical samples. Moreover, high levels of SRPX2 expression in the PCa tissues were significantly associated with Gleason score (P=0.008), lymph node metastasis (P=0.009), and distant metastasis (P=0.021). Furthermore, higher levels of SRPX2 expression in the PCa tissues were significantly associated with shorter overall survival (OS) (P<0.001). Conclusion Our results demonstrate that SRPX2 is highly expressed in aggressive PCa cells in vitro, and its protein expression in PCa is significantly associated with malignant clinical features and shorter OS, strongly indicating its prognostic value in prostate cancers. PMID:29881288

  15. Long interspersed element-1 protein expression is a hallmark of many human cancers.

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    Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

    2014-05-01

    Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. CIP2A protein expression in high-grade, high-stage bladder cancer

    International Nuclear Information System (INIS)

    Huang, Lisa P; Savoly, Diana; Sidi, Abraham A; Adelson, Martin E; Mordechai, Eli; Trama, Jason P

    2012-01-01

    Bladder cancer is one of the most common cancers in the United States. Numerous markers have been evaluated for suitability of bladder cancer detection and surveillance. However, few of them are acceptable as a routine tool. Therefore, there exists a continuing need for an assay that detects the presence of bladder cancer in humans. It would be advantageous to develop an assay with a protein that is associated with the development of bladder cancer. We have identified the cancerous inhibitor of PP2A (CIP2A) protein as a novel bladder cancer biomarker. In this study, Western blot analysis was used to assess the expression level of CIP2A protein in bladder cancer cell lines and bladder cancer patient tissues (n = 43). Our studies indicated CIP2A protein was abundantly expressed in bladder cancer cell lines but not in nontumor epithelial cell lines. Furthermore, CIP2A was specifically expressed in transitional cell carcinoma (TCC) of the bladder tumor tissues but not in adjacent nontumor bladder tissue. Our data showed that CIP2A protein detection in high-grade TCC tissues had a sensitivity of 65%, which is 3.4-fold higher than that seen in low-grade TCC tissues (19%). The level of CIP2A protein expression increased with the stage of disease (12%, 27%, 67%, and 100% for pTa, pT1, pT2, and pT3 tumor, respectively). In conclusion, our studies suggest that CIP2A protein is specifically expressed in human bladder tumors. CIP2A is preferentially expressed in high-grade and high-stage TCC tumors, which are high-risk and invasive tumors. Our studies reported here support the role of CIP2A in bladder cancer progression and its usefulness for the surveillance of recurrence or progression of human bladder cancer

  17. Prokineticin 1 protein expression is a useful new prognostic factor for human sporadic colorectal cancer.

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    Nakazawa, Toshiyuki; Goi, Takanori; Hirono, Yasuo; Yamaguchi, Akio

    2015-05-01

    Hematogenous metastasis, regarded as closely related to angiogenic growth factors, is associated with colorectal cancer prognosis. The angiogenic growth factor prokineticin 1 (PROK1) has been cloned from endocrine cells. However, its protein expression in human malignant tumors has not been studied. The current study established the anti-PROK1 monoclonal antibody (mAb) and examined the relationship between the expression of PROK1 protein and human colorectal cancer. The expression of PROK1 protein was assessed in 620 resected sporadic colorectal cancer tissue samples by immunohistochemical staining with in-house-developed human PROK1 mAb to investigate the relationship of PROK1 expression to clinicopathologic factors, recurrence, and survival rate and to evaluate its prognostic significance. The expression of PROK1 protein was detected in 36 % (223/620) of human primary colorectal cancer lesions but no in the healthy mucosa adjacent to the colorectal cancer lesions. According to the clinicopathologic examinations, the frequency of positive PROK1 expression was significantly higher in cases with serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, liver metastasis, hematogenous metastasis, and higher stage disease. The recurrence rate and prognosis for patients with PROK1 expression-positive lesions were significantly worse. In the Cox proportional hazard model, PROK1 expression was an independent prognostic factor. The expression of PROK1 protein was identified for the first time as a new prognostic factor in colorectal cancer.

  18. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

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    Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

    2006-05-01

    Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

  19. S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

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    Suzuki Sayo

    2011-12-01

    Full Text Available Abstract Background Individual responses to oxaliplatin (L-OHP-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC cell lines. We performed expression difference mapping (EDM analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS, and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF. Results Of the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations (P R2 = 0.80. We identified this protein as Protein S100-A10 (S100A10 by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells. Conclusions By proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

  20. Correlation between quantitative HER-2 protein expression and risk for brain metastases in HER-2+ advanced breast cancer patients receiving trastuzumab-containing therapy.

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    Duchnowska, Renata; Biernat, Wojciech; Szostakiewicz, Barbara; Sperinde, Jeff; Piette, Fanny; Haddad, Mojgan; Paquet, Agnes; Lie, Yolanda; Czartoryska-Arłukowicz, Bogumiła; Wysocki, Piotr; Jankowski, Tomasz; Radecka, Barbara; Foszczynska-Kłoda, Małgorzata; Litwiniuk, Maria; Debska, Sylwia; Weidler, Jodi; Huang, Weidong; Buyse, Marc; Bates, Michael; Jassem, Jacek

    2012-01-01

    Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this

  1. Variation of ATM protein expression in response to irradiation of lymphocytes in lung cancer patients and controls

    International Nuclear Information System (INIS)

    Lou Jianlin; He Jiliang; Jin Lifen; Zheng Wei; Chen Zhijian; Chen Shijie; Xu Shijie

    2006-01-01

    The aim of this research work was to study the cellular response to ionizing radiation (IR) and its relationship with the ATM protein expression levels in lung cancer patients. Heparinized blood samples were collected from 22 controls and 22 lung cancer patients. Each sample was divided into two parts: non-irradiated sample and irradiated sample, which was exposed to 3 Gy X-ray. The spontaneous and IR-induced genetic damage in both lung cancer patients and controls was measured with comet assay and micronucleus (MN) assay, and the ATM protein expression levels of non-irradiated samples in lung cancer patients and controls were detected by Western blotting. The results indicated that the baseline values of average mean tail moment (MTM) and micronucleus rate (MNR) in lung cancer patients were 0.86 and 11.41 per mille , respectively, which was significantly higher than those (0.64 and 6.77 per mille ) of controls (P < 0.05 for MTM, P < 0.01 for MNR). The IR-induced average MTM and MNR in lung cancer patients were 1.23 and 77.64 per mille , respectively, which was also significantly higher than those (0.71 and 66.05 per mille ) of controls (P < 0.05 for MTM, P < 0.01 for MNR). The results of Western blotting showed that the ATM protein expression levels in lung cancer patients and controls were 0.64 and 1.71, respectively, and there was significant (P < 0.01) difference between lung cancer patients and controls. In present investigation, it was found that the genetic instability measured with comet assay and MN assay in lung cancer patients were significantly higher than those in controls, on the contrary, ATM protein expression level in lung cancer patients were significantly lower than that in controls. However, no good correlation was found either between ATM protein expression and IR-induced MTM or between ATM protein expression and IR-induced MNR in lung cancer patients

  2. Differences in IGF-axis protein expression and survival among multiethnic breast cancer patients

    International Nuclear Information System (INIS)

    Hernandez, Brenda Y; Wilkens, Lynne R; Le Marchand, Loïc; Horio, David; Chong, Clayton D; Loo, Lenora W M

    2015-01-01

    There is limited knowledge about the biological basis of racial/ethnic disparities in breast cancer outcomes. Aberrations in IGF signaling induced by obesity and other factors may contribute to these disparities. This study examines the expression profiles of the insulin-like growth factor (IGF)-axis proteins and the association with breast cancer survival across a multiethnic population. We examined the expression profiles of the IGF1, IGF1R, IGFBP2 (IGF-binding proteins), and IGFBP3 proteins in breast tumor tissue and their relationships with all-cause and breast cancer-specific survival up to 17 years postdiagnosis in a multiethnic series of 358 patients in Hawaii, USA. Native Hawaiians, Caucasians, and Japanese were compared. Covariates included demographic and clinical factors and ER/PR/HER2 (estrogen receptor/progesterone receptor/human epidermal growth factor receptor-2) status. In Native Hawaiian patients, IGFBP2 and IGFBP3 expression were each independently associated with overall and breast cancer mortality (IGFB2: HR mort = 10.96, 95% CI: 2.18–55.19 and HR mort = 35.75, 95% CI: 3.64–350.95, respectively; IGFBP3: HR mort = 5.16, 95% CI: 1.27–20.94 and HR mort = 8.60, 95% CI: 1.84–40.15, respectively). IGF1R expression was also positively associated with all-cause mortality in Native Hawaiians. No association of IGF-axis protein expression and survival was observed in Japanese or Caucasian patients. The interaction of race/ethnicity and IGFBP3 expression on mortality risk was significant. IGF-axis proteins may have variable influence on breast cancer progression across different racial/ethnic groups. Expression of binding proteins and receptors in breast tumors may influence survival in breast cancer patients by inducing aberrations in IGF signaling and/or through IGF-independent mechanisms. Additional studies to evaluate the role of the IGF-axis in breast cancer are critical to improve targeted breast cancer treatment strategies

  3. Chemokine (C-X-C) ligand 1 (CXCL1) protein expression is increased in aggressive bladder cancers

    International Nuclear Information System (INIS)

    Miyake, Makito; Lawton, Adrienne; Goodison, Steve; Urquidi, Virginia; Gomes-Giacoia, Evan; Zhang, Ge; Ross, Shanti; Kim, Jeongsoon; Rosser, Charles J

    2013-01-01

    Chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), may regulate tumor epithelial-stromal interactions that facilitate tumor growth and invasion. Studies have linked CXCL1 expression to gastric, colon and skin cancers, but limited studies to date have described CXCL1 protein expression in human bladder cancer (BCa). CXCL1 protein expression was examined in 152 bladder tissue specimens (142 BCa) by immunohistochemical staining. The expression of CXCL1 was scored by assigning a combined score based on the proportion of cells staining and intensity of staining. CXCL1 expression patterns were correlated with clinicopathological features and follow-up data. CXCL1 protein expression was present in cancerous tissues, but was entirely absent in benign tissue. CXCL1 combined immunostaining score was significantly higher in high-grade tumors relative to low-grade tumors (p = 0.012). Similarly, CXCL1 combined immunostaining score was higher in high stage tumors (T2-T4) than in low stage tumors (Ta-T1) (p < 0.0001). An increase in the combined immunostaining score of CXCL1 was also associated with reduced disease-specific survival. To date, this is the largest study describing increased CXCL1 protein expression in more aggressive phenotypes in human BCa. Further studies are warranted to define the role CXCL1 plays in bladder carcinogenesis and progression

  4. Correlation of human epidermal growth factor receptor protein expression and colorectal cancer.

    Science.gov (United States)

    Yang, Wen-Juan; Shen, Xing-Jie; Ma, Xiao-Xia; Tan, Zhi-Gang; Song, Yan; Guo, Yi-Tong; Yuan, Mei

    2015-07-28

    To investigate the correlation between human epidermal growth factor receptor (HER-2) protein expression and colorectal cancer (CRC) using a case-control study and meta-analysis. Tumor tissue specimens from 162 CRC patients were selected for the case group. Fifty cases were randomly selected, and normal CRC tissue at least 10 cm away from the tumor margins of these cases was used to generate the control group. The expression of the HER-2 protein in the 162 CRC tissue samples and the 50 adjacent normal mucosa tissue samples was detected via immunohistochemistry. The experimental data were analyzed using SPSS 18.0 software, and R software version 3.1.0 was utilized for further verification. The expression of HER-2 protein in the 162 CRC tissue samples was significantly higher than in the normal tissue specimens. The data showed that the expression of HER-2 in CRC was related to the Dukes' stage, the depth of invasion and lymph node metastasis. The HER-2-positive patients had lower 3- and 5-year OS rates than the HER-2-negative patients, but there was no significant difference. However, there was a statistically significant difference in the 3- and 5-year disease-free survival (DFS) rates of HER-2-positive and HER-2-negative patients. The results of the meta-analysis showed that the expression of HER-2 in CRC patients was statistically significantly increased over that of healthy people. The 3-year DFS rate in HER-2-positive patients was markedly lower than that in HER-2-negative patients. Down-regulation of HER-2 expression might be a dependable strategy for CRC therapy.

  5. Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

    Science.gov (United States)

    Kubota, Eiji; Williamson, Christopher T; Ye, Ruiqiong; Elegbede, Anifat; Peterson, Lars; Lees-Miller, Susan P; Bebb, D Gwyn

    2014-01-01

    Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption. PMID:24841718

  6. MAGE-C2/CT10 protein expression is an independent predictor of recurrence in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Lotta von Boehmer

    Full Text Available The cancer-testis (CT family of antigens is expressed in a variety of malignant neoplasms. In most cases, no CT antigen is found in normal tissues, except in testis, making them ideal targets for cancer immunotherapy. A comprehensive analysis of CT antigen expression has not yet been reported in prostate cancer. MAGE-C2/CT-10 is a novel CT antigen. The objective of this study was to analyze extent and prognostic significance of MAGE-C2/CT10 protein expression in prostate cancer. 348 prostate carcinomas from consecutive radical prostatectomies, 29 castration-refractory prostate cancer, 46 metastases, and 45 benign hyperplasias were immunohistochemically analyzed for MAGE-C2/CT10 expression using tissue microarrays. Nuclear MAGE-C2/CT10 expression was identified in only 3.3% primary prostate carcinomas. MAGE-C2/CT10 protein expression was significantly more frequent in metastatic (16.3% positivity and castration-resistant prostate cancer (17% positivity; p<0.001. Nuclear MAGE-C2/CT10 expression was identified as predictor of biochemical recurrence after radical prostatectomy (p = 0.015, which was independent of preoperative PSA, Gleason score, tumor stage, and surgical margin status in multivariate analysis (p<0.05. MAGE-C2/CT10 expression in prostate cancer correlates with the degree of malignancy and indicates a higher risk for biochemical recurrence after radical prostatectomy. Further, the results suggest MAGE-C2/CT10 as a potential target for adjuvant and palliative immunotherapy in patients with prostate cancer.

  7. High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

    International Nuclear Information System (INIS)

    Egerod, Frederikke Lihme; Bartels, Annette; Fristrup, Niels; Borre, Michael; Ørntoft, Torben F; Oleksiewicz, Martin B; Brünner, Nils; Dyrskjøt, Lars

    2009-01-01

    Egr-1 (early growth response-1 transcription factor) has been proposed to be involved in invasion and metastasis processes of human bladder cancer, but Egr-1 protein expression levels in human bladder cancer have not been investigated. In the present study we investigated the expression levels of Egr-1 protein in early stages of human bladder cancer and correlated it to later progression. Expression of Egr-1 protein in human bladder cancer was examined by immunohistochemistry, on a tissue microarray constructed from tumors from 289 patients with non-muscle invasive urothelial bladder cancer. The frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling were found to localize at the tumor front in some of the tumor biopsies. The results from this study support a potential involvement of Egr-1 in the progression from non-muscle invasive bladder cancers to muscle invasive bladder cancer

  8. The effect of curcumol on protein expression of JAK2/STAT3 signaling pathway in human ovarian cancer line SKOV3

    Directory of Open Access Journals (Sweden)

    Feng-juan HAN

    2013-12-01

    Full Text Available Objective: To study the effects of curcumol on the protein expression of JAK2 and STAT3 in SKOV3 and to investigate its treatment on molecular mechanism of ovarian cancer. Methods: Choose curcumol of different concentrations to act on human ovarian cancer cell line SKOV3, and extract the corresponding cell protein, and detect the protein expression of JAK2 and STAT3 by western blotting. Results: The protein expression of JAK2 and STAT3 in SKOV3 are significantly inhabited by curcumol, and its strength will enhance with the increase in drug concentration, and it shows in a dose-dependent manner. Conclusion: Curcumol can significantly inhabit the proliferation of SKOV3 cells, and induce apoptosis, and achieve its mechanism by regulating the protein expression of JAK2 and STAT3.

  9. Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

    Science.gov (United States)

    Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

    2012-08-01

    Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

  10. Protein expression of P13K and P53 in prediction of response to radiotherapy in cervical cancer

    International Nuclear Information System (INIS)

    Teja Kisnanto; Devita Tetriana; Iin Kurnia; Sudiono S; Mellova Amir; Budiningsih Siregar; Ramli; Andrijono; Setiawan Soetopo; Irwan; Tjahya Kurjana; Bethy S Hernowo; Maringan DL Tobing

    2015-01-01

    Cervical cancer is a malignant disease that is common in women and is the first order of malignant disease in Indonesia. Radiotherapy is the main treatment on cervical cancer, especially at an advanced stage (IIB-IIB). P13K and P-53 protein plays a role in the regulation of apoptosis (programmed cell death). The purpose of this study was to determine the protein expression of P13K and P-53 in the prediction of response to radiotherapy action in patients with cervical cancer. Microscopic preparations obtained from biopsy tissue cancer (IIB-IIIB) to 20 patients from RSCM and RSHS. The method used is the method of immunohistochemistry using P13K and P-53 protein biomarkers in cervical cancer tissue preparations. P13K protein expression value obtained by the method of immuno reactive Score (IRS). P13K protein positive expression marked in blue on the cell cytoplasm and P-53 protein is characterized by brown or dark colors contained in the cell nucleus. Results showed that IRS value by 10% a negative P13K, P13K IRS weaker by 70%, IRS P13K was at 15%, and the IRS P13K stronger by 5%. While the index positive P-53 was obtained by 75% and negative P-53 index by 5%. Radiotherapy response analysis showed that there were 75% good response and 25% a bad response. The conclusion from this study is the expression of the protein P13K and P-53 for response prediction of radiotherapy IRS P13K values obtained in response to both radiotherapy is higher compared with radiotherapy response is bad, and the P-53 protein is not found differences in response to radiotherapy between positive and negative expressions. (author)

  11. ERG protein expression over time

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Brasso, Klaus; Thomsen, Frederik Birkebæk

    2015-01-01

    AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed by immunohistochem......AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed...

  12. Effect of 17-allylamino-17-demethoxygeldanamycin (17-AAG) on Akt protein expression is more effective in head and neck cancer cell lineages that retain PTEN protein expression.

    Science.gov (United States)

    Pontes, Flávia Sirotheau C; Pontes, Hélder A R; de Souza, Lucas L; de Jesus, Adriana S; Joaquim, Andrea M C; Miyahara, Ligia A N; Fonseca, Felipe P; Pinto Junior, Décio S

    2018-03-01

    The aim of this study was to evaluate the expression of Akt, PTEN, Mdm2 and p53 proteins in three different head and neck squamous cell carcinoma (HNSCC) cell lines (HN6, HN19 and HN30), all of them treated with epidermal growth factor (EGF) and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 protein. Immunofluorescence and western blot were performed in order to analyze the location and quantification, respectively, of proteins under the action 17-AAG and EGF. Treatment with EGF resulted in increased levels of Akt, PTEN and p53 in all cell lineages. The expression of Mdm2 was constant in HN30 and HN6 lineages, while in HN19 showed slightly decreased expression. Under the action 17-AAG, in HN6 and HN19, the expression of PTEN and p53 proteins was suppressed, while Akt and Mdm2 expression was reduced. Finally, in the HN30 cell lineage were absolute absence of expression of Akt, Mdm2 and p53 and decreased expression of PTEN. These data allow us to speculate on the particular utility of 17-AAG for HNSCC treatment through the inhibition of Akt protein expression, especially in the cases that retain the expression of PTEN protein. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. The ErbB signalling pathway : protein expression and prognostic value in epithelial ovarian cancer

    NARCIS (Netherlands)

    de Graeff, P.; Crijns, A.P.; ten Hoor, K.A.; Klip, H.G.; Hollema, H.; Oien, K.; Bartlett, J.M.; Wisman, G.B.; de Bock, G.H.; de Vries, E.G.; de Jong, S.; van der Zee, A.G.

    2008-01-01

    Ovarian cancer is the most frequent cause of death from gynaecological cancer in the Western world. Current prognostic factors do not allow reliable prediction of response to chemotherapy and survival for individual ovarian cancer patients. Epidermal growth factor receptor (EGFR) and HER-2/neu are

  14. Trade-Offs Associated with Photoprotective Green Fluorescent Protein Expression as Potential Drivers of Balancing Selection for Color Polymorphism in Reef Corals

    Directory of Open Access Journals (Sweden)

    Cathryn Quick

    2018-02-01

    Full Text Available Photodamage of symbiotic algae exposed to thermal stress is involved in mass coral bleaching, a major cause of reef decline. Photoprotection is therefore a vital part of coral stress physiology. Corals produce a variety of green fluorescent protein (GFP-like proteins, some of which screen the symbiotic algae from excess sun light. Different tissue concentrations of these GFP-like proteins distinguish color morphs that are characteristic for many coral species. The question arises whether these pigmentation differences may diversify the niches that can be occupied by corals along the steep light gradient that structures coral reef communities. We assessed the implications of GFP-like protein expression in two color morphs of the symbiotic coral Hydnophora grandis, both associated with the same Symbiodinium sp. (subclade C40. The color morphs of this species (high fluorescent, HF; and low fluorescent, LF, characterized by markedly different contents of a cyan fluorescent protein, were exposed to different quantities of blue light (470 nm that matched the major absorption band of the host pigment (473 nm. High intensities of blue light caused less photodamage to the symbiotic algae of the HF morph and resulted in higher growth rates of these corals compared to representatives of the LF morph. In contrast, under low intensities of blue light, the HF morph showed lower growth rates than the LF morph, indicating that trade-offs are associated with high levels of fluorescent protein expression under this condition. Both morphs showed highest growth rates at medium light intensities with no obvious influence of the tissue pigmentation. Reef coral color polymorphism caused by photoprotective GFP-like proteins may therefore be a product of balancing selection in which high pigment contents may be beneficial at the upper and detrimental at the lower end of the depth distribution range of symbiotic corals. Conversely, color morphs with GFP-like proteins

  15. Candidate Genes for Testicular Cancer Evaluated by In Situ Protein Expression Analyses on Tissue Microarrays

    Directory of Open Access Journals (Sweden)

    Rolf I. Skotheim

    2003-09-01

    Full Text Available By the use of high-throughput molecular technologies, the number of genes and proteins potentially relevant to testicular germ cell tumor (TGCT and other diseases will increase rapidly. In a recent transcriptional profiling, we demonstrated the overexpression of GRB7 and JUP in TGCTs, confirmed the reported overexpression of CCND2. We also have recent evidences for frequent genetic alterations of FHIT and epigenetic alterations of MGMT. To evaluate whether the expression of these genes is related to any clinicopathological variables, we constructed a tissue microarray with 510 testicular tissue cores from 279 patients diagnosed with TGCT, covering various histological subgroups and clinical stages. By immunohistochemistry, we found that JUP, GRB7, CCND2 proteins were rarely present in normal testis, but frequently expressed at high levels in TGCT. Additionally, all premalignant intratubular germ cell neoplasias were JUP-immunopositive. MGMT and FHIT were expressed by normal testicular tissues, but at significantly lower frequencies in TGCT. Except for CCND2, the expressions of all markers were significantly associated with various TGCT subtypes. In summary, we have developed a high-throughput tool for the evaluation of TGCT markers, utilized this to validate five candidate genes whose protein expressions were indeed deregulated in TGCT.

  16. Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using Fluorescence in situ Hybridization (FISH) and immunohistochemistry

    International Nuclear Information System (INIS)

    Sato-Kuwabara, Yukie; Neves, José I; Fregnani, José HTG; Sallum, Rubens A; Soares, Fernando A

    2009-01-01

    Esophageal squamous cell carcinoma (ESCC) is the sixth most frequent neoplasia in Brazil. It is usually associated with a poor prognosis because it is often at an advanced stage when diagnosed and there is a high frequency of lymph node metastases. It is important to know what prognostic factors can facilitate diagnosis, optimize therapeutic decisions, and improve the survival of these patients. A member of the epidermal growth factor receptor (EGFR) family, c-erbB-2, has received much attention because of its therapeutic implications; however, few studies involving fluorescence in situ hybridization (FISH) analysis of HER-2/neu gene amplification and protein expression in ESCC have been conducted. The aim of this study was to verify the presence of HER-2/neu gene amplification using FISH, and to correlate the results with immunohistochemical expression and clinical-pathological findings. One hundred and ninety-nine ESCC cases were evaluated using the Tissue Microarray (TMA) technique. A polyclonal antibody against c-erbB-2 was used for immunohistochemistry. Analyses were based on the membrane staining pattern. The results were classified according to the Herceptest criteria (DAKO): negative (0/1+), potential positive (2+) and positive (3+). The FISH reactions were performed according to the FISH HER2 PharmDx (DAKO) protocol. In each case, 100 tumor nuclei were evaluated. Cases showing a gene/CEN17 fluorescence ratio ≥ 2 were considered positive for gene amplification. The c-erbB-2 expression was negative in 117/185 cases (63.2%) and positive in 68 (36.8%), of which 56 (30.3%) were 2+ and 12 (6.5%) were 3+. No significant associations were found among protein expression, clinicopathological data and overall survival. Among the 47 cases analyzed, 38 (80.9%) showed no gene amplification while 9 (19.1%) showed amplification, as demonstrated by FISH. Cases that were negative (0/1+) and potential positive (2+) for c-erbB-2 expression by immunohistochemistry showed no

  17. Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

    Science.gov (United States)

    Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2007-12-01

    A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

  18. Dynamic Change in p63 Protein Expression during Implantation of Urothelial Cancer Clusters

    Directory of Open Access Journals (Sweden)

    Takahiro Yoshida

    2015-07-01

    Full Text Available Although the dissemination of urothelial cancer cells is supposed to be a major cause of the multicentricity of urothelial tumors, the mechanism of implantation has not been well investigated. Here, we found that cancer cell clusters from the urine of patients with urothelial cancer retain the ability to survive, grow, and adhere. By using cell lines and primary cells collected from multiple patients, we demonstrate that △Np63α protein in cancer cell clusters was rapidly decreased through proteasomal degradation when clusters were attached to the matrix, leading to downregulation of E-cadherin and upregulation of N-cadherin. Decreased △Np63α protein level in urothelial cancer cell clusters was involved in the clearance of the urothelium. Our data provide the first evidence that clusters of urothelial cancer cells exhibit dynamic changes in △Np63α expression during attachment to the matrix, and decreased △Np63α protein plays a critical role in the interaction between cancer cell clusters and the urothelium. Thus, because △Np63α might be involved in the process of intraluminal dissemination of urothelial cancer cells, blocking the degradation of △Np63α could be a target of therapy to prevent the dissemination of urothelial cancer.

  19. Green fluorescent protein expression from recombinant lettuce infectious yellows virus-defective RNAs originating from RNA 2.

    Science.gov (United States)

    Yeh, H H; Tian, T; Medina, V; Falk, B W

    2001-10-10

    Lettuce infectious yellows virus (LIYV) RNA 2 defective RNAs (D RNAs) were compared in protoplasts for their ability to replicate and to express the green fluorescent protein (GFP) from recombinant D RNA constructs. Initially four LIYV D RNAs of different genetic composition were compared, but only two (LIYV D RNA M5 and M18) replicated to high levels. Both of these contained at least two complete ORFs, one being the 3'-terminal ORF encoding P26. Northern hybridization analysis using probes corresponding to 3' regions of LIYV RNA 2 detected the P26 subgenomic RNA from protoplasts infected with LIYV RNAs 1 and 2 or protoplasts inoculated only with RNA 1 plus either the LIYV D RNA M5 or M18, suggesting that these LIYV D RNAs served as templates to generate the P26 subgenomic RNA. The GFP coding region was inserted as an in-frame insertion into the P26 coding region of the LIYV M5 and M18 D RNAs, yielding M5gfp and M18gfp. When transcripts of M5gfp and M18gfp were used to inoculate protoplasts, bright fluorescence was seen only when they were co-inoculated with LIYV RNA 1. The percentage of fluorescent protoplasts ranged from experiment to experiment, but was as high as 5.8%. Time course analyses showed that fluorescence was not detected before 48 h pi, and this correlated with the timing of LIYV RNA 2 and RNA 2 D RNA accumulation, but not with that of LIYV RNA 1. Copyright 2001 Academic Press.

  20. Unique protein expression signatures of survival time in kidney renal clear cell carcinoma through a pan-cancer screening.

    Science.gov (United States)

    Han, Guangchun; Zhao, Wei; Song, Xiaofeng; Kwok-Shing Ng, Patrick; Karam, Jose A; Jonasch, Eric; Mills, Gordon B; Zhao, Zhongming; Ding, Zhiyong; Jia, Peilin

    2017-10-03

    In 2016, it is estimated that there will be 62,700 new cases of kidney cancer in the United States, and 14,240 patients will die from the disease. Because the incidence of kidney renal clear cell carcinoma (KIRC), the most common type of kidney cancer, is expected to continue to increase in the US, there is an urgent need to find effective diagnostic biomarkers for KIRC that could help earlier detection of and customized treatment strategies for the disease. Accordingly, in this study we systematically investigated KIRC's prognostic biomarkers for survival using the reverse phase protein array (RPPA) data and the high throughput sequencing data from The Cancer Genome Atlas (TCGA). With comprehensive data available in TCGA, we systematically screened protein expression based survival biomarkers in 10 major cancer types, among which KIRC presented many protein prognostic biomarkers of survival time. This is in agreement with a previous report that expression level changes (mRNAs, microRNA and protein) may have a better performance for prognosis of KIRC. In this study, we also identified 52 prognostic genes for KIRC, many of which are involved in cell-cycle and cancer signaling, as well as 15 tumor-stage-specific prognostic biomarkers. Notably, we found fewer prognostic biomarkers for early-stage than for late-stage KIRC. Four biomarkers (the RPPA protein IDs: FASN, ACC1, Cyclin_B1 and Rad51) were found to be prognostic for survival based on both protein and mRNA expression data. Through pan-cancer screening, we found that many protein biomarkers were prognostic for patients' survival in KIRC. Stage-specific survival biomarkers in KIRC were also identified. Our study indicated that these protein biomarkers might have potential clinical value in terms of predicting survival in KIRC patients and developing individualized treatment strategies. Importantly, we found many biomarkers in KIRC at both the mRNA expression level and the protein expression level. These

  1. A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

    Science.gov (United States)

    Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

    2017-01-01

    Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P < 0.0001). According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

  2. Cell-cycle protein expression in a population-based study of ovarian and endometrial cancers

    Directory of Open Access Journals (Sweden)

    Ashley S. Felix

    2015-02-01

    Full Text Available Aberrant expression of cyclin-dependent kinase (CDK inhibitors is implicated in the carcinogenesis of many cancers, including ovarian and endometrial cancers. We examined associations between CDK inhibitor expression, cancer risk factors, tumor characteristics, and survival outcomes among ovarian and endometrial cancer patients enrolled in a population-based case control study. Expression (negative vs. positive of three CDK inhibitors (p16, p21, p27 and ki67 was examined with immunohistochemical staining of tissue microarrays. Logistic regression was used to estimate adjusted odds ratios (ORs and 95% confidence intervals (CIs for associations between biomarkers, risk factors, and tumor characteristics. Survival outcomes were available for ovarian cancer patients and examined using Kaplan-Meier plots and Cox proportional hazards regression. Among ovarian cancer patients (n=175, positive p21 expression was associated with endometrioid tumors (OR=12.22, 95% CI=1.45-102.78 and higher overall survival (log-rank p=0.002. In Cox models adjusted for stage, grade, and histology, the association between p21 expression and overall survival was borderline significant (hazard ratio=0.65, 95% CI=0.42-1.05. Among endometrial cancer patients (n=289, positive p21 expression was inversely associated with age (OR ≥ 65 years of age=0.25, 95% CI=0.07-0.84 and current smoking status (OR: 0.33, 95% CI 0.15, 0.72 compared to negative expression. Our study showed heterogeneity in expression of cell-cycle proteins associated with risk factors and tumor characteristics of gynecologic cancers. Future studies to assess these markers of etiological classification and behavior may be warranted.

  3. Retinoblastoma protein expression and radiation response in muscle-invasive bladder cancer

    International Nuclear Information System (INIS)

    Pollack, Alan; Czerniak, Bogdan; Zagars, Gunar K.; Hu Shixue; Wu, Catherine S.; Dinney, Colin P.N.; Chyle, Valerian; Benedict, William F.

    1997-01-01

    survival in Stage T3b patients. Patient outcome was best when RB negative and p53 negative staining were seen. Conclusion: Our results indicate that loss of RB function as measured by immunohistochemical staining is the strongest correlate of radiation response thus far recognized. Loss of RB expression also predicted for poor outcome in Stage T3b patients, which appeared to compliment the finding of normal p53 expression. While normal RB protein expression is usually associated with better patient outcome, other series have not examined patients treated with radiotherapy. The absence of pRB may be a useful marker for selecting patients for bladder preservation with radiotherapy, particularly when wild-type p53 is present

  4. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Jakobsen, Anders

    2009-01-01

    : Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian cancer were used for immunohistochemical staining for the ERCC1 protein. All patients received carboplatin-paclitaxel combination chemotherapy. RESULTS: Excision repair cross-complementation group 1 enzyme...

  5. Association of ERCC1 protein expression to platinum resistance in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Jakobsen, Anders

    was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Methods: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian...

  6. Association of p53 protein expression with clinical outcome in advanced supraglottic cancer

    International Nuclear Information System (INIS)

    Kang, Jin Oh; Hong, Seong Eon

    1998-01-01

    To determine the incidence and prognostic effect of p53 expression in patients with advanced supraglottic cancer. Twenty-one cases of total 48 advanced supraglottic cancer patients who received postoperative adjuvant radiation therapy were evaluated by immunohistochemical staining employing p53 monoclonal antibody. Three out of six stage III patients and four out of fifteen stage IV patients showed p53 expression without statistically significant difference (p=0.608). Five year survival rates are 93% in p53 negative, 86% in p53 positive patients and there was no significant difference(p=0.776). p53 expression does not show statistically significant correlation with primary tumor status(p=0.877), lymph node status(p=0.874) and age(p=0.64). There was no statistically significant correlation between traditionally known risk factors and p53 expression

  7. Significance of glycolytic metabolism-related protein expression in colorectal cancer, lymph node and hepatic metastasis

    International Nuclear Information System (INIS)

    Martins, Sandra Fernandes; Amorim, Ricardo; Viana-Pereira, Marta; Pinheiro, Céline; Costa, Ricardo Filipe Alves; Silva, Patrícia; Couto, Carla; Alves, Sara; Fernandes, Sara; Vilaça, Sónia; Falcão, Joaquim; Marques, Herlander; Pardal, Fernando; Rodrigues, Mesquita; Preto, Ana; Reis, Rui Manuel; Longatto-Filho, Adhemar; Baltazar, Fátima

    2016-01-01

    Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide. Most cancer cells display high rates of glycolysis with production of lactic acid, which is then exported to the microenvironment by monocarboxylate transporters (MCTs). The main aim of this study was to evaluate the significance of MCT expression in a comprehensive series of primary CRC cases, lymph node and hepatic metastasis. Expressions of MCT1, MCT4, CD147 and GLUT1 were studied in human samples of CRC, lymph node and hepatic metastasis, by immunohistochemistry. All proteins were overexpressed in primary CRC, lymph node and hepatic metastasis, when compared with non-neoplastic tissue, with exception of MCT1 in lymph node and hepatic metastasis. MCT1 and MCT4 expressions were associated with CD147 and GLUT1 in primary CRC. These markers were associated with clinical pathological features, reflecting the putative role of these metabolism-related proteins in the CRC setting. These findings provide additional evidence for the pivotal role of MCTs in CRC maintenance and progression, and support the use of MCTs as biomarkers and potential therapeutic targets in primary and metastatic CRC

  8. Extracellular Matrix Proteins Expression Profiling in Chemoresistant Variants of the A2780 Ovarian Cancer Cell Line

    Directory of Open Access Journals (Sweden)

    Radosław Januchowski

    2014-01-01

    Full Text Available Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly—over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis.

  9. Coffee induces breast cancer resistance protein expression in Caco-2 cells.

    Science.gov (United States)

    Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

    2011-01-01

    Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates.

  10. Limited prognostic value of tissue protein expression levels of cyclin E in Danish ovarian cancer patients

    DEFF Research Database (Denmark)

    Heeran, Mel C; Høgdall, Claus K; Kjaer, Susanne K

    2012-01-01

    The primary objective of this study was to assess the expression of cyclin E in tumour tissues from 661 patients with epithelial ovarian tumours. The second was to evaluate whether cyclin E tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using...... tissue arrays (TA), we analysed the cyclin E expression levels in tissues from 168 women with borderline ovarian tumours (BOT) (147 stage I, 4 stage II, 17 stage III) and 493 Ovarian cancer (OC) patients (127 stage I, 45 stage II, 276 stage III, 45 stage IV). Using a 10% cut-off level for cyclin E......-off value showed that cyclin E had no independent prognostic value. In conclusion, we found cyclin E expression in tumour tissue to be of limited prognostic value to Danish OC patients....

  11. CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

    DEFF Research Database (Denmark)

    Caldeira, José Roberto F; Prando, Erika C; Quevedo, Francisco C

    2006-01-01

    prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression. METHODS: The aim of our study was to assess...... not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association......BACKGROUND: The E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor...

  12. Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

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    Brendan T. McKeown

    2015-01-01

    Full Text Available Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer. Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro. Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase, PI3K (phosphatidylinositol 3-kinase, NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells and AP-1 (activator protein 1 cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased. Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.

  13. Low ERCC1 mRNA and protein expression are associated with worse survival in cervical cancer patients treated with radiation alone

    International Nuclear Information System (INIS)

    Doll, Corinne M.; Prystajecky, Michael; Eliasziw, Misha; Klimowicz, Alexander C.; Petrillo, Stephanie K.; Craighead, Peter S.; Hao, Desiree; Diaz, Roman; Lees-Miller, Susan P.; Magliocco, Anthony M.

    2010-01-01

    Purpose: To evaluate the association of excision repair cross-complementation group 1 (ERCC1) expression, using both mRNA and protein expression analysis, with clinical outcome in cervical cancer patients treated with radical radiation therapy (RT). Experimental design: Patients (n = 186) with locally advanced cervical cancer, treated with radical RT alone from a single institution were evaluated. Pre-treatment FFPE biopsy specimens were retrieved from 112 patients. ERCC1 mRNA level was determined by real-time PCR, and ERCC1 protein expression (FL297, 8F1) was measured using quantitative immunohistochemistry (AQUA (registered) ). The association of ERCC1 status with local response, 10-year disease-free (DFS) and overall survival (OS) was analyzed. Results: ERCC1 protein expression levels using both FL297 and 8F1 antibodies were determined for 112 patients; mRNA analysis was additionally performed in 32 patients. Clinical and outcome factors were comparable between the training and validation sets. Low ERCC1 mRNA expression status was associated with worse OS (17.9% vs 50.1%, p = 0.046). ERCC1 protein expression using the FL297 antibody, but not the 8F1 antibody, was significantly associated with both OS (p = 0.002) and DFS (p = 0.010). After adjusting for pre-treatment hemoglobin in a multivariate analysis, ERCC1 FL297 expression status remained statistically significant for OS [HR 1.9 (1.1-3.3), p = 0.031]. Conclusions: Pre-treatment tumoral ERCC1 mRNA and protein expression, using the FL297 antibody, are predictive factors for survival in cervical cancer patients treated with RT, with ERCC1 FL297 expression independently associated with survival. These results identify a subset of patients who may derive the greatest benefit from the addition of cisplatin chemotherapy.

  14. FLUORESCENCE DIAGNOSIS FOR RECURRENT BLADDER CANCER

    Directory of Open Access Journals (Sweden)

    R. V. Ulyanov

    2017-01-01

    Full Text Available The clinical case of successful use of local fluorescence spectroscopy combined with fluorescence imaging during cystoscopy for diagnosis of recurrent bladder cancer is represented in the article. Histological study of fluorescent foci confirmed tumor growth (urothelial carcinoma in all areas with high levels of diagnostic parameter. In the fluorescent focus with low diagnostic parameter inflammation was detected.

  15. Effect of aqueous media on the copper-ion-mediated phototoxicity of CuO nanoparticles toward green fluorescent protein-expressing Escherichia coli.

    Science.gov (United States)

    Shang, Enxiang; Li, Yang; Niu, Junfeng; Guo, Huiyuan; Zhou, Yijing; Liu, Han; Zhang, Xinqi

    2015-12-01

    Quantitative comparison of different aqueous media on the phototoxicity of copper oxide nanoparticles (CuO NPs) is crucial for understanding their ecological effects. In this study, the phototoxicity of CuO NPs toward the green fluorescent protein-expressing Escherichia coli (GFP-E. coli) under UV irradiation (365 nm) was investigated in Luria-Bertani medium (LB), NaCl solution, deionized water (DI) and phosphate-buffered saline (PBS). The phototoxicity of CuO NPs toward GFP-E. coli decreased in the order of DI>NaCl>PBS>LB because of different released concentrations of Cu(2+). The 3h released Cu(2+) concentrations by 10mg/L CuO NPs in DI water, NaCl solution, LB medium, and PBS were 1946.3 ± 75.6, 1242.5 ± 47.6, 1023.4 ± 41.2, and 1162.1 ± 41.9 μg/L, respectively. Transmission electron microscope and laser scanning confocal microscope images of E. coli exposed to CuO NPs demonstrated that the released Cu(2+) resulted in fragmentation of bacterial cell walls, leakage of intracellular components, and finally death of bacteria in four media after UV light irradiation. In each medium, the bacterial mortality rate logarithmically increased with the releasing concentrations of Cu(2+) by CuO NPs (R(2)>0.90) exposed to 3h UV light. This study highlights the importance of taking into consideration of water chemistry when the phototoxicity of CuO NPs is assessed in nanotoxicity research. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Angiogenesis-related protein expression in bevacizumab-treated metastatic colorectal cancer: NOTCH1 detrimental to overall survival

    International Nuclear Information System (INIS)

    Paiva, Tadeu Ferreira Jr.; Jesus, Victor Hugo Fonseca de; Marques, Raul Amorim; Costa, Alexandre André Balieiro Anastácio da; Macedo, Mariana Petaccia de; Peresi, Patricia Maria; Damascena, Aline; Rossi, Benedito Mauro; Begnami, Maria Dirlei; Lima, Vladmir Cláudio Cordeiro de

    2015-01-01

    The development of targeted therapies has undoubtedly broadened therapeutic options for patients with colorectal cancer (CRC). The use of bevacizumab to reduce angiogenesis has been associated with improved clinical outcomes. However, an urgent need for prognostic/predictive biomarkers for anti-angiogenic therapies still exists. Clinical data of 105 CRC patients treated with bevacizumab in conjunction with chemotherapy were analyzed. The expression of vascular endothelial growth factor (VEGF) receptors, NOTCH1 receptor and its ligand DLL4 were determined by immunohistochemistry. Tumor samples were arranged on a tissue microarray. The association between protein expression and clinicopathological characteristics and outcomes was determined. Bevacizumab was administered as a first-line of treatment in 70.5 % of our cases. The median progression-free survival (PFS) was 10.2 months. The median overall survival (OS) of the total cohort was 24.4 months. Bevacizumab, as the first-line of treatment, and the presence of liver metastasis were independently associated with objective response rate. Membrane VEGFR1 and VEGFR3 expressions were associated with the presence of lung metastasis; interestingly, VEGFR3 was associated with less liver metastasis. NOTCH1 expression was associated with lymph node metastasis. There was a trend toward association between improved PFS and lower NOTCH1 expression (p = 0.06). Improved OS was significantly associated with lower NOTCH1 expression (p = 0.01). In a multivariate analysis, ECOG (Eastern Cooperative Oncology Group) performance status, liver metastasis, histological grade, and NOTCH1 expression were independently associated with OS. Our findings illustrated the expression profile of angiogenesis-related proteins and their association with clinicopathological characteristics and outcomes. NOTCH1 expression is a detrimental prognostic factor in metastatic CRC patients treated with chemotherapy plus bevacizumab. The online version of

  17. Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

    International Nuclear Information System (INIS)

    Liao, Christopher CL; Ward, Nicholas; Marsh, Simon; Arulampalam, Tan; Norton, John D

    2010-01-01

    /23 cases (P = < 0.001; ROC error, 0.105). A similar analysis of normal mucosa spectra correctly predicted 11/14 patients with, and 15/19 patients without lymph node involvement (P = 0.001; ROC error, 0.212). Protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value in studies aimed at improved molecular classification of this disease. Further studies, with longer follow-up times and larger patient cohorts, that would permit independent validation of supervised classification models, would be required to confirm the predictive value of tumour spectra for disease recurrence/patient survival

  18. Mass spectrometry protein expression profiles in colorectal cancer tissue associated with clinico-pathological features of disease

    Directory of Open Access Journals (Sweden)

    Liao Christopher CL

    2010-08-01

    Full Text Available Abstract Background Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS. Methods Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the 'GenePattern' suite of computational tools (Broad Institute, MIT, USA. Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR test statistic was used to identify and rank differentially expressed marker peaks. The k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, 'leave-one-out' cross-validation method. Results 73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour verses adjacent normal mucosa tissue (P ≤ 0.01, false discovery rate ≤ 0.05. Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100% sensitivity and specificity (95% confidence interval: 67.9-99.2%. Supervised prediction using 'leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours (P = P = P = 0.001; ROC error, 0.212. Conclusions Protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value in studies aimed at improved molecular

  19. Gene promoter methylation and protein expression of BRMS1 in uterine cervix in relation to high-risk human papilloma virus infection and cancer.

    Science.gov (United States)

    Panagopoulou, Maria; Lambropoulou, Maria; Balgkouranidou, Ioanna; Nena, Evangelia; Karaglani, Makrina; Nicolaidou, Christina; Asimaki, Anthi; Konstantinidis, Theocharis; Constantinidis, Theodoros C; Kolios, George; Kakolyris, Stylianos; Agorastos, Theodoros; Chatzaki, Ekaterini

    2017-04-01

    Cervical cancer is strongly related to certain high-risk types of human papilloma virus infection. Breast cancer metastasis suppressor 1 (BRMS1) is a tumor suppressor gene, its expression being regulated by DNA promoter methylation in several types of cancers. This study aims to evaluate the methylation status of BRMS1 promoter in relation to high-risk types of human papilloma virus infection and the development of pre-cancerous lesions and describe the pattern of BRMS1 protein expression in normal, high-risk types of human papilloma virus-infected pre-cancerous and malignant cervical epithelium. We compared the methylation status of BRMS1 in cervical smears of 64 women with no infection by high-risk types of human papilloma virus to 70 women with proven high-risk types of human papilloma virus infection, using real-time methylation-specific polymerase chain reaction. The expression of BRMS1 protein was described by immunohistochemistry in biopsies from cervical cancer, pre-cancerous lesions, and normal cervices. Methylation of BRMS1 promoter was detected in 37.5% of women with no high-risk types of human papilloma virus infection and was less frequent in smears with high-risk types of human papilloma virus (11.4%) and in women with pathological histology (cervical intraepithelial neoplasia) (11.9%). Methylation was detected also in HeLa cervical cancer cells. Immunohistochemistry revealed nuclear BRMS1 protein staining in normal high-risk types of human papilloma virus-free cervix, in cervical intraepithelial neoplasias, and in malignant tissues, where staining was occasionally also cytoplasmic. In cancer, expression was stronger in the more differentiated cancer blasts. In conclusion, BRMS1 promoter methylation and aberrant protein expression seem to be related to high-risk types of human papilloma virus-induced carcinogenesis in uterine cervix and is worthy of further investigation.

  20. Protein expression levels of carcinoembryonic antigen (CEA) in Danish ovarian cancer patients: from the Danish 'MALOVA'ovarian cancer study

    DEFF Research Database (Denmark)

    Hogdall, E.V.; Christensen, L.; Blaakaer, J.

    2008-01-01

    from 189 women diagnosed with low malignant potential ovarian tumours (LMP, borderline ovarian tumours) and 571 women diagnosed with ovarian cancer (OC). RESULTS: Using 30% as the cut-off level for CEA over-expression, 18% of LMPs and 4% of OCs were positive. A higher proportion of mucinous tumours...... (I to IV), the highest CEA expression compared with no expression was found to be a prognostic factor (level 3 versus negative: HR = 2.12, 95%CI 1.11-4.05). FIGO stage, residual tumour after primary surgery, age at diagnosis, other histological types versus serous adenocarcinoma and low versus high...

  1. PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

    Science.gov (United States)

    Diaz-Cueto, Laura; Arechavaleta-Velasco, Fabian; Diaz-Arizaga, Adriana; Dominguez-Lopez, Pablo; Robles-Flores, Martha

    2012-07-01

    Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

  2. Fluorescence lifetime imaging of skin cancer

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

    2011-03-01

    Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

  3. Analysis of post-operative changes in serum protein expression profiles from colorectal cancer patients by MALDI-TOF mass spectrometry: a pilot methodological study

    Directory of Open Access Journals (Sweden)

    Marsh Simon

    2010-04-01

    Full Text Available Abstract Background Mass spectrometry-based protein expression profiling of blood sera can be used to discriminate colorectal cancer (CRC patients from unaffected individuals. In a pilot methodological study, we have evaluated the changes in protein expression profiles of sera from CRC patients that occur following surgery to establish the potential of this approach for monitoring post-surgical response and possible early prediction of disease recurrence. Methods In this initial pilot study, serum specimens from 11 cancer patients taken immediately prior to surgery and at approximately 6 weeks following surgery were analysed alongside 10 normal control sera by matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS. Using a two-sided t-test the top 20 ranked protein peaks that discriminate normal from pre-operative sera were identified. These were used to classify post-operative sera by hierarchical clustering analysis (Spearman's Rank correlation and, as an independent 'test' dataset, by k-nearest neighbour and weighted voting supervised learning algorithms. Results Hierarchical cluster analysis classified post-operative sera from all six early Dukes' stage (A and B patients as normal. The remaining five post-operative sera from more advanced Dukes' stages (C1 and C2 were classified as cancer. Analysis by supervised learning algorithms similarly grouped all advanced Dukes' stages as cancer, with four of the six post-operative sera from early Dukes' stages being classified as normal (P = 0.045; Fisher's exact test. Conclusions The results of this pilot methodological study illustrate the proof-of-concept of using protein expression profiling of post-surgical blood sera from individual patients to monitor disease course. Further validation on a larger patient cohort and using an independent post-operative sera dataset would be required to evaluate the potential clinical relevance of this approach. Prospective

  4. c-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

    Science.gov (United States)

    La Rosa, Stefano; Bernasconi, Barbara; Vanoli, Alessandro; Sciarra, Amedeo; Notohara, Kenji; Albarello, Luca; Casnedi, Selenia; Billo, Paola; Zhang, Lizhi; Tibiletti, Maria Grazia; Sessa, Fausto

    2018-05-02

    The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

  5. Effect of X-irradiation on the protein expression of P57kip2 and TGF-β1 in lung cancer cell stain A549

    International Nuclear Information System (INIS)

    Zou Huawei; Tan Yonggang; Zhang Heying

    2008-01-01

    Objective: To analyze the effect of X-irradiation on the proteins expression of p57 kip2 and TGF-β1 in lung cancer cell stain A549 and its clinical significance. Methods: Lung cancer cell stain A549 was cultivated and cell, protein was extracted at 6,12,24,36 and 48 hours after X-irradiation by different doses(2,4, 8 and 12 Gy). The expression of p57 kip2 and TGF-β1 proteins were examined by Western blot. Results: The expression of p57 kip2 in lung cancer cell stain A549 was very low before X-irradiation, and increased significantly after irradiation with different doses and reached the peak level at 12 hours after irradiation (P kip2 and TGF-β1 proteins which increased with certain doses, p57 kip2 and TGF-β1 could be used to predict the damage degree of cancer cells by X-ray. (authors)

  6. High frequency of tumor cells with nuclear Egr-1 protein expression in human bladder cancer is associated with disease progression

    DEFF Research Database (Denmark)

    Egerod, Frederikke N S Lihme; Bartels, Annette; Fristrup, Niels

    2009-01-01

    bladder cancer. RESULTS: The frequency of tumor cells with nuclear Egr-1 immunolabelling correlated to bladder cancer stage, grade and to later progression to muscle-invasive bladder cancer (T2-4). Stage T1 tumors exhibited significantly higher frequencies of tumor cells with nuclear Egr-1 immunolabelling...... than Ta tumors (P = 0.001). Furthermore, Kaplan-Meier survival analysis showed that a high frequency of tumor cells with nuclear Egr-1 immunolabelling was significantly associated with a higher risk of progression to stage T2-4 (log-rank test, P = 0.035). Tumor cells with nuclear Egr-1 immunolabelling...

  7. Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.

    Directory of Open Access Journals (Sweden)

    Chul Hee Choi

    Full Text Available The recent introduction of "oxygen-independent" flavin mononucleotide (FMN-based fluorescent proteins (FbFPs is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP- to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms.

  8. Genetic transformation of an obligate anaerobe, P. gingivalis for FMN-green fluorescent protein expression in studying host-microbe interaction.

    Science.gov (United States)

    Choi, Chul Hee; DeGuzman, Jefferson V; Lamont, Richard J; Yilmaz, Özlem

    2011-04-15

    The recent introduction of "oxygen-independent" flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP) revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs) were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP-) to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms.

  9. PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer

    International Nuclear Information System (INIS)

    Knostman, Katherine AB; McCubrey, James A; Morrison, Carl D; Zhang, Zhaoxia; Capen, Charles C; Jhiang, Sissy M

    2007-01-01

    The sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer. NIS expression, subcellular localization, and function were analyzed in MCF-7 human breast cancer cells and MCF-7 cells stably or transiently expressing PI3K p110alpha subunit using Western blot of whole cell lysate, cell surface biotinylation Western blot and immunofluorescence, and radioiodide uptake assay, respectively. NIS localization was determined in a human breast cancer tissue microarray using immunohistochemical staining (IHC) and was correlated with pre-existing pAkt IHC data. Statistical analysis consisted of Student's t-test (in vitro studies) or Fisher's Exact Test (in vivo correlational studies). In this study, we demonstrate that PI3K activation in MCF-7 human mammary carcinoma cells leads to expression of underglycosylated NIS lacking cell surface trafficking necessary for iodide uptake ability. PI3K activation also appears to interfere with cell surface trafficking of exogenous NIS as well as all-trans retinoic acid-induced endogenous NIS. A correlation between NIS expression and upregulation of PI3K signaling was found in a human breast cancer tissue microarray. Thus, the PI3K pathway likely plays a major role in the discordance between NIS expression and iodide uptake in breast cancer patients. Further study is warranted to realize the application of NIS-mediated radioiodide ablation in breast cancer

  10. Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

    Directory of Open Access Journals (Sweden)

    Verena Natalie Schreier

    Full Text Available Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac-His-Asp-Trp-Lys(Da  = Aoa-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl. This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

  11. Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Rasmussen, Anders Aamann; Byriel, Lene

    2013-01-01

    In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim...... of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management....

  12. Retinoblastoma protein expression is an independent predictor of both radiation response and survival in muscle invasive bladder cancer

    DEFF Research Database (Denmark)

    Agerbæk, Mads; Alsner, Jan; Marcussen, Niels

    2003-01-01

    The objective of the study was to investigate the predictive value of various clinical, biochemical, and histopathological parameters, with special emphasis on the expression of the retinoblastoma protein (pRB), on the radiation response in bladder cancer. In order to obtain a truly objective...

  13. Thymidylate synthase protein expression levels remain stable during paclitaxel and carboplatin treatment in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Jakobsen, Jan Nyrop; Santoni-Rugiu, Eric; Sørensen, Jens Benn

    2014-01-01

    BACKGROUND: Thymidylate synthase (TS) is a potential predictive marker for efficacy of treatment with pemetrexed. The current study aimed at investigating whether TS expression changes during non-pemetrexed chemotherapy of non-small cell lung cancer (NSCLC), thus making rebiopsy necessary for dec...

  14. c-erbB2 and topoisomerase IIα protein expression independently predict poor survival in primary human breast cancer: a retrospective study

    International Nuclear Information System (INIS)

    Fritz, Peter; Cabrera, Cristina M; Dippon, Jürgen; Gerteis, Andreas; Simon, Wolfgang; Aulitzky, Walter E; Kuip, Heiko van der

    2005-01-01

    c-erbB2 (also known as HER-2/neu) and topoisomerase IIα are frequently overexpressed in breast cancer. The aim of the study was to analyze retrospectively whether the expression of c-erbB2 and topoisomerase IIα protein influences the long-term outcome of patients with primary breast cancer. In this study c-erbB2 and topoisomerase IIα protein were evaluated by immunohistochemistry in formalin-fixed paraffin-embedded tissue from 225 samples of primary breast cancer, obtained between 1986 and 1998. The prognostic value of these markers was analyzed. Of 225 primary breast tumor samples, 78 (34.7%) showed overexpression of either c-erbB2 (9.8%) or topoisomerase IIα protein (24.9%), whereas in 21 tumors (9.3%) both proteins were found to be overexpressed. Patients lacking both c-erbB2 and topoisomerase IIα overexpression had the best long-term survival. Overexpression of either c-erbB2 or topoisomerase IIα was associated with shortened survival, whereas patients overexpressing both c-erbB2 and topoisomerase IIα showed the worst disease outcome (P < 0.0001). Treatment with anthracyclines was not capable of reversing the negative prognostic impact of topoisomerase IIα or c-erbB2 overexpression. The results of this exploratory study suggest that protein expression of c-erbB2 and topoisomerase IIα in primary breast cancer tissues are independent prognostic factors and are not exclusively predictive factors for anthracycline response in patients with primary breast cancer

  15. Genetic Transformation of an Obligate Anaerobe, P. gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe Interaction

    OpenAIRE

    Choi, Chul Hee; DeGuzman, Jefferson V.; Lamont, Richard J.; Yilmaz, Özlem

    2011-01-01

    The recent introduction of "oxygen-independent" flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial pr...

  16. GRP78 Protein Expression as Prognostic Values in Neoadjuvant Chemoradiotherapy and Laparoscopic Surgery for Locally Advanced Rectal Cancer.

    Science.gov (United States)

    Lee, Hee Yeon; Jung, Ji-Han; Cho, Hyun-Min; Kim, Sung Hwan; Lee, Kang-Moon; Kim, Hyung-Jin; Lee, Jong Hoon; Shim, Byoung Yong

    2015-10-01

    We investigated the relationships between biomarkers related to endoplasmic reticulum stress proteins (glucose-regulated protein of molecular mass 78 [GRP78] and Cripto-1 [teratocarcinoma-derived growth factor 1 protein]), pathologic response, and prognosis in locally advanced rectal cancer. All clinical stage II and III rectal cancer patients received 50.4 Gy over 5.5 weeks, plus 5-fluorouracil (400 mg/m(2)/day) and leucovorin (20 mg/m(2)/day) bolus on days 1 to 5 and 29 to 33, and surgery was performed at 7 to 10 weeks after completion of all therapies. Expression of GRP78 and Cripto-1 proteins was determined by immunohistochemistry and was assessed in 101 patients with rectal cancer treated with neoadjuvant chemoradiotherapy (CRT). High expression of GRP78 and Cripto-1 proteins was observed in 86 patients (85.1%) and 49 patients (48.5%), respectively. Low expression of GRP78 protein was associated with a significantly high rate of down staging (80.0% vs. 52.3%, respectively; p=0.046) and a significantly low rate of recurrence (0% vs. 33.7%, respectively; p=0.008) compared with high expression of GRP78 protein. Mean recurrence-free survival according to GRP78 expression could not be estimated because the low expression group did not develop recurrence events but showed a significant correlation with time to recurrence, based on the log rank method (p=0.007). GRP78 also showed correlation with overall survival, based on the log rank method (p=0.045). GRP78 expression is a predictive and prognostic factor for down staging, recurrence, and survival in rectal cancer patients treated with 5-fluorouracil and leucovorin neoadjuvant CRT.

  17. Protein expression profile and prevalence pattern of the molecular classes of breast cancer - a Saudi population based study

    International Nuclear Information System (INIS)

    Al Tamimi, Dalal M; Shawarby, Mohamed A; Ahmed, Ayesha; Hassan, Ammar K; AlOdaini, Amal A

    2010-01-01

    Breast cancer is not a single entity but a diverse group of entities. Advances in gene expression profiling and immunohistochemistry as its surrogate marker have led to the unmasking of new breast cancer molecular subtypes, resulting in the emergence of more elaborate classification systems that are therapeutically and prognostically more predictive. Molecular class distribution across various ethnic groups may also reveal variations that can lead to different clinical outcomes in different populations. We aimed to analyze the spectrum of molecular subtypes present in the Saudi population. ER, PR, HER2, EGFR and CK5/6 were used as surrogate markers for gene expression profiling to classify 231 breast cancer specimens. Correlation of each molecular class with Ki-67 proliferation index, p53 mutation status, histologic type and grade of the tumor was also carried out. Out of 231 cases 9 (3.9%) were classified as luminal A (strong ER +ve, PR +ve or -ve), 37 (16%) as luminal B (weak to moderate ER +ve, and/or PR +ve), 40 (17.3%) as HER2+ (strong or moderately positive HER 2 with confirmation by silver enhanced in-situ hybridization) and 23 (10%) as basal (CK5/6 or EGFR +ve). Co-positivity of different markers in varied patterns was seen in 23 (10%) of cases which were grouped into a hybrid category comprising luminal B-HER2, HER2-basal and luminal-basal hybrids. Ninety nine (42.8%) of the tumors were negative for all five immunohistochemical markers and were labelled as unclassified (penta negative). A high Ki-67 proliferation index was seen in basal (p = 0.007) followed by HER2+ class. Overexpression of p53 was predominantly seen in HER2 + (p = 0.001) followed by the basal group of tumors. A strong correlation was noted between invasive lobular carcinoma and hormone receptor expression with 8 out of 9 lobular carcinoma cases (88.9%) classifiable as luminal cancers. Otherwise, there was no association between the molecular class and the histologic type or grade of the

  18. Merkel cell polyomavirus detection in Merkel cell cancer tumors in Northern Germany using PCR and protein expression.

    Science.gov (United States)

    Leitz, Miriam; Stieler, Kristin; Grundhoff, Adam; Moll, Ingrid; Brandner, Johanna M; Fischer, Nicole

    2014-10-01

    Merkel cell carcinoma is a highly malignant skin cancer which predominantly occurs in elderly and immunocompromised persons. The identification of the Merkel cell polyomavirus (MCPyV) has inaugurated a new understanding of Merkel cell carcinoma pathogenesis. The frequent detection of the virus in Merkel cell carcinoma tissue (70-90%), its monoclonal integration in the tumor cells and the expression of viral oncogenes highly suggest that MCPyV is causally linked to the pathogenesis of the majority of Merkel cell cancer (MCC) cases. Using qualitative and quantitative PCR together with immunohistochemical staining this study aimed at characterizing the presence of MCPyV sequences and viral early gene expression in a cohort of MCC cases (n = 32) selected in Northern Germany. 40-57% of the cases were identified as MCPyV positive with 40.6% of the cases positive by immunohistochemical staining and 51.6-57.6% positive by PCR. Interestingly, in the majority (64%) of LT-Antigen positive tumors only 25-50% of tumor cells express LT-Antigen. These data are in accord with published studies describing heterogeneity in MCPyV viral loads and suggest that detection of MCPyV in Merkel cell carcinoma by PCR should be undertaken using multiple primer pairs. © 2013 Wiley Periodicals, Inc.

  19. Technetium-99m-hexakis-2-methoxyisobutylisonitrile scintigraphy and multidrug resistance-related protein expression in human primary lung cancer

    International Nuclear Information System (INIS)

    Duan Xiaoyi; Wang Jiansheng; Liu Min; Guo Youmin

    2008-01-01

    The occurrence of multidrug resistance (MDR) is a major cause of resistance to chemotherapeutic agents in patients with lung cancer, in part owing to the overexpression of MDR-related proteins. Technetium-99m-hexakis-2-methoxyisobutylisonitrile ( 99m Tc-MIBI) has been shown to be a substrate for some MDR-related proteins. The aim of this study is to evaluate the role of 99m Tc-MIBI scintigraphy for functional imaging of MDR-related protein phenotypes. To determine the correlation between 99m Tc-MIBI scintigraphy and the expression level of P-glycoprotein (Pgp), multidrug-resistance protein (MRP), and glutathione-S-transferase Pi (GSTπ), 26 patients (17 men and 9 women, median age 57.5 years) with primary lung cancer were investigated. Following intravenous administration of 925 MBq 99m Tc-MIBI, single-photon emission computed tomography (SPECT) and computed tomography (CT) were performed at 15 min and 2 h. On the basis of the fused images, tumor to background (T/B) ratio of both early and delayed images, and washout rate (WR%) of 99m Tc-MIBI were calculated. The immunohistochemical staining of Pgp, MRP, and GSTπ was performed, and the expression level was semiquantitated using a pathoimage analysis system. The imaging results were compared with the status of Pgp, MRP, and GSTπ expression. The WR% of 99m Tc-MIBI showed a significant positive correlation with Pgp expression (r=0.560, P=0.003), as no correlation was observed between WR% and MRP or GSTπ (r=0.354, P=0.076; r=0.324, P=0.106). Neither early T/B nor delayed T/B correlated with the expression level of Pgp, MRP, and GSTπ. WR%, Pgp, and GSTπ expression showed significant differences between squamous cell carcinoma (group A) and adenocarcinoma (group B). There was no significant difference among Pgp, MRP, and GSTπ expression levels in any cases (P>0.05). Our data confirmed that 99m Tc-MIBI scintigraphy is useful for determining the MDR caused by Pgp in patients with primary lung cancer. (author)

  20. In vivo monitoring of intranuclear p27{sup kip1} protein expression in breast cancer cells during trastuzumab (Herceptin) therapy

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, Bart [Division of Nuclear Medicine, University Health Network, Toronto, ON, Canada M5S 3E2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); MRC/CRUK Gray Institute for Radiation Oncology and Biology, Oxford University, OX3 7LJ Oxford (United Kingdom)], E-mail: bart.cornelissen@rob.ox.ac.uk; Kersemans, Veerle; McLarty, Kristin [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3E2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Tran, Lara [Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Vallis, Katherine A. [MRC/CRUK Gray Institute for Radiation Oncology and Biology, Oxford University, OX3 7LJ Oxford (United Kingdom); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3E2 (Canada); Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3E2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada)

    2009-10-15

    Introduction: Trastuzumab, a humanized antibody directed against the Her2 receptor, induces the expression of p27{sup kip1}, an intranuclear cyclin-dependent kinase inhibitor in some breast cancer cells. The aim of this study was to develop a radioimmunoconjugate (RIC) to monitor trastuzumab-induced p27{sup kip1} protein up-regulation in vivo. Materials and Methods: Anti-p27{sup kip1} IgG was purified, and conjugated to diethylenetriaminopentaacetate, to allow radiolabeling with {sup 111}In for in vivo detection. Then tat peptide (GRKKRRQRRRPPQGYG), containing a nuclear localization sequence (underlined), was conjugated to the Fc-domain of IgG, using NaIO{sub 4} oxidation of carbohydrates and the resulting Schiff base stabilized with NaCNBH{sub 3}. The conjugate was radiolabeled with {sup 111}In, yielding [{sup 111}In]-anti-p27{sup kip1}-tat. {sup 111}In labeling efficiency, purity and p27{sup kip1} binding were measured. Trastuzumab-induced p27{sup kip1} up-regulation was assessed in a panel of breast cancer cell lines by Western blot analysis. Uptake and retention of [{sup 111}In]-anti-p27{sup kip1}-tat were measured in MDA-MB-361 and SKBr3 cells after exposure to trastuzumab. Uptake of [{sup 111}In]-anti-p27{sup kip1}-tat was determined at 72 h postintravenous injection in MDA-MB-361 xenografts in athymic mice treated with trastuzumab or saline. Results: [{sup 111}In]-anti-p27{sup kip1}-tat was synthesized to 97% purity. The RIC was able to bind to p27{sup kip1} protein and internalized in the cells and was transported to the nuclei of MDA-MB-361 cells. The level of p27{sup kip1} protein in MDA-MB-361 cells was increased after exposure to clinically relevant doses of trastuzumab for 3 days. Trastuzumab-mediated induction of p27{sup kip1} was not associated with increased cellular uptake or nuclear localization of [{sup 111}In]-anti-p27{sup kip1}-tat (6.53{+-}0.61% vs. 6.98{+-}1.36% internalized into trastuzumab-treated vs. control cells, respectively). However

  1. Digital PCR assessment of MGMT promoter methylation coupled with reduced protein expression optimises prediction of response to alkylating agents in metastatic colorectal cancer patients.

    Science.gov (United States)

    Sartore-Bianchi, Andrea; Pietrantonio, Filippo; Amatu, Alessio; Milione, Massimo; Cassingena, Andrea; Ghezzi, Silvia; Caporale, Marta; Berenato, Rosa; Falcomatà, Chiara; Pellegrinelli, Alessio; Bardelli, Alberto; Nichelatti, Michele; Tosi, Federica; De Braud, Filippo; Di Nicolantonio, Federica; Barault, Ludovic; Siena, Salvatore

    2017-01-01

    O(6)-methylguanine-DNA-methyltransferase (MGMT) is a repair protein, and its deficiency makes tumours more susceptible to the cytotoxic effect of alkylating agents. Five clinical trials with temozolomide or dacarbazine have been performed in metastatic colorectal cancer (mCRC) with selection based on methyl-specific PCR (MSP) testing with modest results. We hypothesised that mitigated results are consequences of unspecific patient selection and that alternative methodologies for MGMT testing such as immunohistochemistry (IHC) and digital polymerase chain reaction (PCR) could enhance patient enrolment. Formalin-fixed paraffin embedded archival tumour tissue samples from four phase II studies of temozolomide or dacarbazine in MGMT MSP-positive mCRCs were analysed by IHC for MGMT protein expression and by methyl-BEAMing (MB) for percentage of promoter methylation. Pooled data were then retrospectively analysed according to objective response rate, progression-free survival (PFS) and overall survival (OS). One hundred and five patients were included in the study. Twelve had achieved partial response (PR) (11.4%), 24 stable disease (SD; 22.9%) and 69 progressive disease (PD; 65.7%). Patients with PR/SD had lower IHC scores and higher MB levels than those with PD. MGMT expression by IHC was negatively and MB levels positively associated with PFS (p alkylating agents. Their combination could enhance patient selection in this setting. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Quantitative fluorescence nanoscopy for cancer biomedicine

    Science.gov (United States)

    Huang, Tao; Nickerson, Andrew; Peters, Alec; Nan, Xiaolin

    2015-08-01

    Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.

  3. Immunohistochemistry of connexin 43 throughout anterior pituitary gland in a transgenic rat with green fluorescent protein-expressing folliculo-stellate cells.

    Science.gov (United States)

    Horiguchi, Kotaro; Fujiwara, Ken; Kouki, Tom; Kikuchi, Motoshi; Yashiro, Takashi

    2008-12-01

    Folliculo-stellate (FS) cells in the anterior pituitary gland have been speculated to possess multifunctional properties. Because gap junctions (GJ) have been identified between FS cells, FS cells may be interconnected electrophysiologically by GJ and serve as signal transmission networks to modulate hormone release in the anterior pituitary gland. But whether GJ are localized among FS cells from the pars tuberalis through the pars distalis is unclear. The S100b-GFP transgenic rat has recently been generated, which expresses green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary. This model is expected to be a powerful tool for studies of FS cells. The purpose of the present paper was therefore to examine the localization of GJ on connexin 43 immunohistochemistry throughout the anterior pituitary gland of S100b-GFP rats under confocal laser microscopy. The localization patterns of FS cells was also observed in primary culture of anterior pituitary cells and the question of whether GJ between FS cells are reconstructed in vitro was investigated. In vivo studies showed that GJ were present specifically between FS cells from the pars tuberalis to the pars distalis in the anterior pituitary gland. The appearance of FS cells was distinguished into two types, with localization of GJ differing between types. In vitro, it was observed for the first time that FS cells in primary culture could be categorized into two types. In vivo localization of GJ between FS cells was reconstructed in vitro. These morphological observations are consistent with the hypothesis that FS cells form an electrophysiological network throughout the anterior pituitary for signal transmission.

  4. Effects of HGF gene polymorphisms and protein expression on transhepatic arterial chemotherapeutic embolism efficacy and prognosis in patients with primary liver cancer

    Directory of Open Access Journals (Sweden)

    Chen HY

    2017-02-01

    Full Text Available Hai-Yong Chen,1,2 Yao-Min Chen,3 Jian Wu,1,2 Fu-Chun Yang,1,2 Zhen Lv,1,2 Yi-Gang Qian,1,2 Shu-Sen Zheng1,2 1Department of Surgery, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China Objective: To investigate the correlations of two hepatocyte growth factor (HGF gene polymorphisms (rs5745652 and rs2074725 and their protein expression levels with the efficacy of transhepatic arterial chemotherapeutic embolism (TACE and prognosis in patients with primary liver cancer (PLC. Methods: From March 2011 to June 2012, 109 PLC patients (the case group who chose TACE as primary treatment and 80 healthy people (the control group who had undergone physical examination in The First Affiliated Hospital, Zhejiang University were selected during the same period. Gene polymorphisms of HGF rs5745652 and HGF rs2074725 were detected. Serum HGF level, treating efficacy, survival quality, and 3-year survival rate for PLC patients who received TACE were observed. Results: There were significant differences in genotype and allele frequencies of HGF rs5745652 and HGF rs2074725, between the case and control groups (all P<0.05. Compared with CT+TT genotype of HGF rs5745652, patients carrying CC genotype had lower serum HGF levels, higher efficacy, better survival quality, and prolonged 3-year survival rate (all P<0.05. In rs2074725, patients carrying CA+AA genotype had lower serum HGF levels, higher efficacy, better survival quality, and prolonged 3-year survival rate compared with patients carrying rs2074725 CC genotype (all P<0.05. Gene polymorphisms of HGF rs5745652 and HGF rs2074725, tumor size, and Barcelona Clinic Liver Cancer stage were independent prognostic factors for PLC (P<0.05. Conclusion: Our

  5. Survivin protein expression is involved in the progression of non-small cell lung cancer in Asians: a meta-analysis

    International Nuclear Information System (INIS)

    Duan, Liang; Hu, Xuefei; Jin, Yuxing; Liu, Ruijun; You, Qingjun

    2016-01-01

    Surviving expression might serve as a prognostic biomarker predicting the clinical outcome of non-small cell lung cancer (NSCLC). The study was conducted to explore the potential correlation of survivin protein expression with NSCLC and its clinicopathologic characteristics. PubMed, Medline, Cochrane Library, CNKI and Wanfang database were searched through January 2016 with a set of inclusion and exclusion criteria. Data was extracted from these articles and all statistical analysis was conducted by using Stata 12.0. A total of 28 literatures (14 studies in Chinese and 14 studies in English) were enrolled in this meta-analysis, including 3206 NSCLC patients and 816 normal controls. The result of meta-analysis demonstrated a significant difference of survivin positive expression between NSCLC patients and normal controls (RR = 7.16, 95 % CI = 4.63-11.07, P < 0.001). To investigate the relationship of survivin expression and clinicopathologic characteristics, we performed a meta-analysis in NSCLC patients. Our results indicates survivin expression was associated with histological differentiation, tumor-node-metastasis (TNM) stage and lymph node metastasis (LNM) (RR = 0.80, 95 % CI = 0.73-0.87, P < 0.001; RR = 0.75, 95 % CI = 0.67-0.84, P < 0.001; RR = 1.14, 95 % CI = 1.01-1.29, P = 0.035, respectively), but not pathological type and tumor size. (RR = 1.00, 95 % CI = 0.93-1.07, P = 0.983; RR = 0.95, 95 % CI = 0.86-1.05, P = 0.336, respectively). Higher expression of survivin in NSCLC patients was found when compared to normal controls. Survivin expression was associated with the clinicopathologic characteristics of NSCLC and may serves as an important biomarker for NSCLC progression

  6. Imaging characteristics of stage I non-small cell lung cancer on CT and FDG-PER; Relationship with epidermal growth factor receptor protein expression status and survival

    International Nuclear Information System (INIS)

    Lee, Youkyung; Lee, Hyun Ju; Kim, Young Tae; Kang, Chang Hyun; Goo, Jin Mo; Park, Chang Min; Paeng, Jin Chul; Chung, Doo Hyun; Jeon, Yoon Kyung

    2013-01-01

    To identify CT and FDG-PET features associated with epidermal growth factor receptor (EGFR) protein overexpression, and to evaluate whether imaging features and EGFR-overexpression can help predict clinical outcome. In 214 patients (M : F = 129 : 85; mean age, 63.2) who underwent curative resection of stage I non-small cell lung cancer, EGFR protein expression status was determined through immunohistochemical analysis. Imaging characteristics on CT and FDG-PET was assessed in relation to EGFR-overexpression. Imaging features and EGFR-overexpression were also evaluated for clinical outcome by using the Cox proportional hazards model. EGFR-overexpression was found in 51 patients (23.8%). It was significantly more frequent in tumors with an SUVmax > 5.0 (p 2.43 cm (p 5.0 (OR, 3.113; 95% CI, 1.375-7.049; p = 0.006) and diameter > 2.43 cm (OR, 2.799; 95% CI, 1.285-6.095; p = 0.010) were independent predictors of EGFR overexpression. Multivariate analysis showed that SUVmax > 4.0 (hazard ratio, 10.660; 95% CI, 1.370-82.966; p = 0.024), and the presence of cavitation within a tumor (hazard ratio, 3.122; 95% CI, 1.143-8.532; p = 0.026) were factors associated with poor prognosis. EGFR-overexpression is associated with high SUVmax, large tumor diameter, and small GGO proportion. CT and FDG-PET findings, which are closely related to EGFR overexpression, can be valuable in the prediction of clinical outcome.

  7. Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

    Science.gov (United States)

    Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

    2014-09-01

    The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients. © 2014 UICC.

  8. Improving fluorescence diagnosis of cancer by SLIM

    Science.gov (United States)

    Rück, Angelika; Dolp, Frank; Kinzler, Ingrid; Hauser, Carmen; Scalfi-Happ, Claudia

    2006-02-01

    Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting of a laser scanning microscope equipped with a 16 channel array for time-correlated single photon counting (TCSPC) and a spectrograph in front of the array. A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during

  9. Restriction of GAGE protein expression to subpopulations of cancer cells is independent of genotype and may limit the use of GAGE proteins as targets for cancer immunotherapy

    DEFF Research Database (Denmark)

    Gjerstorff, M F; Johansen, L E; Nielsen, O

    2006-01-01

    The GAGE cancer testis antigen gene family encodes products that can be recognized by autologous T cells, and GAGE proteins have been suggested as potential targets for cancer immunotherapy. Analysis of GAGE expression in tumours has primarily been performed at the level of gene transcription, wh...

  10. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  11. Low Ki67/high ATM protein expression in malignant tumors predicts favorable prognosis in a retrospective study of early stage hormone receptor positive breast cancer.

    Science.gov (United States)

    Feng, Xiaolan; Li, Haocheng; Kornaga, Elizabeth N; Dean, Michelle; Lees-Miller, Susan P; Riabowol, Karl; Magliocco, Anthony M; Morris, Don; Watson, Peter H; Enwere, Emeka K; Bebb, Gwyn; Paterson, Alexander

    2016-12-27

    This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC.

  12. The predictive value of ERG protein expression for development of castration-resistant prostate cancer in hormone-naïve advanced prostate cancer treated with primary androgen deprivation therapy

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Røder, Martin A; Thomsen, Frederik B

    2015-01-01

    BACKGROUND: Biomarkers predicting response to primary androgen deprivation therapy (ADT) and risk of castration-resistant prostate cancer (CRPC) is lacking. We aimed to analyse the predictive value of ERG expression for development of CRPC. METHODS: In total, 194 patients with advanced and....../or metastatic prostate cancer (PCa) treated with first-line castration-based ADT were included. ERG protein expression was analysed in diagnostic specimens using immunohistochemistry (anti-ERG, EPR3864). Time to CRPC was compared between ERG subgroups using multiple cause-specific Cox regression stratified......-negative group, respectively. Compared to a model omitting ERG-status, the ERG-stratified model showed comparable AUC values 1 year (77.6% vs. 78.0%, P = 0.82), 2 years (71.7% vs. 71.8%, P = 0.85), 5 years (68.5% vs. 69.9%, P = 0.32), and 8 years (67.9% vs. 71.4%, P = 0.21) from ADT initiation. No differences...

  13. Laser-induced fluorescence of oral mucosa cancer

    Science.gov (United States)

    Jaliashvili, Z. V.; Medoidze, T. D.; Melikishvili, Z. G.; Gogilashvili, K. T.

    2017-10-01

    The laser-induced fluorescence (LIF) spectra have been measured for cancer-infused and control mice mucosa tissues. It was established that there is quite a difference between their LIF spectral shapes. These spectral shapes are used to express the diagnostic of different states of tissues: from normal to cancer.

  14. PD-L1 protein expression assessed by immunohistochemistry is neither prognostic nor predictive of benefit from adjuvant chemotherapy in resected non-small cell lung cancer.

    Science.gov (United States)

    Tsao, M-S; Le Teuff, G; Shepherd, F A; Landais, C; Hainaut, P; Filipits, M; Pirker, R; Le Chevalier, T; Graziano, S; Kratze, R; Soria, J-C; Pignon, J-P; Seymour, L; Brambilla, E

    2017-04-01

    The expression of programmed death (PD) ligand 1 (PD-L1) protein expression assessed by immunohistochemistry (IHC) has been correlated with response and survival benefit from anti-PD-1/PD-L1 immune checkpoint inhibitor therapies in advanced non-small cell lung carcinoma (NSCLC). The efficacy of several agents appears correlated with PD-L1 expression. It remains controversial whether PD-L1 is prognostic in NSCLC. We assessed the prognostic value of PD-L1 IHC and its predictive role for adjuvant chemotherapy in early stage NSCLC. Tumor sections from three pivotal adjuvant chemotherapy trials (IALT, JBR.10, CALGB 9633) using the E1L3N antibody were studied in this pooled analysis. PD-L1 staining intensity and percentage in both tumor cells (TCs) and immune cells (ICs) were scored by two pathologists. The average or consensus PD-L1 expression levels across intensities and/or percent cells stained were correlated with clinicopathological and molecular features, patient survivals and potential benefit of adjuvant chemotherapy. Results from 982 patients were available for analysis. Considering staining at any intensities for overall PD-L1 expression, 314 (32.0%), 204 (20.8%) and 141 (14.3%) tumor samples were positive for PD-L1 staining on TCs using cut-offs at ≥1%, ≥10% and ≥25%, respectively. For PD-L1 expressing ICs, 380 (38.7%), 308 (31.4%) and 148 (15.1%) were positive at ≥ 1%, ≥10% and 25% cut-offs, respectively. Positive PD-L1 was correlated with squamous histology, intense lymphocytic infiltrate, and KRAS but not with TP53 mutation. EGFR mutated tumors showed statistically non-significant lower PD-L1 expression. PD-L1 expression was neither prognostic with these cut-offs nor other exploratory cut-offs, nor were predictive for survival benefit from adjuvant chemotherapy. PD-L1 IHC is not a prognostic factor in early stage NSCLC patients. It is also not predictive for adjuvant chemotherapy benefit in these patients. © The Author 2017. Published by

  15. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    Science.gov (United States)

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    -quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ. We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system(6). Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index(7). To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

  16. Inhibition of bcl-2 and cox-2 Protein Expression after Local Application of a New Carmustine-Loaded Clinoptilolite-Based Delivery System in a Chemically Induced Skin Cancer Model in Mice

    Directory of Open Access Journals (Sweden)

    Cristina Mihaela Ghiciuc

    2017-11-01

    Full Text Available Our research has focused on in vitro and in vivo evaluations of a new Carmustine (BCNU-loaded clinoptilolite-based delivery system. Two clinoptilolite ionic forms—hydrogen form (HCLI and sodium form (NaCLI—were prepared, allowing a loading degree of about 5–6 mg BCNU/g of zeolite matrix due to the dual porous feature of clinoptilolite. Clinoptilolite-based delivery systems released 35.23% of the load in 12 h for the BCNU@HCLI system and only 10.82% for the BCNU@NaCLI system. The BCNU@HCLI system was chosen to develop gel and cream semisolid dosage forms. The cream (C_BCNU@HCLI released 29.6% of the loaded BCNU after 12 h in the Nylon synthetic membrane test and 31.6% in the collagen membrane test, higher by comparison to the gel. The new cream was evaluated in vivo in a chemically induced model of skin cancer in mice. Quantitative immunohistochemistry analysis showed stronger inhibition of B-cell lymphoma-2 (bcl-2 and cyclooxygenase 2 (cox-2 protein expression, known markers for cancer survival and aggressiveness, after the treatment with C_BCNU@HCLI by comparison to all the control treatment types, including an off-label magistral formula commercially available Carmustine cream as reference, bringing evidence that a clinoptilolite-based delivery systems could be used as a cancer drug carriers and controlled release systems (skin-targeted topical delivery systems.

  17. Tafazzin protein expression is associated with tumorigenesis and radiation response in rectal cancer: a study of Swedish clinical trial on preoperative radiotherapy.

    Directory of Open Access Journals (Sweden)

    Surajit Pathak

    Full Text Available Tafazzin (TAZ, a transmembrane protein contributes in mitochondrial structural and functional modifications through cardiolipin remodeling. TAZ mutations are associated with several diseases, but studies on the role of TAZ protein in carcinogenesis and radiotherapy (RT response is lacking. Therefore we investigated the TAZ expression in rectal cancer, and its correlation with RT, clinicopathological and biological variables in the patients participating in a clinical trial of preoperative RT.140 rectal cancer patients were included in this study, of which 65 received RT before surgery and the rest underwent surgery alone. TAZ expression was determined by immunohistochemistry in primary cancer, distant, adjacent normal mucosa and lymph node metastasis. In-silico protein-protein interaction analysis was performed to study the predictive functional interaction of TAZ with other oncoproteins.TAZ showed stronger expression in primary cancer and lymph node metastasis compared to distant or adjacent normal mucosa in both non-RT and RT patients. Strong TAZ expression was significantly higher in stages I-III and non-mucinious cancer of non-RT patients. In RT patients, strong TAZ expression in biopsy was related to distant recurrence, independent of gender, age, stages and grade (p = 0.043, HR, 6.160, 95% CI, 1.063-35.704. In silico protein-protein interaction study demonstrated that TAZ was positively related to oncoproteins, Livin, MAC30 and FXYD-3.Strong expression of TAZ protein seems to be related to rectal cancer development and RT response, it can be a predictive biomarker of distant recurrence in patients with preoperative RT.

  18. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

    International Nuclear Information System (INIS)

    Martínez-Galán, Joaquina; Ríos, Sandra; Delgado, Juan Ramón; Torres-Torres, Blanca; Núñez, María Isabel; López-Peñalver, Jesús; Del Moral, Rosario; Ruiz De Almodóvar, José Mariano; Menjón, Salomón; Concha, Ángel; Chamorro, Clara

    2014-01-01

    Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment

  19. Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

    Science.gov (United States)

    Welti, Jonathan; Rodrigues, Daniel Nava; Sharp, Adam; Sun, Shihua; Lorente, David; Riisnaes, Ruth; Figueiredo, Ines; Zafeiriou, Zafeiris; Rescigno, Pasquale; de Bono, Johann S; Plymate, Stephen R

    2016-10-01

    The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; pprostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  20. Targeting pancreatic cancer with magneto-fluorescent theranostic gold nanoshells.

    Science.gov (United States)

    Chen, Wenxue; Ayala-Orozco, Ciceron; Biswal, Nrusingh C; Perez-Torres, Carlos; Bartels, Marc; Bardhan, Rizia; Stinnet, Gary; Liu, Xian-De; Ji, Baoan; Deorukhkar, Amit; Brown, Lisa V; Guha, Sushovan; Pautler, Robia G; Krishnan, Sunil; Halas, Naomi J; Joshi, Amit

    2014-01-01

    We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase-associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the near-infrared (NIR) dye indocyanine green, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. Anti-NGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2-weighted MRI with higher tumor contrast than can be obtained using long-circulating, but nontargeted, PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. TGNS with embedded NIR and magnetic resonance contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy.

  1. Dynamic fluorescence imaging with molecular agents for cancer detection

    Science.gov (United States)

    Kwon, Sun Kuk

    Non-invasive dynamic optical imaging of small animals requires the development of a novel fluorescence imaging modality. Herein, fluorescence imaging is demonstrated with sub-second camera integration times using agents specifically targeted to disease markers, enabling rapid detection of cancerous regions. The continuous-wave fluorescence imaging acquires data with an intensified or an electron-multiplying charge-coupled device. The work presented in this dissertation (i) assessed dose-dependent uptake using dynamic fluorescence imaging and pharmacokinetic (PK) models, (ii) evaluated disease marker availability in two different xenograft tumors, (iii) compared the impact of autofluorescence in fluorescence imaging of near-infrared (NIR) vs. red light excitable fluorescent contrast agents, (iv) demonstrated dual-wavelength fluorescence imaging of angiogenic vessels and lymphatics associated with a xenograft tumor model, and (v) examined dynamic multi-wavelength, whole-body fluorescence imaging with two different fluorescent contrast agents. PK analysis showed that the uptake of Cy5.5-c(KRGDf) in xenograft tumor regions linearly increased with doses of Cy5.5-c(KRGDf) up to 1.5 nmol/mouse. Above 1.5 nmol/mouse, the uptake did not increase with doses, suggesting receptor saturation. Target to background ratio (TBR) and PK analysis for two different tumor cell lines showed that while Kaposi's sarcoma (KS1767) exhibited early and rapid uptake of Cy5.5-c(KRGDf), human melanoma tumors (M21) had non-significant TBR differences and early uptake rates similar to the contralateral normal tissue regions. The differences may be due to different compartment location of the target. A comparison of fluorescence imaging with NIR vs. red light excitable fluorescent dyes demonstrates that NIR dyes are associated with less background signal, enabling rapid tumor detection. In contrast, animals injected with red light excitable fluorescent dyes showed high autofluorescence. Dual

  2. Threshold Analysis and Biodistribution of Fluorescently Labeled Bevacizumab in Human Breast Cancer

    NARCIS (Netherlands)

    Koch, Maximilian; de Jong, Johannes S.; Glatz, Juergen; Symvoulidis, Panagiotis; Lamberts, Laetitia E.; Adams, Arthur L. L.; Kranendonk, Mariette E. G.; Terwisscha Van Scheltinga, Anton; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N.; Schroder, Carolien P.; Jorritsma-Smit, Annelies; Linssen, Matthijs D.; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B.; Elias, Sjoerd G.; Oliveira, Sabrina; Witkamp, Arjen J.; Mali, Willem P. T. M.; Van der Wall, Elsken; Garcia-Allende, P. Beatriz; van Diest, Paul J.; de Vries, Elisabeth G. E.; Walch, Axel; van Dam, Gooitzen M.; Ntziachristos, Vasilis

    2017-01-01

    In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant andnonmalignant tissues are usually distinguished on fluorescence images by applying

  3. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Science.gov (United States)

    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  4. Breast cancer: in vitro measurements of native fluorescence

    Science.gov (United States)

    Lohmann, Wolfgang; Bohle, Rainer M.; Dreyer, Thomas; Haas, Sabine; Wallenfels, Heike; Schwemmle, Konrad; Schill, Wolf-Bernhard

    1996-12-01

    Unfixed, HE stained cryosections of breast tissue obtained from 67 patients during surgery were illuminated with 395 - 440 nm and their fluorescence response as well as the 2- dimensional fluorophore distribution were measured. The histological evaluation of the same cryosection, illuminated as usual with a transmitted light obtained from a halogen lamp, revealed 9 patients with healthy tissue, 11 with benign epithelial hyperplasia, 4 with ductal carcinoma in situ, 35 with invasive ductal carcinoma, 7 with invasive lobular carcinoma, and one with invasive tubular carcinoma. A comparison between the fluorescence and the HE images shows that both match very nicely and that the fluorescence images are also characteristic for the different pathological condition of the biopsy sample. Moreover, benign tumors e.g. fibroadenomas, exhibit a fluorescence response different from cancer and healthy tissue.

  5. Role of hexaminolevulinate-guided fluorescence cystoscopy in bladder cancer

    DEFF Research Database (Denmark)

    Malmström, Per-Uno; Grabe, Magnus; Haug, Erik Skaaheim

    2012-01-01

    Hexaminolevulinate (HAL) is an optical imaging agent used with fluorescence cystoscopy (FC) for the detection of non-muscle-invasive bladder cancer (NMIBC). Guidelines from the European Association of Urology (EAU) and a recent, more detailed European expert consensus statement agree that HAL...

  6. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  7. Quantification of tumor fluorescence during intraoperative optical cancer imaging.

    Science.gov (United States)

    Judy, Ryan P; Keating, Jane J; DeJesus, Elizabeth M; Jiang, Jack X; Okusanya, Olugbenga T; Nie, Shuming; Holt, David E; Arlauckas, Sean P; Low, Phillip S; Delikatny, E James; Singhal, Sunil

    2015-11-13

    Intraoperative optical cancer imaging is an emerging technology in which surgeons employ fluorophores to visualize tumors, identify tumor-positive margins and lymph nodes containing metastases. This study compares instrumentation to measure tumor fluorescence. Three imaging systems (Spectropen, Glomax, Flocam) measured and quantified fluorescent signal-to-background ratios (SBR) in vitro, murine xenografts, tissue phantoms and clinically. Evaluation criteria included the detection of small changes in fluorescence, sensitivity of signal detection at increasing depths and practicality of use. In vitro, spectroscopy was superior in detecting incremental differences in fluorescence than luminescence and digital imaging (Ln[SBR] = 6.8 ± 0.6, 2.4 ± 0.3, 2.6 ± 0.1, p = 0.0001). In fluorescent tumor cells, digital imaging measured higher SBRs than luminescence (6.1 ± 0.2 vs. 4.3 ± 0.4, p = 0.001). Spectroscopy was more sensitive than luminometry and digital imaging in identifying murine tumor fluorescence (SBR = 41.7 ± 11.5, 5.1 ± 1.8, 4.1 ± 0.9, p = 0.0001), and more sensitive than digital imaging at detecting fluorescence at increasing depths (SBR = 7.0 ± 3.4 vs. 2.4 ± 0.5, p = 0.03). Lastly, digital imaging was the most practical and least time-consuming. All methods detected incremental differences in fluorescence. Spectroscopy was the most sensitive for small changes in fluorescence. Digital imaging was the most practical considering its wide field of view, background noise filtering capability, and sensitivity to increasing depth.

  8. Quantitative measurements of tumoral p95HER2 protein expression in metastatic breast cancer patients treated with trastuzumab: independent validation of the p95HER2 clinical cutoff.

    Science.gov (United States)

    Duchnowska, Renata; Sperinde, Jeff; Chenna, Ahmed; Haddad, Mojgan; Paquet, Agnes; Lie, Yolanda; Weidler, Jodi M; Huang, Weidong; Winslow, John; Jankowski, Tomasz; Czartoryska-Arłukowicz, Bogumiła; Wysocki, Piotr J; Foszczyńska-Kłoda, Małgorzata; Radecka, Barbara; Litwiniuk, Maria M; Zok, Jolanta; Wiśniewski, Michał; Zuziak, Dorota; Biernat, Wojciech; Jassem, Jacek

    2014-05-15

    P95HER2 (p95) is a truncated form of the HER2, which lacks the trastuzumab-binding site and contains a hyperactive kinase domain. Previously, an optimal clinical cutoff of p95 expression for progression-free survival (PFS) and overall survival (OS) was defined using a quantitative VeraTag assay (Monogram Biosciences) in a training set of trastuzumab-treated metastatic breast cancer (MBC) patients. In the current study, the predictive value of the p95 VeraTag assay cutoff established in the training set was retrospectively validated for PFS and OS in an independent series of 240 trastuzumab-treated MBC patients from multiple institutions. In the subset of 190 tumors assessed as HER2-total (H2T)-positive using the quantitative HERmark assay (Monogram Biosciences), p95 VeraTag values above the predefined cutoff correlated with shorter PFS (HR = 1.43; P = 0.039) and shorter OS (HR = 1.94; P = 0.0055) where both outcomes were stratified by hormone receptor status and tumor grade. High p95 expression correlated with shorter PFS (HR = 2.41; P = 0.0003) and OS (HR = 2.57; P = 0.0025) in the hormone receptor-positive subgroup of patients (N = 78), but not in the hormone receptor-negative group. In contrast with the quantitative p95 VeraTag measurements, p95 immunohistochemical expression using the same antibody was not significantly correlated with outcomes. The consistency in the p95 VeraTag cutoff across different cohorts of patients with MBC treated with trastuzumab justifies additional studies using blinded analyses in larger series of patients. ©2014 American Association for Cancer Research.

  9. Image-guided cancer surgery using near-infrared fluorescence

    Science.gov (United States)

    Vahrmeijer, Alexander L.; Hutteman, Merlijn; van der Vorst, Joost R.; van de Velde, C.J.H.; Frangioni, John V.

    2013-01-01

    Paradigm shifts in surgery arise when surgeons are empowered to perform surgery faster, better, and/or less expensively. Optical imaging that exploits invisible near-infrared fluorescent light has the potential to improve cancer surgery outcomes while minimizing anesthesia time and lowering healthcare costs. Because of this, the last few years have witnessed an explosion of proof-of-concept clinical trials in the field. In this review, we introduce the concept of near-infrared fluorescence imaging for cancer surgery, review the clinical trial literature to date, outline the key issues pertaining to imaging system and contrast agent optimization, discuss limitations and leverage, and provide a framework for making the technology available for the routine care of cancer patients in the near future. PMID:23881033

  10. Fluorescent imaging of cancerous tissues for targeted surgery

    Science.gov (United States)

    Bu, Lihong; Shen, Baozhong; Cheng, Zhen

    2014-01-01

    To maximize tumor excision and minimize collateral damage is the primary goal of cancer surgery. Emerging molecular imaging techniques have to “image-guided surgery” developing into “molecular imaging-guided surgery”, which is termed “targeted surgery” in this review. Consequently, the precision of surgery can be advanced from tissue-scale to molecule-scale, enabling “targeted surgery” to be a component of “targeted therapy”. Evidence from numerous experimental and clinical studies has demonstrated significant benefits of fluorescent imaging in targeted surgery with preoperative molecular diagnostic screening. Fluorescent imaging can help to improve intraoperative staging and enable more radical cytoreduction, detect obscure tumor lesions in special organs, highlight tumor margins, better map lymph node metastases, and identify important normal structures intraoperatively. Though limited tissue penetration of fluorescent imaging and tumor heterogeneity are two major hurdles for current targeted surgery, multimodality imaging and multiplex imaging may provide potential solutions to overcome these issues, respectively. Moreover, though many fluorescent imaging techniques and probes have been investigated, targeted surgery remains at a proof-of-principle stage. The impact of fluorescent imaging on cancer surgery will likely be realized through persistent interdisciplinary amalgamation of research in diverse fields. PMID:25064553

  11. Fluorescence photodiagnosis of early stage lung cancer

    International Nuclear Information System (INIS)

    Kato, H.; Sakai, H.; Konaka, C.; Okunaka, T.; Furukawa, K.; Saito, Y.; Aizawa, K.; Hayata, Y.

    1992-01-01

    Sputum cytology examination is the most effective method to detect early stage central type squamous cell carcinoma. As sputum-positive early stage lung cancer usually does not show any abnormal findings on chest X-ray film, fiberoptic bronchoscopy is subsequently performed for localization. However, sometimes cases do not show any abnormal findings of cancer endoscopically because they are very early stage cases. For the purpose of localization of invisible lesions the photodynamic reaction was employed in this study. Photodynamic reaction is achieved by transfer of energy of an excited photo-sensitizer induced by photoradiation of light. This phenomenon was already recognized in the beginning of this century. Study of tumor localization of the bronchial tree using hematoporphyrin derivative (HpD) and a mercury arc lamp was first performed in the Mayo Clinic in 1960s. In 1978, krypton laser was used first as a light source by Profio and Doiron. Authors have been doing research on early localization of such endoscopically occult early lung cancer since 1978. They recently developed an image processing system using an excimer dye laser for early localization of lung cancer. (author). 5 refs., 4 figs., 1 tab

  12. HER-2, p53, p21 and hormonal receptors proteins expression as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination

    International Nuclear Information System (INIS)

    Tiezzi, Daniel G; Andrade, Jurandyr M; Ribeiro-Silva, Alfredo; Zola, Fábio E; Marana, Heitor RC; Tiezzi, Marcelo G

    2007-01-01

    Neoadjuvant chemotherapy has been considered the standard care in locally advanced breast cancer. However, about 20% of the patients do not benefit from this clinical treatment and, predictive factors of response were not defined yet. This study was designed to evaluate the importance of biological markers to predict response and prognosis in stage II and III breast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting. Sixty patients received preoperative docetaxel (75 mg/m 2 ) in combination with epirubicin (50 mg/m 2 ) in i.v. infusion in D1 every 3 weeks after incisional biopsy. They received adjuvant chemotherapy with CMF or FEC, attaining axillary status following definitive breast surgery. Clinical and pathologic response rates were measured after preoperative therapy. We evaluated the response rate to neoadjuvant chemotherapy and the prognostic significance of clinicopathological and immunohistochemical parameters (ER, PR, p51, p21 and HER-2 protein expression). The median patient age was 50.5 years with a median follow up time 48 months after the time of diagnosis. Preoperative treatment achieved clinical response in 76.6% of patients and complete pathologic response in 5%. The clinical, pathological and immunohistochemical parameters were not able to predict response to therapy and, only HER2 protein overexpression was associated with a decrease in disease free and overall survival (P = 0.0007 and P = 0.003) as shown by multivariate analysis. Immunohistochemical phenotypes were not able to predict response to neoadjuvant chemotherapy. Clinical response is inversely correlated with a risk of death in patients submitted to neoadjuvant chemotherapy and HER2 overexpression is the major prognostic factor in stage II and III breast cancer patients treated with a neoadjuvant docetaxel and epirubicin combination

  13. Fluorescent supramolecular micelles for imaging-guided cancer therapy

    Science.gov (United States)

    Sun, Mengmeng; Yin, Wenyan; Dong, Xinghua; Yang, Wantai; Zhao, Yuliang; Yin, Meizhen

    2016-02-01

    A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSM nanomedicine platform as a nanovehicle has great potential for fluorescence imaging-guided cancer therapy.A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth

  14. HER3 protein expression in relation to HER2 positivity in patients with primary colorectal cancer: clinical relevance and prognostic value.

    Science.gov (United States)

    Seo, An Na; Kwak, Yoonjin; Kim, Woo Ho; Kim, Duck-Woo; Kang, Sung-Bum; Choe, Gheeyoung; Lee, Hye Seung

    2015-06-01

    The clinical and prognostic significance of HER3 expression and its relation to HER2 status in primary colorectal cancer (pCRC) were investigated. We retrospectively analysed 365 consecutive cases of pCRC that had been previously evaluated for HER2 status and included their 143 matched lymph node metastases. Immunohistochemistry (IHC) was performed to assess HER3 expression using tissue array methods. Of 364 eligible patients, HER3 overexpression was detected in 251 cases (69 %) (IHC 2+, n = 186 and IHC 3+, n = 65). HER3 overexpression was inversely correlated with histologic grade (p = 0.006), tumour size (p < 0.001), tumour depth (p < 0.001), TNM stage (p = 0.002), lymphatic invasion (p = 0.004), lymph node metastasis (p = 0.013) and distant metastasis (p = 0.039). Moreover, it positively correlated with both HER2 overexpression (p = 0.007) and HER2 gene amplification (p = 0.006). Although HER3 overexpression was associated with longer survival in univariate analysis (p = 0.026), it was not an independent prognostic factor in multivariate analysis (p = 0.359). Moreover, co-alteration of HER3 and HER2 was associated with neither survival nor any clinicopathologic parameter except tumour location in the rectum. Although not an independent prognostic factor for overall survival, HER3 overexpression was associated with several favourable prognostic clinicopathologic parameters. Additionally, HER3 overexpression strongly correlated with HER2 positivity in this cohort of patients.

  15. Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Miralem Mrkonjic

    Full Text Available We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734 and microsatellite unstable (MSI-H colorectal cancers (CRCs in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls and from Seattle (591 cases, 629 controls. Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10(-4 when the SNP was examined alone.The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

  16. Potential actionable targets in appendiceal cancer detected by immunohistochemistry, fluorescent in situ hybridization, and mutational analysis

    Science.gov (United States)

    Millis, Sherri Z.; Kimbrough, Jeffery; Doll, Nancy; Von Hoff, Daniel; Ramanathan, Ramesh K.

    2017-01-01

    Background Appendiceal cancers are rare and consist of carcinoid, mucocele, pseudomyxoma peritonei (PMP), goblet cell carcinoma, lymphoma, and adenocarcinoma histologies. Current treatment involves surgical resection or debulking, but no standard exists for adjuvant chemotherapy or treatment for metastatic disease. Methods Samples were identified from approximately 60,000 global tumors analyzed at a referral molecular profiling CLIA-certified laboratory. A total of 588 samples with appendix primary tumor sites were identified (male/female ratio of 2:3; mean age =55). Sixty-two percent of samples were adenocarcinomas (used for analysis); the rest consisted of 9% goblet cell, 15% mucinous; 6% pseudomyxoma, and less than 5% carcinoids and 2% neuroendocrine. Tests included sequencing [Sanger, next generation sequencing (NGS)], protein expression/immunohistochemistry (IHC), and gene amplification [fluorescent in situ hybridization (FISH) or CISH]. Results Profiling across all appendiceal cancer histological subtypes for IHC revealed: 97% BRCP, 81% MRP1, 81% COX-2, 71% MGMT, 56% TOPO1, 5% PTEN, 52% EGFR, 40% ERCC1, 38% SPARC, 35% PDGFR, 35% TOPO2A, 25% RRM1, 21% TS, 16% cKIT, and 12% for TLE3. NGS revealed mutations in the following genes: 50.4% KRAS, 21.9% P53, 17.6% GNAS, 16.5% SMAD4, 10% APC, 7.5% ATM, 5.5% PIK3CA, 5.0% FBXW7, and 1.8% BRAF. Conclusions Appendiceal cancers show considerable heterogeneity with high levels of drug resistance proteins (BCRP and MRP1), which highlight the difficulty in treating these tumors and suggest an individualized approach to treatment. The incidence of low TS (79%) could be used as a backbone of therapy (using inhibitors such as 5FU/capecitabine or newer agents). Therapeutic options includeTOPO1 inhibitors (irinotecan/topotecan), EGFR inhibitors (erlotinib, cetuximab), PDGFR antagonists (regorafenib, axitinib), MGMT (temozolomide). Clinical trials targeting pathways involving KRAS, p53, GNAS, SMAD4, APC, ATM, PIK3CA, FBXW7, and

  17. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Protein expression dynamics observed in Experiment, Synchronous and. Asynchronous simulation. .... molecular basis for T cell suppression by IL-10: CD28-asso- ciated IL-10 receptor inhibits CD28 tyrosine ...

  18. Fluorescently labeled bevacizumab in human breast cancer: defining the classification threshold

    Science.gov (United States)

    Koch, Maximilian; de Jong, Johannes S.; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E.; Adams, Arthur L. L.; Kranendonk, Mariëtte E. G.; Terwisscha van Scheltinga, Anton G. T.; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N.; Schröder, Carolien P.; Jorritsma-Smit, Annelies; Linssen, Matthijs D.; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B.; Elias, Sjoerd G.; Oliveira, Sabrina; Witkamp, Arjen J.; Mali, Willem P. Th. M.; Van der Wall, Elsken; Garcia-Allende, P. Beatriz; van Diest, Paul J.; de Vries, Elisabeth G. E.; Walch, Axel; van Dam, Gooitzen M.; Ntziachristos, Vasilis

    2017-07-01

    In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

  19. Intrinsic fluorescence of the clinically approved multikinase inhibitor nintedanib reveals lysosomal sequestration as resistance mechanism in FGFR-driven lung cancer.

    Science.gov (United States)

    Englinger, Bernhard; Kallus, Sebastian; Senkiv, Julia; Heilos, Daniela; Gabler, Lisa; van Schoonhoven, Sushilla; Terenzi, Alessio; Moser, Patrick; Pirker, Christine; Timelthaler, Gerald; Jäger, Walter; Kowol, Christian R; Heffeter, Petra; Grusch, Michael; Berger, Walter

    2017-09-07

    Studying the intracellular distribution of pharmacological agents, including anticancer compounds, is of central importance in biomedical research. It constitutes a prerequisite for a better understanding of the molecular mechanisms underlying drug action and resistance development. Hyperactivated fibroblast growth factor receptors (FGFRs) constitute a promising therapy target in several types of malignancies including lung cancer. The clinically approved small-molecule FGFR inhibitor nintedanib exerts strong cytotoxicity in FGFR-driven lung cancer cells. However, subcellular pharmacokinetics of this compound and its impact on therapeutic efficacy remain obscure. 3-dimensional fluorescence spectroscopy was conducted to asses cell-free nintedanib fluorescence properties. MTT assay was used to determine the impact of the lysosome-targeting agents bafilomycin A1 and chloroquine combined with nintedanib on lung cancer cell viability. Flow cytometry and live cell as well as confocal microscopy were performed to analyze uptake kinetics as well as subcellular distribution of nintedanib. Western blot was conducted to investigate protein expression. Cryosections of subcutaneous tumor allografts were generated to detect intratumoral nintedanib in mice after oral drug administration. Here, we report for the first time drug-intrinsic fluorescence properties of nintedanib in living and fixed cancer cells as well as in cryosections derived from allograft tumors of orally treated mice. Using this feature in conjunction with flow cytometry and confocal microscopy allowed to determine cellular drug accumulation levels, impact of the ABCB1 efflux pump and to uncover nintedanib trapping into lysosomes. Lysosomal sequestration - resulting in an organelle-specific and pH-dependent nintedanib fluorescence - was identified as an intrinsic resistance mechanism in FGFR-driven lung cancer cells. Accordingly, combination of nintedanib with agents compromising lysosomal acidification

  20. Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials

    International Nuclear Information System (INIS)

    Fountzilas, George; Tsolaki, Eleftheria; Televantou, Despina; Timotheadou, Eleni; Koutras, Angelos; Klouvas, George; Samantas, Epaminontas; Pisanidis, Nikolaos; Karanikiotis, Charisios; Sfakianaki, Ioanna; Pavlidis, Nicholas; Dafni, Urania; Gogas, Helen; Linardou, Helena; Kalogeras, Konstantine T; Pectasides, Dimitrios; Dimopoulos, Meletios A; Bobos, Mattheos; Kotoula, Vassiliki; Batistatou, Anna; Xanthakis, Ioannis; Papadimitriou, Christos; Kostopoulos, Ioannis; Koletsa, Triantafillia

    2013-01-01

    The HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy. Formalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated. HER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death. HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy. Australian New Zealand Clinical

  1. Parts Characterization for Tunable Protein Expression

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Sommer, Morten Otto Alexander

    2018-01-01

    Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Construc......Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression...

  2. Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer

    NARCIS (Netherlands)

    Koch, Maximillian; de Jong, Johannes S; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E; Adams, Arthur; Kranendonk, Mariëtte E G; Terwisscha van Scheltinga, Anton G T; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N; Schröder, Carolina P; Jorritsma-Smit, Annelies; Linssen, Matthijs D; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B; Elias, Sjoerd G; Oliveira, Sabrina; Witkamp, Arjen; Mali, Willem P Th M; van der Wall, Elsken; Garcia-Allende, Beatriz P; Van Diest, Paul J; de Vries, Elisabeth G; Walch, Axel; van Dam, Gooitzen M; Ntziachristos, Vasilis

    2017-01-01

    In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying

  3. Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer

    NARCIS (Netherlands)

    Koch, Maximillian; de Jong, Johannes S; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E; Adams, Arthur; Kranendonk, Mariëtte E G; Terwisscha van Scheltinga, Anton G T; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N; Schröder, Carolina P; Jorritsma-Smit, Annelies; Linssen, Matthijs D; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B; Elias, Sjoerd G; Oliveira, Sabrina|info:eu-repo/dai/nl/304841455; Witkamp, Arjen; Mali, Willem P Th M; van der Wall, Elsken; Garcia-Allende, Beatriz P; Van Diest, Paul J; de Vries, Elisabeth G; Walch, Axel; van Dam, Gooitzen M; Ntziachristos, Vasilis

    2016-01-01

    In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying

  4. Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics

    Science.gov (United States)

    Liu, Quan; Grant, Gerald; Li, Jianjun; Zhang, Yan; Hu, Fangyao; Li, Shuqin; Wilson, Christy; Chen, Kui; Bigner, Darell; Vo-Dinh, Tuan

    2011-03-01

    We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivty to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection.

  5. Rare earth fluorescent nanoparticles for specific cancer cell targeting

    International Nuclear Information System (INIS)

    Stefanakis, Dimitrios; Ghanotakis, Demetrios F.

    2016-01-01

    Terbium layered hydroxide nanoparticles (Tb_2(OH)_5NO_3) were synthesized by a one-pot coprecipitation method. The characterization of this preparation revealed highly oriented fluorescent nanoparticles. An attempt to improve the properties of Tb_2(OH)_5NO_3 resulted in the preparation of two optimized nanoparticles. In particular, Tb_2(OH)_5NO_3:Eu and Tb_2(OH)_5NO_3-FA were prepared when Tb_2(OH)_5NO_3 was doped with Europium and when the surface was modified with folic acid (FA), respectively. The size of the above nanoparticles was below 100 nm, and thus they have the potential to be used for biomedical applications. The interaction of nanoparticles with human cells was studied using confocal microscopy. This study revealed that only the nanoparticles modified with folic acid have the ability to be targeted to HeLa cells. This specific identification of cancer cells, in combination with the fluorescent properties of Tb_2(OH)_5NO_3, could render these nanoparticles appropriate for biomedical applications.

  6. Rare earth fluorescent nanoparticles for specific cancer cell targeting

    Energy Technology Data Exchange (ETDEWEB)

    Stefanakis, Dimitrios; Ghanotakis, Demetrios F., E-mail: ghanotakis@uoc.gr [University of Crete, Department of Chemistry (Greece)

    2016-07-15

    Terbium layered hydroxide nanoparticles (Tb{sub 2}(OH){sub 5}NO{sub 3}) were synthesized by a one-pot coprecipitation method. The characterization of this preparation revealed highly oriented fluorescent nanoparticles. An attempt to improve the properties of Tb{sub 2}(OH){sub 5}NO{sub 3} resulted in the preparation of two optimized nanoparticles. In particular, Tb{sub 2}(OH){sub 5}NO{sub 3}:Eu and Tb{sub 2}(OH){sub 5}NO{sub 3}-FA were prepared when Tb{sub 2}(OH){sub 5}NO{sub 3} was doped with Europium and when the surface was modified with folic acid (FA), respectively. The size of the above nanoparticles was below 100 nm, and thus they have the potential to be used for biomedical applications. The interaction of nanoparticles with human cells was studied using confocal microscopy. This study revealed that only the nanoparticles modified with folic acid have the ability to be targeted to HeLa cells. This specific identification of cancer cells, in combination with the fluorescent properties of Tb{sub 2}(OH){sub 5}NO{sub 3}, could render these nanoparticles appropriate for biomedical applications.

  7. Fluorescence spectroscopy for throat cancer detection using human saliva

    Science.gov (United States)

    Kumar, Pavan; Singh, Ashutosh; Zaffar, Mohammad; Pradhan, Asima

    2018-02-01

    Throat precancer detection using fluorescence from human saliva is reported here. It may be noted that accessing the throat for investigation is cumbersome and use of saliva as a diagnostic medium may ease the process. The study has been conducted on three groups of patients: oral squamous cell carcinoma (OSCC), dysplasia, and normal (control). An in-house developed compact set-up has been used for fluorescence measurements. The compact system consist of a 375 nm laser diode, collimating lens, long pass filter, fibers, and cuvette holder. Major and minor bands of flavin adenine dinucleotide (FAD) and porphyrin are observed in the spectra. A receiver operating characteristic (ROC) analysis has been used to evaluate the diagnostic performance. Area under the spectra has been chosen for discrimination among the groups and is able to differentiate OSCC to normal, dysplasia to normal, and OSCC to dysplasia with sensitivities 100% (48/48), 92% (32/35), 77% (37/48), and specificities 96% (50/52), 96% (50/52), 89% (31/35) with the accuracy of 98%, 94% and 82% respectively. Sensitivity and specificity, when differentiating OSCC to normal and dysplasia to normal, are significantly large, which indicates that human saliva may be an excellent diagnostic medium for early detection of throat cancer.

  8. Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in humans

    Science.gov (United States)

    Corlu, Alper; Choe, Regine; Durduran, Turgut; Rosen, Mark A.; Schweiger, Martin; Arridge, Simon R.; Schnall, Mitchell D.; Yodh, Arjun G.

    2007-05-01

    We present three-dimensional (3D) in vivo images of human breast cancer based on fluorescence diffuse optical tomography (FDOT). To our knowledge, this work represents the first reported 3D fluorescence tomography of human breast cancer in vivo. In our protocol, the fluorophore Indocyanine Green (ICG) is injected intravenously. Fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using laser sources at 786 nm and spectrally filtered CCD detection. Phantom and in vivo studies confirm the signals are due to ICG fluorescence, rather than tissue autofluorescence and excitation light leakage. Fluorescence images of breast tumors were in good agreement with those of MRI, and with DOT based on endogenous contrast. Tumorto- normal tissue contrast based on ICG fluorescence was two-to-four-fold higher than contrast based on hemoglobin and scattering parameters. In total the measurements demonstrate that FDOT of breast cancer is feasible and promising.

  9. Fluorescent polymer-based post-translational differentiation and subtyping of breast cancer cells.

    Science.gov (United States)

    Scott, Michael D; Dutta, Rinku; Haldar, Manas K; Wagh, Anil; Gustad, Thomas R; Law, Benedict; Friesner, Daniel L; Mallik, Sanku

    2012-12-07

    Herein, we report the application of synthesized fluorescent, water soluble polymers for post-translational subtyping and differentiation of breast cancer cells in vitro. The fluorescence emission spectra from these polymers were modulated differently in the presence of conditioned cell culture media from various breast cancer cells. These polymers differentiate at a post-translation level possibly due to their ability to interact with extracellular enzymes that are over-expressed in cancerous conditions.

  10. Pathological diagnosis of bladder cancer by image analysis of hypericin induced fluorescence cystoscopic images

    Science.gov (United States)

    Kah, James C. Y.; Olivo, Malini C.; Lau, Weber K. O.; Sheppard, Colin J. R.

    2005-08-01

    Photodynamic diagnosis of bladder carcinoma based on hypericin fluorescence cystoscopy has shown to have a higher degree of sensitivity for the detection of flat bladder carcinoma compared to white light cystoscopy. The potential of the photosensitizer hypericin-induced fluorescence in performing non-invasive optical biopsy to grade bladder cancer in vivo using fluorescence cystoscopic image analysis without surgical resection for tissue biopsy is investigated in this study. The correlation between tissue fluorescence and histopathology of diseased tissue was explored and a diagnostic algorithm based on fluorescence image analysis was developed to classify the bladder cancer without surgical resection for tissue biopsy. Preliminary results suggest a correlation between tissue fluorescence and bladder cancer grade. By combining both the red-to-blue and red-to-green intensity ratios into a 2D scatter plot yields an average sensitivity and specificity of around 70% and 85% respectively for pathological cancer grading of the three different grades of bladder cancer. Therefore, the diagnostic algorithm based on colorimetric intensity ratio analysis of hypericin fluorescence cystoscopic images developed in this preliminary study shows promising potential to optically diagnose and grade bladder cancer in vivo.

  11. Early Detection of Breast Cancer by Fluorescence Molecular Tomography

    National Research Council Canada - National Science Library

    Ntziachristos, Vasilis

    2007-01-01

    .... We have successfully completed all goals and achieved the three major aims of the proposal, i.e. i) the development of appropriate fluorescence imaging methods for highly reliable and quantitative fluorescence imaging ii...

  12. Enhancing early bladder cancer detection with fluorescence-guided endoscopic optical coherence tomography

    Science.gov (United States)

    Pan, Y. T.; Xie, T. Q.; Du, C. W.; Bastacky, S.; Meyers, S.; Zeidel, M. L.

    2003-12-01

    We report an experimental study of the possibility of enhancing early bladder cancer diagnosis with fluorescence-image-guided endoscopic optical coherence tomography (OCT). After the intravesical instillation of a 10% solution of 5-aminolevulinic acid, simultaneous fluorescence imaging (excitation of 380-420 nm, emission of 620-700 nm) and OCT are performed on rat bladders to identify the photochemical and morphological changes associated with uroepithelial tumorigenesis. The preliminary results of our ex vivo study reveal that both fluorescence and OCT can identify early uroepithelial cancers, and OCT can detect precancerous lesions (e.g., hyperplasia) that fluorescence may miss. This suggests that a cystoscope combining 5-aminolevulinic acid fluorescence and OCT imaging has the potential to enhance the efficiency and sensitivity of early bladder cancer diagnosis.

  13. Stimulation of Protein Expression Through the Harmonic Resonance of Frequency-Specific Music.

    Science.gov (United States)

    Orhan, Ibrahim Y; Gulbahar, Burak A

    2016-12-01

    The use of specific frequencies for specific individual amino acids may increase the potential energy of protein molecules in the medium [1]. The resonance would also increase the movement of particles in the cytosol, increasing the collisions necessary for the conduction of protein expression. The clash of two waves that share frequencies will exhibit an increase in energy through an increase in amplitude [2]. The increase in energy would in turn increase the number of collisions forming the tRNA-amino acid, increasing the amino acid acquiry for ribosomes to improve intracellular efficiency in gene expression. To test the hypothesis, Red Fluorescent Protein (RFP) in transformated BL-21 strains of E. coli and p53 protein of MCF-7 were examined after exposure to sounds of specific frequencies. Through the exposure of the experimental systems to a sequence of sounds that match the frequencies of specific amino acids, the levels of RFP exhibition respective to the control groups in the bacterial medium increased two-fold in terms of RFU. The experiments that targeted the p53 protein with the 'music' showed a decrease in the cell prevalence in the MCF-7 type breast cancer cells by 28%, by decreasing the speed of tumour formation. Exposure to 'music' that was designed through assigning a musical note for every single one of the twenty unique amino acids, produced both an analytical and a visible shift in protein synthesis, making it as potential tool for reducing procedural time uptake.

  14. Bladder cancer diagnosis with fluorescence-image-guided optical coherence tomography

    Science.gov (United States)

    Wang, Z. G.; Durand, D. B.; Adler, H.; Pan, Y. T.

    2006-02-01

    A fluorescence-image-guided OCT (FIG-OCT) system is described, and its ability to enhance the sensitivity and specificity is examined in an animal bladder cancer model. Total 97 specimens were examined by fluorescence imaging, OCT and histological microscopy. The sensitivity and specificity of FIG-OCT is 100% and 93% respectively, compared to 79% and 53% for fluorescence imaging, while the OCT examination time has been dramatically decreased by 3~4 times. In combination of endoscopic OCT, FIG-OCT is a promising technique for effective early bladder cancer diagnosis.

  15. Mechanistic background and clinical applications of indocyanine green fluorescence imaging of hepatocellular carcinoma.

    Science.gov (United States)

    Ishizawa, Takeaki; Masuda, Koichi; Urano, Yasuteru; Kawaguchi, Yoshikuni; Satou, Shouichi; Kaneko, Junichi; Hasegawa, Kiyoshi; Shibahara, Junji; Fukayama, Masashi; Tsuji, Shingo; Midorikawa, Yutaka; Aburatani, Hiroyuki; Kokudo, Norihiro

    2014-02-01

    Although clinical applications of intraoperative fluorescence imaging of liver cancer using indocyanine green (ICG) have begun, the mechanistic background of ICG accumulation in the cancerous tissues remains unclear. In 170 patients with hepatocellular carcinoma cells (HCC), the liver surfaces and resected specimens were intraoperatively examined by using a near-infrared fluorescence imaging system after preoperative administration of ICG (0.5 mg/kg i.v.). Microscopic examinations, gene expression profile analysis, and immunohistochemical staining were performed for HCCs, which showed ICG fluorescence in the cancerous tissues (cancerous-type fluorescence), and HCCs showed fluorescence only in the surrounding non-cancerous liver parenchyma (rim-type fluorescence). ICG fluorescence imaging enabled identification of 273 of 276 (99%) HCCs in the resected specimens. HCCs showed that cancerous-type fluorescence was associated with higher cancer cell differentiation as compared with rim-type HCCs (P Fluorescence microscopy identified the presence of ICG in the canalicular side of the cancer cell cytoplasm, and pseudoglands of the HCCs showed a cancerous-type fluorescence pattern. The ratio of the gene and protein expression levels in the cancerous to non-cancerous tissues for Na(+)/taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide 8 (OATP8), which are associated with portal uptake of ICG by hepatocytes that tended to be higher in the HCCs that showed cancerous-type fluorescence than in those that showed rim-type fluorescence. Preserved portal uptake of ICG in differentiated HCC cells by NTCP and OATP8 with concomitant biliary excretion disorders causes accumulation of ICG in the cancerous tissues after preoperative intravenous administration. This enables highly sensitive identification of HCC by intraoperative ICG fluorescence imaging.

  16. Synthesis and characterization of near IR fluorescent albumin nanoparticles for optical detection of colon cancer

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, Sarit; Pellach, Michal [Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900 (Israel); Kam, Yossi [Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120 (Israel); Grinberg, Igor; Corem-Salkmon, Enav [Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900 (Israel); Rubinstein, Abraham [Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120 (Israel); Margel, Shlomo, E-mail: shlomo.margel@mail.biu.ac.il [Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900 (Israel)

    2013-03-01

    Near IR (NIR) fluorescent human serum albumin (HSA) nanoparticles hold great promise as contrast agents for tumor diagnosis. HSA nanoparticles are considered to be biocompatible, non-toxic and non-immunogenic. In addition, NIR fluorescence properties of these nanoparticles are important for in vivo tumor diagnostics, with low autofluorescence and relatively deep penetration of NIR irradiation due to low absorption of biomatrices. The present study describes the synthesis of new NIR fluorescent HSA nanoparticles, by entrapment of a NIR fluorescent dye within the HSA nanoparticles, which also significantly increases the photostability of the dye. Tumor-targeting ligands such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibodies (anti-CEA) were covalently conjugated to the NIR fluorescent albumin nanoparticles, increasing the potential fluorescent signal in tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent HSA nanoparticles was demonstrated in a chicken embryo model and a rat model. In future work we also plan to encapsulate cancer drugs such as doxorubicin within the NIR fluorescent HSA nanoparticles for both colon cancer imaging and therapy. - Highlights: Black-Right-Pointing-Pointer Near IR human serum albumin nanoparticles were synthesized and characterized. Black-Right-Pointing-Pointer Nanoparticles were shown to be physically and chemically stable and photostable. Black-Right-Pointing-Pointer Tumor-targeting ligands were covalently conjugated to the nanoparticles. Black-Right-Pointing-Pointer Specific colon cancer tumor detection was demonstrated in chicken-embryo and rat models.

  17. Synthesis and characterization of near IR fluorescent albumin nanoparticles for optical detection of colon cancer

    International Nuclear Information System (INIS)

    Cohen, Sarit; Pellach, Michal; Kam, Yossi; Grinberg, Igor; Corem-Salkmon, Enav; Rubinstein, Abraham; Margel, Shlomo

    2013-01-01

    Near IR (NIR) fluorescent human serum albumin (HSA) nanoparticles hold great promise as contrast agents for tumor diagnosis. HSA nanoparticles are considered to be biocompatible, non-toxic and non-immunogenic. In addition, NIR fluorescence properties of these nanoparticles are important for in vivo tumor diagnostics, with low autofluorescence and relatively deep penetration of NIR irradiation due to low absorption of biomatrices. The present study describes the synthesis of new NIR fluorescent HSA nanoparticles, by entrapment of a NIR fluorescent dye within the HSA nanoparticles, which also significantly increases the photostability of the dye. Tumor-targeting ligands such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibodies (anti-CEA) were covalently conjugated to the NIR fluorescent albumin nanoparticles, increasing the potential fluorescent signal in tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent HSA nanoparticles was demonstrated in a chicken embryo model and a rat model. In future work we also plan to encapsulate cancer drugs such as doxorubicin within the NIR fluorescent HSA nanoparticles for both colon cancer imaging and therapy. - Highlights: ► Near IR human serum albumin nanoparticles were synthesized and characterized. ► Nanoparticles were shown to be physically and chemically stable and photostable. ► Tumor-targeting ligands were covalently conjugated to the nanoparticles. ► Specific colon cancer tumor detection was demonstrated in chicken-embryo and rat models.

  18. Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer

    Directory of Open Access Journals (Sweden)

    Cohen Sarit

    2012-08-01

    Full Text Available Abstract Background The use of near-infrared (NIR fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. Methods The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR. Tumor-targeting ligands such as peanut agglutinin (PNA, anti-carcinoembryonic antigen antibodies (anti-CEA and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72 were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. Results and discussion Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. Conclusions These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA

  19. Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer.

    Science.gov (United States)

    Cohen, Sarit; Margel, Shlomo

    2012-08-14

    The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon

  20. Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

    Science.gov (United States)

    Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

    2016-11-01

    The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

  1. Oral cancer detection based on fluorescence polarization of blood plasma at excitation wavelength 405 nm

    Science.gov (United States)

    Pachaiappan, Rekha; Prakasarao, Aruna; Manoharan, Yuvaraj; Dornadula, Koteeswaran; Singaravelu, Ganesan

    2017-02-01

    During metabolism the metabolites such as hormones, proteins and enzymes were released in to the blood stream by the cells. These metabolites reflect any change that occurs due to any disturbances in normal metabolic function of the human system. This was well observed with the altered spectral signatures observed with fluorescence spectroscopic technique. Previously many have reported on the significance of native fluorescence spectroscopic method in the diagnosis of cancer. As fluorescence spectroscopy is sensitive and simple, it has complementary techniques such as excitation-emission matrix, synchronous and polarization. The fluorescence polarization measurement provides details about any association or binding reactions and denaturing effects that occurs due to change in the micro environment of cells and tissues. In this study, we have made an attempt in the diagnosis of oral cancer at 405 nm excitation using fluorescence polarization measurement. The fluorescence anisotropic values calculated from polarized fluorescence spectral data of normal and oral cancer subjects yielded a good accuracy when analyzed with linear discriminant analysis based artificial neural network. The results will be discussed in detail.

  2. The effects of visual fluorescence marking induced by 5-aminolevulinic acid for endoscopic diagnosis of urinary bladder cancer

    Science.gov (United States)

    Daniltchenko, Dmitri I.; Koenig, Frank; Schnorr, Dietmar; Valdman, Alexander; Al-Shukri, Salman; Loening, Stefan A.

    2003-10-01

    During cystoscopy procedure, fluorescence diagnostics induced by 5-ALA improves visual detection of the bladder cancer. Macroscopic ALA-fluorescence allows visualizing of small flat tumors, carcinoma in situ, true neoplasm margins and dysplasias of the bladder. Following ALA instillation, cystoscopy has been performed under both standard and blue light illumination. Totally, 153 biopsies have been carried out at 53 patients with suspicion of bladder cancer. The results were compared to ALA-fluorescence data. In 13% of the patients, bladder cancer and dysplasia were found out in addition, due to red fluorescence. The sensitivity and specificity of ALA-fluorescence technique aggregated 96% and 52% respectively. The sensitivity and specificity of 5-ALA-fluorescent detection exceeded standard endoscopy under white light on 20%. The new method does not exclude a false positive and a false negative fluorescent luminescence. The ALA-based fluorescence detection system enhances the diagnosis of malignant/dysplastic bladder lesions significantly.

  3. Fluorescence cystoscopy in patients with non-muscle invasive bladder cancer

    Directory of Open Access Journals (Sweden)

    I. G. Rusakov

    2015-01-01

    Full Text Available The main challenge of treating non-muscle invasive bladder cancer is multifocal tumors. Current methods of diagnosis are failed to detect all superficial flat tumor lesions in bladder mucosa. The use of fluorescence imaging with 5-aminolevulinic acid (5-ALA allows to improve the sensibility of routine cystoscopy, but low specificity decreases its diagnostic accuracy. The method of fluorescence imaging combined with local fluorescence spectroscopy developed in P.A. Herzen MCRI has been shown to increase the specificity from 71% to 84%. Thus, local fluorescence spectroscopy in visible fluorescence of 5-ALA-induced protoporphyrin allows to perform guided biopsy and decrease the rate of diagnostic mistakes. 

  4. Design of a smartphone-camera-based fluorescence imaging system for the detection of oral cancer

    Science.gov (United States)

    Uthoff, Ross

    Shown is the design of the Smartphone Oral Cancer Detection System (SOCeeDS). The SOCeeDS attaches to a smartphone and utilizes its embedded imaging optics and sensors to capture images of the oral cavity to detect oral cancer. Violet illumination sources excite the oral tissues to induce fluorescence. Images are captured with the smartphone's onboard camera. Areas where the tissues of the oral cavity are darkened signify an absence of fluorescence signal, indicating breakdown in tissue structure brought by precancerous or cancerous conditions. With this data the patient can seek further testing and diagnosis as needed. Proliferation of this device will allow communities with limited access to healthcare professionals a tool to detect cancer in its early stages, increasing the likelihood of cancer reversal.

  5. Protein expression analysis of inflammation-related colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Yasui Yumiko

    2009-01-01

    Full Text Available Background: Chronic inflammation is a risk factor for colorectal cancer (CRC development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM and dextran sodium sulfate (DSS using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight, followed by 2% (w/v DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins. Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.

  6. Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.

    Science.gov (United States)

    Bobek, Vladimir; Plachy, Jiri; Pinterova, Daniela; Kolostova, Katarina; Boubelik, Michael; Jiang, Ping; Yang, Meng; Hoffman, Robert M

    2004-01-01

    The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.

  7. Hybrid phosphorescence and fluorescence native spectroscopy for breast cancer detection.

    Science.gov (United States)

    Alimova, Alexandra; Katz, A; Sriramoju, Vidyasagar; Budansky, Yuri; Bykov, Alexei A; Zeylikovich, Roman; Alfano, R R

    2007-01-01

    Fluorescence and phosphorescence measurements are performed on normal and malignant ex vivo human breast tissues using UV LED and xenon lamp excitation. Tryptophan (trp) phosphorescence intensity is higher in both normal glandular and adipose tissue when compared to malignant tissue. An algorithm based on the ratio of trp fluorescence intensity at 345 nm to phosphorescence intensity at 500 nm is successfully used to separate normal from malignant tissue types. Normal specimens consistently exhibited a low I(345)I(500) ratio (15). The ratio analysis correlates well with histopathology. Intensity ratio maps with a spatial resolution of 0.5 mm are generated in which local regions of malignancy could be identified.

  8. Laser-induced fluorescence for the detection of esophageal and skin cancer

    Science.gov (United States)

    Vo-Dinh, Tuan; Panjehpour, Masoud; Overholt, Bergein F.; Julius, Clark E.; Overholt, Suzanne; Phan, Mary N.

    2003-07-01

    Laser-induced fluorescence (LIF) is used for in-vivo cancer diagnosis of the esophagus and skin cancer. For esophageal measurements a fiberoptic probe inserted through an endoscope was used. Autofluorescence of normal and malignant tissues were measured directly on patient skin without requiring an endoscope. Measurement of the fluorescence signal from the tissue was performed using laser excitation at 410 nm. The methodology was applied to differentiate normal and malignant tumors of the esophagus and malignant skin lesions. The results of this LIF approach were compared with histopathology results of the biopsy samples and indicated excellent agreement in the classification of normal and malignant tumors for the samples investigated.

  9. Fluorescence background subtraction technique for hybrid fluorescence molecular tomography/x-ray computed tomography imaging of a mouse model of early stage lung cancer.

    Science.gov (United States)

    Ale, Angelique; Ermolayev, Vladimir; Deliolanis, Nikolaos C; Ntziachristos, Vasilis

    2013-05-01

    The ability to visualize early stage lung cancer is important in the study of biomarkers and targeting agents that could lead to earlier diagnosis. The recent development of hybrid free-space 360-deg fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) imaging yields a superior optical imaging modality for three-dimensional small animal fluorescence imaging over stand-alone optical systems. Imaging accuracy was improved by using XCT information in the fluorescence reconstruction method. Despite this progress, the detection sensitivity of targeted fluorescence agents remains limited by nonspecific background accumulation of the fluorochrome employed, which complicates early detection of murine cancers. Therefore we examine whether x-ray CT information and bulk fluorescence detection can be combined to increase detection sensitivity. Correspondingly, we research the performance of a data-driven fluorescence background estimator employed for subtraction of background fluorescence from acquisition data. Using mice containing known fluorochromes ex vivo, we demonstrate the reduction of background signals from reconstructed images and sensitivity improvements. Finally, by applying the method to in vivo data from K-ras transgenic mice developing lung cancer, we find small tumors at an early stage compared with reconstructions performed using raw data. We conclude with the benefits of employing fluorescence subtraction in hybrid FMT-XCT for early detection studies.

  10. Genetic Polymorphisms in the EGFR (R521K and Estrogen Receptor (T594T Genes, EGFR and ErbB-2 Protein Expression, and Breast Cancer Risk in Tunisia

    Directory of Open Access Journals (Sweden)

    Imen Kallel

    2009-01-01

    Full Text Available We evaluated the association of epidermal growth factor receptor (EGFR 142285G>A (R521K and estrogen receptor alpha (ESR1 2014G>A (T594T single nucleotide polymorphisms with breast cancer risk and prognosis in Tunisian patients. EGFR 142285G>A and ESR1 2014G>A were genotyped in a sample of 148 Tunisian breast cancer patients and 303 controls using PCR-RFLP method. Immunohistochemitsry was used to evaluate the expression levels of EGFR, HER2, ESR1, progesterone receptor and BCL2 in tumors. We found no evidence for an association between EGFR R521K polymorphism and breast cancer risk. However, we found that the homozygous GG (Arg genotype was more prevalent in patients with lymph node metastasis (=.03 and high grade tumors (=.011. The ESR1 2014G allele showed significant association with breast cancer risk (=.025. The GG genotype was associated with HER2 overexpression and this association withstood univariate and multivariate analyses (=.009; =.021, resp.. These data suggest that the R521K might be a prognostic factor, because it correlates with both tumor grade and nodule status. The higher expression of HER2 in ESR1 T594T GG patients suggests the possibility that ESR1 gene polymorphisms accompanied by HER2 expression might influence the pathogenesis of breast cancers.

  11. In-vivo optical detection of cancer using chlorin e6 – polyvinylpyrrolidone induced fluorescence imaging and spectroscopy

    International Nuclear Information System (INIS)

    Chin, William WL; Thong, Patricia SP; Bhuvaneswari, Ramaswamy; Soo, Khee Chee; Heng, Paul WS; Olivo, Malini

    2009-01-01

    Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM) and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer

  12. Use of fluorescent proteins and color-coded imaging to visualize cancer cells with different genetic properties.

    Science.gov (United States)

    Hoffman, Robert M

    2016-03-01

    Fluorescent proteins are very bright and available in spectrally-distinct colors, enable the imaging of color-coded cancer cells growing in vivo and therefore the distinction of cancer cells with different genetic properties. Non-invasive and intravital imaging of cancer cells with fluorescent proteins allows the visualization of distinct genetic variants of cancer cells down to the cellular level in vivo. Cancer cells with increased or decreased ability to metastasize can be distinguished in vivo. Gene exchange in vivo which enables low metastatic cancer cells to convert to high metastatic can be color-coded imaged in vivo. Cancer stem-like and non-stem cells can be distinguished in vivo by color-coded imaging. These properties also demonstrate the vast superiority of imaging cancer cells in vivo with fluorescent proteins over photon counting of luciferase-labeled cancer cells.

  13. Molecular Detection of Bladder Cancer by Fluorescence Microsatellite Analysis and an Automated Genetic Analyzing System

    Directory of Open Access Journals (Sweden)

    Sarel Halachmi

    2007-01-01

    Full Text Available To investigate the ability of an automated fluorescent analyzing system to detect microsatellite alterations, in patients with bladder cancer. We investigated 11 with pathology proven bladder Transitional Cell Carcinoma (TCC for microsatellite alterations in blood, urine, and tumor biopsies. DNA was prepared by standard methods from blood, urine and resected tumor specimens, and was used for microsatellite analysis. After the primers were fluorescent labeled, amplification of the DNA was performed with PCR. The PCR products were placed into the automated genetic analyser (ABI Prism 310, Perkin Elmer, USA and were subjected to fluorescent scanning with argon ion laser beams. The fluorescent signal intensity measured by the genetic analyzer measured the product size in terms of base pairs. We found loss of heterozygocity (LOH or microsatellite alterations (a loss or gain of nucleotides, which alter the original normal locus size in all the patients by using fluorescent microsatellite analysis and an automated analyzing system. In each case the genetic changes found in urine samples were identical to those found in the resected tumor sample. The studies demonstrated the ability to detect bladder tumor non-invasively by fluorescent microsatellite analysis of urine samples. Our study supports the worldwide trend for the search of non-invasive methods to detect bladder cancer. We have overcome major obstacles that prevented the clinical use of an experimental system. With our new tested system microsatellite analysis can be done cheaper, faster, easier and with higher scientific accuracy.

  14. In-vivo cancer diagnosis of the esophagus using laser-induced fluorescence

    Science.gov (United States)

    Vo-Dinh, Tuan; Panjehpour, Masoud; Overholt, Bergein F.; Buckley, Paul F., II; Edwards, Donna H.

    1995-04-01

    Laser-induced fluorescence (LIF) was used for direct in-vivo cancer diagnosis of the esophagus without requiring biopsy. The methodology was applied to differentiate normal and malignant tumors of the esophagus. Endogenous fluorescence of normal and malignant tissues were measured directly using a fiberoptic probe inserted through an endoscope. The measurements were performed in vivo during routine endoscopy. Detection of the fluorescence signal from the tissue was performed using laser excitation. The results of this LIF approach were compared with histopathology results of the biopsy samples and indicated excellent agreement in the classification of normal and malignant tumors for the samples investigated. The LIF procedure could lead to the development of a rapid and cost-effective technique for cancer diagnosis.

  15. A small molecular pH-dependent fluorescent probe for cancer cell imaging in living cell.

    Science.gov (United States)

    Ma, Junbao; Li, Wenqi; Li, Juanjuan; Shi, Rongguang; Yin, Gui; Wang, Ruiyong

    2018-05-15

    A novel pH-dependent two-photon fluorescent molecular probe ABMP has been prepared based on the fluorophore of 2, 4, 6-trisubstituted pyridine. The probe has an absorption wavelength at 354 nm and corresponding emission wavelength at 475 nm with the working pH range from 2.20 to 7.00, especially owning a good liner response from pH = 2.40 to pH = 4.00. ABMP also has excellent reversibility, photostability and selectivity which promotes its ability in analytical application. The probe can be excited with a two-photon fluorescence microscopy and the fluorescence cell imaging indicated that the probe can distinguish Hela cancer cells out of normal cells with a two-photon fluorescence microscopy which suggested its potential application in tumor cell detection. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Biomarkers for ALK and ROS1 in Lung Cancer: Immunohistochemistry and Fluorescent In Situ Hybridization.

    Science.gov (United States)

    Luk, Peter P; Selinger, Christina I; Mahar, Annabelle; Cooper, Wendy A

    2018-06-14

    - A small proportion of non-small cell lung cancers harbor rearrangements of ALK or ROS1 genes, and these tumors are sensitive to targeted tyrosine kinase inhibitors. It is crucial for pathologists to accurately identify tumors with these genetic alterations to enable patients to access optimal treatments and avoid unnecessary side effects of less effective agents. Although a number of different techniques can be used to identify ALK- and ROS1-rearranged lung cancers, immunohistochemistry and fluorescence in situ hybridization are the mainstays. - To review the role of immunohistochemistry in assessment of ALK and ROS1 rearrangements in lung cancer, focusing on practical issues in comparison with other modalities such as fluorescence in situ hybridization. - This manuscript reviews the current literature on ALK and ROS1 detection using immunohistochemistry and fluorescence in situ hybridization as well as current recommendations. - Although fluorescence in situ hybridization remains the gold standard for detecting ALK and ROS1 rearrangement in non-small cell lung cancer, immunohistochemistry plays an important role and can be an effective screening method for detection of these genetic alterations, or a diagnostic test in the setting of ALK.

  17. Current Concepts and Future Perspectives on Intraoperative Fluorescence Imaging in Cancer : Clinical Need

    NARCIS (Netherlands)

    van Dam, Gooitzen M.; Ntziachristos, Vasilis

    Progress with technology and regulatory approvals has recently allowed the successful clinical translation of fluorescence molecular imaging to intra-operative applications. Initial studies have demonstrated a promising outlook for imaging cancer micro-foci, margins and lymph-nodes. However, not all

  18. Dual-Color Fluorescence Imaging of Magnetic Nanoparticles in Live Cancer Cells Using Conjugated Polymer Probes

    Science.gov (United States)

    Sun, Minjie; Sun, Bin; Liu, Yun; Shen, Qun-Dong; Jiang, Shaojun

    2016-01-01

    Rapid growth in biological applications of nanomaterials brings about pressing needs for exploring nanomaterial-cell interactions. Cationic blue-emissive and anionic green-emissive conjugated polymers are applied as dual-color fluorescence probes to the surface of negatively charged magnetic nanoparticles through sequentially electrostatic adsorption. These conjugated polymers have large extinction coefficients and high fluorescence quantum yield (82% for PFN and 62% for ThPFS). Thereby, one can visualize trace amount (2.7 μg/mL) of fluorescence-labeled nanoparticles within cancer cells by confocal laser scanning microscopy. Fluorescence labeling by the conjugated polymers is also validated for quantitative determination of the internalized nanoparticles in each individual cell by flow cytometry analysis. Extensive overlap of blue and green fluorescence signals in the cytoplasm indicates that both conjugated polymer probes tightly bind to the surface of the nanoparticles during cellular internalization. The highly charged and fluorescence-labeled nanoparticles non-specifically bind to the cell membranes, followed by cellular uptake through endocytosis. The nanoparticles form aggregates inside endosomes, which yields a punctuated staining pattern. Cellular internalization of the nanoparticles is dependent on the dosage and time. Uptake efficiency can be enhanced three-fold by application of an external magnetic field. The nanoparticles are low cytotoxicity and suitable for simultaneously noninvasive fluorescence and magnetic resonance imaging application. PMID:26931282

  19. HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

    Science.gov (United States)

    Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

    2014-05-01

    The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

  20. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  1. Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

    Science.gov (United States)

    Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

    2015-03-01

    With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

  2. Calibrating the imaging and therapy performance of magneto-fluorescent gold nanoshells for breast cancer

    Science.gov (United States)

    Dowell, Adam; Chen, Wenxue; Biswal, Nrusingh; Ayala-Orozco, Ciceron; Giuliano, Mario; Schiff, Rachel; Halas, Naomi J.; Joshi, Amit

    2012-03-01

    Gold nanoshells with NIR plasmon resonance can be modified to simultaneously enhance conjugated NIR fluorescence dyes and T2 contrast of embedded iron-oxide nanoparticles, and molecularly targeted to breast and other cancers. We calibrated the theranostic performance of magneto-fluorescent nanoshells, and contrasted the performance of molecularly targeted and untargeted nanoshells for breast cancer therapy, employing MCF-7L and their HER2 overexpressing derivative MCF-7/HER2-18 breast cancer cells as in vitro model systems. Silica core gold nanoshells with plasmon resonance on ~810 nm were doped with NIR dye ICG and ~10 nm iron-oxide nanoparticles in a ~20 nm epilayer of silica. A subset of nanoshells was conjugated to antibodies targeting HER2. Cell viability with varying laser power levels in presence and absence of bare and HER2-targeted nanoshells was assessed by calcein and propidium iodide staining. For MCF-7L cells, increasing power resulted in increased cell death (F=5.63, p=0.0018), and bare nanoshells caused more cell death than HER2-targeted nanoshells or laser treatment alone (F=30.13, pmagneto-fluorescent nanocomplexes for imaging and therapy of breast cancer cells, and the advantages of targeting receptors unique to cancer cells.

  3. In vivo fluorescence enhanced optical tomography reconstruction of lung cancer of non immersed small animals

    Science.gov (United States)

    Hervé, L.; Koenig, A.; Da Silva, A.; Berger, M.; Boutet, J.; Dinten, J. M.; Peltié, P.; Rizo, P.

    2007-02-01

    Fluorescence enhanced diffuse optical tomography (fDOT) is envisioned to be useful to collect functional information from small animal models. For oncology applications, cancer-targeted fluorescent markers can be used as a surrogate of the cancer activity. We are developing a continuous wave fDOT bench intended to be integrated in systems dedicated to whole body small animal fluorescence analyses. The focus is currently put on the reconstruction of non immersed small animals imaged by a CCD camera. The reconstruction stage already corrects the tissue heterogeneity artifacts through the computation of an optical heterogeneity map. We will show how this formalism coupled with the determination of the animal boundaries performed by a laser scanner, can be used to manage non contact acquisitions. The time of reconstruction for a 10 × 9 laser source positions, 45 × 40 detector elements and 14 × 11 × 14 mesh voxels is typically 10 minutes on a 3GHz PCs corresponding to the acquisition time allowing the two tasks to be performed in parallel. The system is validated on an in vivo experiment performed on three healthy nude mice and a mouse bearing a lung tumor at 10, 12 and 14 days after implantation allowing the follow up of the disease. The 3D fluorescence reconstructions of this mouse are presented and the total fluorescence amounts are compared.

  4. Optical diagnostic of breast cancer using Raman, polarimetric and fluorescence spectroscopy

    Science.gov (United States)

    Anwar, Shahzad; Firdous, Shamaraz; Rehman, Aziz-ul; Nawaz, Muhammed

    2015-04-01

    We presented the optical diagnostic of normal and cancerous human breast tissues using Raman, polarimetric and fluorescence spectroscopic techniques. Breast cancer is the second leading cause of cancer death among women worldwide. Optical diagnostics of cancer offered early intervention and the greatest chance of cure. Spectroscopic data were collected from freshly excised surgical specimens of normal tissues with Raman bands at 800, 1171 and 1530 cm-1 arising mainly by lipids, nucleic acids, proteins, carbohydrates and amino acids. For breast cancer, Raman bands are observed at 1070, 1211, 1495, 1583 and 1650 cm-1. Results demonstrate that the spectra of normal tissue are dominated by lipids and amino acids. Polarization decomposition of the Mueller matrix and confocal microscopic fluorescence provides detailed description of cancerous tissue and distinguishes between the normal and malignant one. Based on these findings, we successfully differentiate normal and malignant breast tissues at an early stage of disease. There is a need to develop a new tool for noninvasive, real-time diagnosis of tissue abnormalities and a test procedure for detecting breast cancer at an early stage.

  5. Aptamer conjugated paclitaxel and magnetic fluid loaded fluorescently tagged PLGA nanoparticles for targeted cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Aravind, Athulya; Nair, Remya; Raveendran, Sreejith; Veeranarayanan, Srivani; Nagaoka, Yutaka; Fukuda, Takahiro; Hasumura, Takahashi; Morimoto, Hisao; Yoshida, Yasuhiko; Maekawa, Toru; Sakthi Kumar, D., E-mail: sakthi@toyo.jp

    2013-10-15

    Controlled and targeted drug delivery is an essential criterion in cancer therapy to reduce the side effects caused by non-specific drug release and toxicity. Targeted chemotherapy, sustained drug release and optical imaging have been achieved using a multifunctional nanocarrier constructed from poly (D, L-lactide-co-glycolide) nanoparticles (PLGA NPs), an anticancer drug paclitaxel (PTX), a fluorescent dye Nile red (NR), magnetic fluid (MF) and aptamers (Apt, AS1411, anti-nucleolin aptamer). The magnetic fluid and paclitaxel loaded fluorescently labeled PLGA NPs (MF-PTX-NR-PLGA NPs) were synthesized by a single-emulsion technique/solvent evaporation method using a chemical cross linker bis (sulfosuccinimidyl) suberate (BS3) to enable binding of aptamer on to the surface of the nanoparticles. Targeting aptamers were then introduced to the particles through the reaction with the cross linker to target the nucleolin receptors over expressed on the cancer cell surface. Specific binding and uptake of the aptamer conjugated magnetic fluid loaded fluorescently tagged PLGA NPs (Apt-MF-NR-PLGA NPs) to the target cancer cells induced by aptamers was observed using confocal microscopy. Cytotoxicity assay conducted in two cell lines (L929 and MCF-7) confirmed that targeted MCF-7 cancer cells were killed while control cells were unharmed. In addition, aptamer mediated delivery resulting in enhanced binding and uptake to the target cancer cells exhibited increased therapeutic effect of the drug. Moreover, these aptamer conjugated magnetic polymer vehicles apart from actively transporting drugs into specifically targeted tumor regions can also be used to induce hyperthermia or for facilitating magnetic guiding of particles to the tumor regions. - Highlights: • Aptamer escorted, theranostic biodegradable PLGA carriers were developed. • Can target cancer cells, control drug release, image and magnetically guide. • Highly specific to the targeted cancer cells thus delivering

  6. Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

    Science.gov (United States)

    Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

    2014-01-01

    The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

  7. Colorectal cancer detection by hyperspectral imaging using fluorescence excitation scanning

    Science.gov (United States)

    Leavesley, Silas J.; Deal, Joshua; Hill, Shante; Martin, Will A.; Lall, Malvika; Lopez, Carmen; Rider, Paul F.; Rich, Thomas C.; Boudreaux, Carole W.

    2018-02-01

    Hyperspectral imaging technologies have shown great promise for biomedical applications. These techniques have been especially useful for detection of molecular events and characterization of cell, tissue, and biomaterial composition. Unfortunately, hyperspectral imaging technologies have been slow to translate to clinical devices - likely due to increased cost and complexity of the technology as well as long acquisition times often required to sample a spectral image. We have demonstrated that hyperspectral imaging approaches which scan the fluorescence excitation spectrum can provide increased signal strength and faster imaging, compared to traditional emission-scanning approaches. We have also demonstrated that excitation-scanning approaches may be able to detect spectral differences between colonic adenomas and adenocarcinomas and normal mucosa in flash-frozen tissues. Here, we report feasibility results from using excitation-scanning hyperspectral imaging to screen pairs of fresh tumoral and nontumoral colorectal tissues. Tissues were imaged using a novel hyperspectral imaging fluorescence excitation scanning microscope, sampling a wavelength range of 360-550 nm, at 5 nm increments. Image data were corrected to achieve a NIST-traceable flat spectral response. Image data were then analyzed using a range of supervised and unsupervised classification approaches within ENVI software (Harris Geospatial Solutions). Supervised classification resulted in >99% accuracy for single-patient image data, but only 64% accuracy for multi-patient classification (n=9 to date), with the drop in accuracy due to increased false-positive detection rates. Hence, initial data indicate that this approach may be a viable detection approach, but that larger patient sample sizes need to be evaluated and the effects of inter-patient variability studied.

  8. Multi-spectral and fluorescence diffuse optical tomography of breast cancer

    Science.gov (United States)

    Corlu, Alper

    Multi-spectral and fluorescence diffuse optical tomography (DOT) techniques are explored and applied to image human breast cancer in vivo. Image reconstruction algorithms that utilize first and second order gradient information are described in detail. Breast DOT requires large computational memory and long run times. To this end, parallel computation techniques were developed appropriate to each reconstruction algorithm. A parallel plate DOT instrument developed for breast cancer imaging is described. The system relies heavily on continuous-wave (CW) transmission measurements and utilizes frequency domain (FD) measurements on the reemission side. However, traditional DOT image reconstruction methods based on CW measurements fail to separate tissue absorption and scattering uniquely. In this manuscript, multi-spectral DOT is shown to be capable of minimizing cross-talk and retrieving spectral parameters almost uniquely when the measurement wavelengths are optimized. A theoretical framework to select optimum wavelengths is provided, and tested with computer simulations. Results from phantom spectroscopy experiments and in vivo patient measurements support the notion that multi-spectral methods are superior to traditional DOT image reconstruction schemes. The same breast DOT instrument is improved and utilized to obtain the first in vivo images of human breast cancer based on fluorescence DOT (FDOT). To this end the fluorophore Indocyanine Green (ICG) is injected intravenously and fluorescence excitation and detection are accomplished in the soft-compression, parallel-plane, transmission geometry using laser sources at 786 nm and spectrally filtered CCD detection. Careful phantom and in vivo measurements are carried on to assure that the signals are due to ICG fluorescence, rather than tissue autofluorescence and excitation light leakage. An in vivo measurement protocol is designed to maximize the ICG contrast by acquiring full fluorescence tomographic scan during

  9. Automated detection of breast cancer in resected specimens with fluorescence lifetime imaging

    Science.gov (United States)

    Phipps, Jennifer E.; Gorpas, Dimitris; Unger, Jakob; Darrow, Morgan; Bold, Richard J.; Marcu, Laura

    2018-01-01

    Re-excision rates for breast cancer lumpectomy procedures are currently nearly 25% due to surgeons relying on inaccurate or incomplete methods of evaluating specimen margins. The objective of this study was to determine if cancer could be automatically detected in breast specimens from mastectomy and lumpectomy procedures by a classification algorithm that incorporated parameters derived from fluorescence lifetime imaging (FLIm). This study generated a database of co-registered histologic sections and FLIm data from breast cancer specimens (N  =  20) and a support vector machine (SVM) classification algorithm able to automatically detect cancerous, fibrous, and adipose breast tissue. Classification accuracies were greater than 97% for automated detection of cancerous, fibrous, and adipose tissue from breast cancer specimens. The classification worked equally well for specimens scanned by hand or with a mechanical stage, demonstrating that the system could be used during surgery or on excised specimens. The ability of this technique to simply discriminate between cancerous and normal breast tissue, in particular to distinguish fibrous breast tissue from tumor, which is notoriously challenging for optical techniques, leads to the conclusion that FLIm has great potential to assess breast cancer margins. Identification of positive margins before waiting for complete histologic analysis could significantly reduce breast cancer re-excision rates.

  10. [Place of indocyanine green coupled with fluorescence imaging in research of breast cancer sentinel node].

    Science.gov (United States)

    Vermersch, Charlotte; Raia Barjat, Tiphaine; Perrot, Marianne; Lima, Suzanne; Chauleur, Céline

    2016-04-01

    The sentinel node has a fundamental role in the management of early breast cancer. Currently, the double detection of blue and radioisotope is recommended. But in common practice, many centers use a single method. However, with a single detection, the risk of false negatives and the identification failure rate increase to a significant extent and the number of sentinel lymph node detected and removed is not enough. Furthermore, the tracers used until now show inconveniences. The purpose of this work is to present a new method of detection, using the green of indocyanine coupled with fluorescence imaging, and to compare it with the already existing methods. The method combined by fluorescence and isotopic is reliable, sure, of fast learning and could constitute a good strategy of detection. The major interest is to obtain a satisfactory number of sentinel nodes. The profit could be even more important for overweight patients. The fluorescence used alone is at the moment not possible. Wide ranging studies are necessary. The FLUOTECH, randomized study of 100 patients, comparing the isotopic method of double isotope technique and fluorescence, is underway to confirm these data. Copyright © 2016 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

  11. Detection of Colorectal Cancer by a Quantitative Fluorescence Determination of DNA Amplification in Stool

    Directory of Open Access Journals (Sweden)

    Daniele Calistri

    2004-09-01

    Full Text Available DNA amplification of exfoliated cells in stool repre sents an inexpensive and rapid test, but has only 50% to 60% sensitivity. A new quantitative method, calle( fluorescence long DNA, was developed and validate( in our laboratory on stool obtained from 86 patient., with primary colorectal cancer and from 62 health individuals. It consists of the amplification of stoo DNA with fluorescence primers and the quantification of the amplification using a standard curve. Results are arbitrarily expressed in nanograms. The potential of thi new method compared to the conventional approact was analyzed in a subgroup of 94 individuals (51 patients and 38 healthy volunteers. In the presen series, DNA amplification analysis showed a specific ity of 97% and a sensitivity of only 50%. Conversely fluorescence DNA evaluation, using the best cutoff o 25 ng, showed a sensitivity of about 76% and a spec ificity of 93%. Similar sensitivity was observed regard less of Dukes stage, tumor location, and size, thu., also permitting the detection of early-stage tumors The present study seems to indicate that quantitative fluorescence DNA determination in stool successfully identifies colorectal cancer patients with a sensitivity comparable, if not superior, to that of multiple gene analysis but at a lower cost and in a shorter time.

  12. Fluorescence lifetime imaging of endogenous molecules in live mouse cancer models (Conference Presentation)

    Science.gov (United States)

    Svindrych, Zdenek; Wang, Tianxiong; Hu, Song; Periasamy, Ammasi

    2017-02-01

    NADH and FAD are important endogenous fluorescent coenzymes participating in key enzymatic reactions of cellular metabolism. While fluorescence intensities of NADH and FAD have been used to determine the redox state of cells and tissues, this simple approach breaks down in the case of deep-tissue intravital imaging due to depth- and wavelength-dependent light absorption and scattering. To circumvent this limitation, our research focuses on fluorescence lifetimes of two-photon excited NADH and FAD emission to study the metabolic state of live tissues. In our custom-built scanning microscope we combine tunable femtosecond Ti:sapphire laser (operating at 740 nm for NADH excitation and 890 nm for FAD excitation), two GaAsP hybrid detectors for registering individual fluorescence photons and two Becker and Hickl time correlator boards for high precision lifetime measurements. Together with our rigorous FLIM analysis approach (including image segmentation, multi-exponential decay fitting and detailed statistical analysis) we are able to detect metabolic changes in cancer xenografts (human pancreatic cancer MPanc96 cells injected subcutaneously into the ear of an immunodeficient nude mouse), relative to surrounding healthy tissue. Advantageously, with the same instrumentation we can also take high-resolution and high-contrast images of second harmonic signal (SHG) originating from collagen fibers of both the healthy skin and the growing tumor. The combination of metabolic measurements (NADH and FAD lifetime) and morphological information (collagen SHG) allows us to follow the tumor growth in live mouse model and the changes in tumor microenvironment.

  13. Automated Analysis of Protein Expression and Gene Amplification within the Same Cells of Paraffin-Embedded Tumour Tissue

    Directory of Open Access Journals (Sweden)

    Timo Gaiser

    2010-01-01

    Full Text Available Background: The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC and Fluorescence in situ Hybridization (FISH negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell.

  14. In-vivo fluorescence detection of breast cancer growth factor receptors by fiber-optic probe

    Science.gov (United States)

    Bustamante, Gilbert; Wang, Bingzhi; DeLuna, Frank; Sun, LuZhe; Ye, Jing Yong

    2018-02-01

    Breast cancer treatment options often include medications that target the overexpression of growth factor receptors, such as the proto-oncogene human epidermal growth factor receptor 2 (HER2/neu) and epidermal growth factor receptor (EGFR) to suppress the abnormal growth of cancerous cells and induce cancer regression. Although effective, certain treatments are toxic to vital organs, and demand assurance that the pursued receptor is present at the tumor before administration of the drug. This requires diagnostic tools to provide tumor molecular signatures, as well as locational information. In this study, we utilized a fiber-optic probe to characterize in vivo HER2 and EGFR overexpressed tumors through the fluorescence of targeted dyes. HER2 and EGFR antibodies were conjugated with ICG-Sulfo-OSu and Alexa Fluor 680, respectively, to tag BT474 (HER2+) and MDA-MB-468 (EGFR+) tumors. The fiber was inserted into the samples via a 30-gauge needle. Different wavelengths of a supercontinuum laser were selected to couple into the fiber and excite the corresponding fluorophores in the samples. The fluorescence from the dyes was collected through the same fiber and quantified by a time-correlated single photon counter. Fluorescence at different antibody-dye concentrations was measured for calibration. Mice with subcutaneous HER2+ and/or EGFR+ tumors received intravenous injections of the conjugates and were later probed at the tumor sites. The measured fluorescence was used to distinguish between tumor types and to calculate the concentration of the antibody-dye conjugates, which were detectable at levels as low as 40 nM. The fiber-optic probe presents a minimally invasive instrument to characterize the molecular signatures of breast cancer in vivo.

  15. Multifunctional fluorescent iron quantum clusters for non-invasive radiofrequency ablationof cancer cells.

    Science.gov (United States)

    Jose, Akhila; Surendran, Mrudula; Fazal, Sajid; Prasanth, Bindhu-Paul; Menon, Deepthy

    2018-05-01

    This work reports the potential of iron quantum clusters (FeQCs) as a hyperthermia agent for cancer, by testing its in-vitro response to shortwave (MHz range), radiofrequency (RF) waves non-invasively. Stable, fluorescent FeQCs of size ∼1 nm prepared by facile aqueous chemistry from endogenous protein haemoglobin were found to give a high thermal response, with a ΔT ∼50 °C at concentrationsas low as165 μg/mL. The as-prepared nanoclusters purified by lyophilization as well as dialysis showed a concentration, power and time-dependent RF response, with the lyophilized FeQCs exhibiting pronounced heating effects. FeQCs were found to be cytocompatible to NIH-3T3 fibroblast and 4T1 cancer cells treated at concentrations upto 1000 μg/mL for 24 h. Upon incubation with FeQCs and exposure to RF waves, significant cancer cell death was observed which proves its therapeutic ability. The fluorescent ability of the clusters could additionally be utilized for imaging cancer cells upon excitation at ∼450 nm. Further, to demonstrate the feasibility of imparting additional functionality such as drug/biomolecule/dye loading to FeQCs, they were self assembled with cationic polymers to form nanoparticles. Self assembly did not alter the RF heating potential of FeQCs and additionally enhanced its fluorescence. The multifunctional fluorescent FeQCs therefore show good promise as a novel therapeutic agent for RF hyperthermia and drug loading. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Designing the nanobiointerface of fluorescent nanodiamonds: highly selective targeting of glioma cancer cells.

    Science.gov (United States)

    Slegerova, Jitka; Hajek, Miroslav; Rehor, Ivan; Sedlak, Frantisek; Stursa, Jan; Hruby, Martin; Cigler, Petr

    2015-01-14

    Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin αvβ3 receptors on glioblastoma cells with high internalization efficacy.

  17. Role of fluorescence study in diagnosis of reccurent skin cancer (case report

    Directory of Open Access Journals (Sweden)

    E. V. Filonenko

    2013-01-01

    Full Text Available The case of diagnosis of recurrent skin cancer using fluorescence methods is reported. The patient who underwent multiple courses of treatment (surgical resection, cryotherapy and laser ablation for multifocal basal cell carcinoma of head. trunk and extremities. The patient represented with multiple whitish scars in the region of surgery, cryotherapy and laser ablation (one of them was in the region of external ear and with multiple foci of hyperemia and skin peeling. The patient underwent fluorescence diagnosis according to developed regimen: alasens was given per os at a dose of 30 mg/kg body weight 4 h before study. In the white light there were no features of recurrent tumor in scar areas. During fluorescence examination there was bright red fluorescence in the area of ear scar, which was intact visually, with no additional fluorescence foci in regions of surgical resection, cryotherapy and laser ablation and other regions. According to cytological study of shave biopsy from fluorescecnse area of ear scar the recurrence of basal cell skin carcinoma was diagnosed. 

  18. In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment.

    Science.gov (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

    2014-07-01

    Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. ©2014 American Association for Cancer Research.

  19. Detection and characterization of stomach cancer and atrophic gastritis with fluorescence and Raman spectroscopy

    Science.gov (United States)

    Li, Xiaozhou; Lin, Junxiu; Jia, Chunde; Wang, Rong

    2003-12-01

    In this paper, we attempt to find a valid method to distinguish gastric cancer and atrophic gastritis. Auto-fluorescence and Raman spectroscopy of laser induced (514.5 nm and 488.0 nm) was measured. The serum spectrum is different between normal and cancer. Average value of diagnosis parameter for normal serum, red shift is less than 12 nm and Raman relative intensity of peak C by 514.5 nm excited is stronger than that of 488.0 nm. To gastric cancer, its red shift of average is bigger than 12 nm and relative intensity of Raman peak C by 514.5 nm excited is weaker than that by 488.0 nm. To atrophic gastritis, the distribution state of Raman peaks is similar with normal serum and auto-fluorescence spectrum's shape is similar to that of gastric cancer. Its average Raman peak red shift is bigger than 12 nm and the relative intensity of peak C by 514.5 excited is stronger than that of by 488.0. We considered it as a criterion and got an accuracy of 85.6% for diagnosis of gastric cancer compared with the result of clinical diagnosis.

  20. Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells

    Directory of Open Access Journals (Sweden)

    Sarah Inwood

    2018-01-01

    Full Text Available The ability to produce recombinant proteins by utilizing different “cell factories” revolutionized the biotherapeutic and pharmaceutical industry. Chinese hamster ovary (CHO cells are the dominant industrial producer, especially for antibodies. Human embryonic kidney cells (HEK, while not being as widely used as CHO cells, are used where CHO cells are unable to meet the needs for expression, such as growth factors. Therefore, improving recombinant protein expression from mammalian cells is a priority, and continuing effort is being devoted to this topic. Non-coding RNAs are RNA segments that are not translated into a protein and often have a regulatory role. Since their discovery, major progress has been made towards understanding their functions. Non-coding RNA has been investigated extensively in relation to disease, especially cancer, and recently they have also been used as a method for engineering cells to improve their protein expression capability. In this review, we provide information about methods used to identify non-coding RNAs with the potential of improving recombinant protein expression in mammalian cell lines.

  1. Engineering of near infrared fluorescent proteinoid-poly(L-lactic acid) particles for in vivo colon cancer detection.

    Science.gov (United States)

    Kolitz-Domb, Michal; Grinberg, Igor; Corem-Salkmon, Enav; Margel, Shlomo

    2014-08-12

    The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer owing to the negligible absorption and autofluorescence of water and other intrinsic biomolecules in this region. The main aim of the present study is to synthesize and characterize novel NIR fluorescent nanoparticles based on proteinoid and PLLA for early detection of colon tumors. The present study describes the synthesis of new proteinoid-PLLA copolymer and the preparation of NIR fluorescent nanoparticles for use in diagnostic detection of colon cancer. These fluorescent nanoparticles were prepared by a self-assembly process in the presence of the NIR dye indocyanine green (ICG), a FDA-approved NIR fluorescent dye. Anti-carcinoembryonic antigen antibody (anti-CEA), a specific tumor targeting ligand, was covalently conjugated to the P(EF-PLLA) nanoparticles through the surface carboxylate groups using the carbodiimide activation method. The P(EF-PLLA) nanoparticles are stable in different conditions, no leakage of the encapsulated dye into PBS containing 4% HSA was detected. The encapsulation of the NIR fluorescent dye within the P(EF-PLLA) nanoparticles improves significantly the photostability of the dye. The fluorescent nanoparticles are non-toxic, and the biodistribution study in a mouse model showed they evacuate from the body over 24 h. Specific colon tumor detection in a chicken embryo model and a mouse model was demonstrated for anti-CEA-conjugated NIR fluorescent P(EF-PLLA) nanoparticles. The results of this study suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent P(EF-PLLA) nanoparticles over colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs such as paclitaxel and/or doxorubicin, within these biodegradable NIR fluorescent P(EF-PLLA) nanoparticles, for both detection and therapy of colon cancer.

  2. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    International Nuclear Information System (INIS)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille; Rochel, Natacha; Mély, Yves; Didier, Pascal; Weiss, Etienne

    2013-01-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins

  3. In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

    Science.gov (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

    2012-01-01

    One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

  4. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

    2013-04-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

  5. In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

    Directory of Open Access Journals (Sweden)

    Yasaman Ardeshirpour

    Full Text Available One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

  6. Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

    Science.gov (United States)

    Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

    2017-08-08

    Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

  7. Targeting of Pancreatic Cancer with Magneto-Fluorescent Theranostic Gold Nanoshells

    Science.gov (United States)

    Chen, Wenxue; Ayala-Orozco, Ciceron; Biswal, Nrusingh C.; Perez-Torres, Carlos; Bartels, Marc; Bardhan, Rizia; Stinnet, Gary; Liu, Xian-De; Ji, Baoan; Deorukhkar, Amit; Brown, Lisa V.; Guha, Sushovan; Pautler, Robia G.; Krishnan, Sunil; Halas, Naomi J; Joshi, Amit

    2014-01-01

    Aim We report a magneto-fluorescent theranostic nanocomplex targeted to neutrophil gelatinase associated lipocalin (NGAL) for imaging and therapy of pancreatic cancer. Materials and Methods Gold nanoshells resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the NIR dye ICG, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL in AsPC-1-derived xenografts in nude mice. Results AntiNGAL-conjugated TGNS specifically targeted pancreatic cancer cells in vitro and in vivo providing contrast for both NIR fluorescence and T2 weighted MR imaging with higher tumor contrast than can be obtained using long-circulating but non-targeted PEGylated nanoparticles. The nanocomplexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. Conclusions Theranostic gold nanoshells with embedded NIR and MR contrasts can be specifically targeted to pancreatic cancer cells with expression of early disease marker NGAL, and enable molecularly targeted imaging and photothermal therapy. PMID:24063415

  8. Tracking and Finding Slow-Proliferating/Quiescent Cancer Stem Cells with Fluorescent Nanodiamonds.

    Science.gov (United States)

    Lin, Hsin-Hung; Lee, Hsiao-Wen; Lin, Ruey-Jen; Huang, Chih-Wei; Liao, Yi-Chun; Chen, Yit-Tsong; Fang, Jim-Min; Lee, Te-Chang; Yu, Alice L; Chang, Huan-Cheng

    2015-09-09

    Quiescent cancer stem cells (CSCs) have long been considered to be a source of tumor initiation. However, identification and isolation of these cells have been hampered by the fact that commonly used fluorescent markers are not sufficiently stable, both chemically and photophysically, to allow tracking over an extended period of time. Here, it is shown that fluorescent nanodiamonds (FNDs) are well suited for this application. Genotoxicity tests of FNDs with comet and micronucleus assays for human fibroblasts and breast cancer cells indicate that the nanoparticles neither cause DNA damage nor impair cell growth. Using AS-B145-1R breast cancer cells as the model cell line for CSC, it is found that the FND labeling outperforms 5-ethynyl-2'-deoxyuridine (EdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) in regards to its long-term tracking capability (>20 d). Moreover, through a quantification of their stem cell activity by measuring mammosphere-forming efficiencies (MFEs) and self-renewal rates, the FND-positive cells are identified to have an MFE twice as high as that of the FND-negative cells isolated from the same dissociated mammospheres. Thus, the nanoparticle-based labeling technique provides an effective new tool for tracking and finding slow-proliferating/quiescent CSCs in cancer research. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Milk-derived multi-fluorescent graphene quantum dot-based cancer theranostic system

    Energy Technology Data Exchange (ETDEWEB)

    Thakur, Mukeshchand, E-mail: mukeshchandthakur@yahoo.com [School of Biotechnology and Bioinformatics, D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra (India); N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Mewada, Ashmi [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Walchand Centre for Research in Nanotechnology and Bio-nanotechnology (wcRnb), Walchand College of Arts and Science, Walchand-Hirachand Marg, Ashok Chowk, Solapur 413006, Maharashtra (India); Pandey, Sunil, E-mail: gurus.spandey@gmail.com [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Bhori, Mustansir, E-mail: mustansyrr@gmail.com [School of Biotechnology and Bioinformatics, D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra (India); Singh, Kanchanlata [School of Biotechnology and Bioinformatics, D.Y. Patil University, Sector 15, CBD Belapur, Navi Mumbai 400 614, Maharashtra (India); Sharon, Maheshwar [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Walchand Centre for Research in Nanotechnology and Bio-nanotechnology (wcRnb), Walchand College of Arts and Science, Walchand-Hirachand Marg, Ashok Chowk, Solapur 413006, Maharashtra (India); Sharon, Madhuri, E-mail: sharonmadhuri@gmail.com [N.S.N. Research Centre for Nanotechnology and Bio-nanotechnology, Jambhul Phata, Ambernath (W) 421 504, Maharashtra (India); Walchand Centre for Research in Nanotechnology and Bio-nanotechnology (wcRnb), Walchand College of Arts and Science, Walchand-Hirachand Marg, Ashok Chowk, Solapur 413006, Maharashtra (India)

    2016-10-01

    An economical green-chemistry approach was used for the synthesis of aqueous soluble graphene quantum dots (GQDs) from cow milk for simultaneous imaging and drug delivery in cancer. The GQDs synthesized using one-pot microwave-assisted heating were multi-fluorescent, spherical in shape having a lateral size of ca. 5 nm. The role of processing parameters such as heating time and ionic strength showed a profound effect on photoluminescence properties of GQDs. The GQDs were N-doped and oxygen-rich as confirmed by X-ray photoelectron spectroscopy (XPS) analysis. Cysteamine hydrochloride (Cys) was used to attach an anti-cancer drug berberine hydrochloride (BHC) on GQDs forming GQDs@Cys-BHC complex with c.a. 88% drug loading efficiency. In vitro drug release was studied at the acidic-basic environment and drug kinetics was studied using pharmacokinetic statistical models. The GQDs were biocompatible on L929 cells whereas theranostic GQDs@Cys-BHC complex showed a potent cytotoxic effect on different cancerous cell line models: cervical cancer cell lines such as HeLa cells and breast cancer cells such as MDA-MB-231 confirmed by Trypan blue and MTT-based cytotoxic assays. Furthermore, multi-excitation based cellular bioimaging was demonstrated using confocal laser scanning microscopy (CLSM) and fluorescence microscopy using GQDs as well as GQDs@Cys-BHC complex. Thus, drug delivery (therapeutic) and bioimaging (diagnostic) properties of GQDs@Cys-BHC complex are thought to have a potential in vitro theranostic application in cancer therapy. - Highlights: • Facile green synthesis of bright dual-florescent GQDs using cow milk as a precursor • Microwave irradiation time and pH have profound effects on fluorescent properties of GQDs. • Decoration of anti-cancer drug BHC onto GQDs via Cys-linker as theranostic platform • A pH responsive in vitro anti-cancer drug release and drug release kinetic study • Multi-photon bioimaging, cell cycle analysis, and apoptosis study

  10. Milk-derived multi-fluorescent graphene quantum dot-based cancer theranostic system

    International Nuclear Information System (INIS)

    Thakur, Mukeshchand; Mewada, Ashmi; Pandey, Sunil; Bhori, Mustansir; Singh, Kanchanlata; Sharon, Maheshwar; Sharon, Madhuri

    2016-01-01

    An economical green-chemistry approach was used for the synthesis of aqueous soluble graphene quantum dots (GQDs) from cow milk for simultaneous imaging and drug delivery in cancer. The GQDs synthesized using one-pot microwave-assisted heating were multi-fluorescent, spherical in shape having a lateral size of ca. 5 nm. The role of processing parameters such as heating time and ionic strength showed a profound effect on photoluminescence properties of GQDs. The GQDs were N-doped and oxygen-rich as confirmed by X-ray photoelectron spectroscopy (XPS) analysis. Cysteamine hydrochloride (Cys) was used to attach an anti-cancer drug berberine hydrochloride (BHC) on GQDs forming GQDs@Cys-BHC complex with c.a. 88% drug loading efficiency. In vitro drug release was studied at the acidic-basic environment and drug kinetics was studied using pharmacokinetic statistical models. The GQDs were biocompatible on L929 cells whereas theranostic GQDs@Cys-BHC complex showed a potent cytotoxic effect on different cancerous cell line models: cervical cancer cell lines such as HeLa cells and breast cancer cells such as MDA-MB-231 confirmed by Trypan blue and MTT-based cytotoxic assays. Furthermore, multi-excitation based cellular bioimaging was demonstrated using confocal laser scanning microscopy (CLSM) and fluorescence microscopy using GQDs as well as GQDs@Cys-BHC complex. Thus, drug delivery (therapeutic) and bioimaging (diagnostic) properties of GQDs@Cys-BHC complex are thought to have a potential in vitro theranostic application in cancer therapy. - Highlights: • Facile green synthesis of bright dual-florescent GQDs using cow milk as a precursor • Microwave irradiation time and pH have profound effects on fluorescent properties of GQDs. • Decoration of anti-cancer drug BHC onto GQDs via Cys-linker as theranostic platform • A pH responsive in vitro anti-cancer drug release and drug release kinetic study • Multi-photon bioimaging, cell cycle analysis, and apoptosis study

  11. Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

    Science.gov (United States)

    Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

    2017-01-01

    Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. PMID:28956810

  12. Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

    Directory of Open Access Journals (Sweden)

    Michael Hausmann

    2017-09-01

    Full Text Available Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2 in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

  13. A novel approach for the detection of early gastric cancer: fluorescence spectroscopy of gastric juice.

    Science.gov (United States)

    Deng, Kai; Zhou, Li Ya; Lin, San Ren; Li, Yuan; Chen, Mo; Geng, Qiu Ming; Li, Yu Wen

    2013-06-01

    This study aimed to investigate the efficacy of fluorescence spectroscopy of gastric juice for early gastric cancer (EGC) screening. Gastric juice was collected from 101 participants who underwent endoscopy in the Outpatient Endoscopy Center of Peking University Third Hospital. The participants were divided into three groups: the normal mucosa or chronic non-atrophic gastritis (NM-CNAG) group (n = 35), advanced gastric cancer (AGC) group (n = 33) and EGC group (n = 33). Fluorescence spectroscopic analysis was performed in all the gastric juice samples and the maximum fluorescence intensity of the first peak (P1 FI) was measured. The mean fluorescence intensity of P1 FI of gastric juice in AGC (92.1 ± 10.7) and EGC (90.8 ± 12.0) groups was significantly higher than that in the NM-CNAG group (55.7 ± 7.5) (AGC vs NM-CNAG, P = 0.006 and EGC vs NM-CNAG, P = 0.015, respectively). The areas under the receiver operating characteristic curves for the detection of AGC and EGC were 0.681 (95% confidence interval [CI] 0.553-0.810, P = 0.010) and 0.655 (95% CI 0.522-0.787, P = 0.028). With the P1 FI of ≥47.7, the sensitivity, specificity and accuracy for detecting EGC were 69.7%, 57.1% and 63.2%, respectively. The enhancement of P1 FI of gastric juice occurs at the early stage of gastric cancer. Fluorescence spectroscopy of gastric juice may be used as a novel screening tool for the early detection of gastric cancer. © 2013 The Authors. Journal of Digestive Diseases © 2013 Wiley Publishing Asia Pty Ltd and Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine.

  14. In situ protein expression in tumour spheres: development of an immunostaining protocol for confocal microscopy

    International Nuclear Information System (INIS)

    Weiswald, Louis-Bastien; Guinebretière, Jean-Marc; Richon, Sophie; Bellet, Dominique; Saubaméa, Bruno; Dangles-Marie, Virginie

    2010-01-01

    Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Protein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9 + cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini

  15. AR-v7 protein expression is regulated by protein kinase and phosphatase

    Science.gov (United States)

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  16. HER-2 protein concentrations in breast cancer cells increase before immunohistochemical and fluorescence in situ hybridization analysis turn positive

    DEFF Research Database (Denmark)

    Olsen, Dorte A; Østergaard, Birthe; Bokmand, Susanne

    2007-01-01

    BACKGROUND: The level of HER-2/neu in breast cancer cells is normally measured by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). It determines whether patients should be treated with trastuzumab (Herceptin). In this study, HER-2 protein in breast cancer tissue...

  17. Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice

    Directory of Open Access Journals (Sweden)

    Wei Xin Cai

    2016-01-01

    Full Text Available Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (n=8. A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells’ tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo.

  18. Fluorescence in situ hybridization evaluation of chromosome deletion patterns in prostate cancer.

    Science.gov (United States)

    Huang, S F; Xiao, S; Renshaw, A A; Loughlin, K R; Hudson, T J; Fletcher, J A

    1996-11-01

    Various nonrandom chromosomal aberrations have been identified in prostate carcinoma. These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm. Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases. However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deleted frequently in other common types of cancer. In this study, we performed dual-color fluorescence in situ hybridization on intact nuclei from touch preparations of 16 prostate cancers. Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chromosome (YAC) and P1 probes. Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer. Other YACs and P1s were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer. Hybridization efficiencies and signal intensities were excellent for both repeat sequence (alpha-satellite) and single, copy (YAC and P1) fluorescence in situ hybridization probes. Of 16 prostate cancers, 11 had clonal aberrations of 1 or more of the 13 chromosome regions evaluated, and 10 cases (62.5%) had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted cells. Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and 1 case, respectively, and each of those cases had a similarly sized cell population with 8p deletion. These studies confirm 8p deletion in the majority of prostate carcinomas. 8p deletions appear to be early events in prostate tumorigenesis, often antedating aneuploidy. Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and

  19. Artificial neural networks for processing fluorescence spectroscopy data in skin cancer diagnostics

    International Nuclear Information System (INIS)

    Lenhardt, L; Zeković, I; Dramićanin, T; Dramićanin, M D

    2013-01-01

    Over the years various optical spectroscopic techniques have been widely used as diagnostic tools in the discrimination of many types of malignant diseases. Recently, synchronous fluorescent spectroscopy (SFS) coupled with chemometrics has been applied in cancer diagnostics. The SFS method involves simultaneous scanning of both emission and excitation wavelengths while keeping the interval of wavelengths (constant-wavelength mode) or frequencies (constant-energy mode) between them constant. This method is fast, relatively inexpensive, sensitive and non-invasive. Total synchronous fluorescence spectra of normal skin, nevus and melanoma samples were used as input for training of artificial neural networks. Two different types of artificial neural networks were trained, the self-organizing map and the feed-forward neural network. Histopathology results of investigated skin samples were used as the gold standard for network output. Based on the obtained classification success rate of neural networks, we concluded that both networks provided high sensitivity with classification errors between 2 and 4%. (paper)

  20. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee

    2015-01-01

    BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

  1. Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

    Science.gov (United States)

    Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

    2013-03-01

    Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

  2. Bcl-2 Protein Expression in Egyptian Acute Myeloid Leukemia

    International Nuclear Information System (INIS)

    El-Shakankiry, N.; El-Sayed, Gh.M.M.; El-Maghraby, Sh.; Moneer, M.M.

    2009-01-01

    Objective: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. Patients and methods: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. Results: The mean bcl-2 protein expression was significantly higher in patients (0.68610.592) compared to controls (0.313±0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups 0»=0.86). Conclusion: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy.

  3. Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles

    Science.gov (United States)

    Wang, Qiu-Yue; Huang, Wei; Jiang, Xing-Lin; Kang, Yan-Jun

    2018-01-01

    In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Stöber method. Streptavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cell-probe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system.

  4. Multimodal fluorescence molecular imaging for in vivo characterization of skin cancer using endogenous and exogenous fluorophores

    Science.gov (United States)

    Miller, Jessica P.; Habimana-Griffin, LeMoyne; Edwards, Tracy S.; Achilefu, Samuel

    2017-06-01

    Similarity of skin cancer with many benign skin pathologies requires reliable methods to detect and differentiate the different types of these lesions. Previous studies have explored the use of disparate optical techniques to identify and estimate the invasive nature of melanoma and basal cell carcinoma with varying outcomes. Here, we used a concerted approach that provides complementary information for rapid screening and characterization of tumors, focusing on squamous cell carcinoma (SCC) of the skin. Assessment of in vivo autofluorescence lifetime (FLT) imaging of endogenous fluorophores that are excitable at longer wavelengths (480 nm) than conventional NADH and FAD revealed a decrease in the short FLT component for SCC compared to normal skin, with mean values of 0.57±0.026 ns and 0.61±0.021 ns, respectively (p=0.004). Subsequent systemic administration of a near-infrared fluorescent molecular probe in SCC bearing mice, followed by the implementation of image processing methods on data acquired from two-dimensional and three-dimensional fluorescence molecular imaging, allowed us to estimate the tumor volume and depth, as well as quantify the fluorescent probe in the tumor. The result suggests the involvement of lipofuscin-like lipopigments and riboflavin in SCC metabolism and serves as a model for staging SCC.

  5. Comparative study of protoporphyrin IX fluorescence image enhancement methods to improve an optical imaging system for oral cancer detection

    Science.gov (United States)

    Jiang, Ching-Fen; Wang, Chih-Yu; Chiang, Chun-Ping

    2011-07-01

    Optoelectronics techniques to induce protoporphyrin IX fluorescence with topically applied 5-aminolevulinic acid on the oral mucosa have been developed to noninvasively detect oral cancer. Fluorescence imaging enables wide-area screening for oral premalignancy, but the lack of an adequate fluorescence enhancement method restricts the clinical imaging application of these techniques. This study aimed to develop a reliable fluorescence enhancement method to improve PpIX fluorescence imaging systems for oral cancer detection. Three contrast features, red-green-blue reflectance difference, R/B ratio, and R/G ratio, were developed first based on the optical properties of the fluorescence images. A comparative study was then carried out with one negative control and four biopsy confirmed clinical cases to validate the optimal image processing method for the detection of the distribution of malignancy. The results showed the superiority of the R/G ratio in terms of yielding a better contrast between normal and neoplastic tissue, and this method was less prone to errors in detection. Quantitative comparison with the clinical diagnoses in the four neoplastic cases showed that the regions of premalignancy obtained using the proposed method accorded with the expert's determination, suggesting the potential clinical application of this method for the detection of oral cancer.

  6. Investigation of relations between skin cancer lesions' images and their fluorescent spectra

    Science.gov (United States)

    Pavlova, P.; Borisova, E.; Avramov, L.; Petkova, El.; Troyanova, P.

    2010-03-01

    This investigation is based on images obtained from healthy tissue and skin cancer lesions and their fluorescent spectra of cutaneous lesions derived after optical stimulation. Our analyses show that the lesions’ spectra of are different of those, obtained from normal tissue and the differences depend on the type of cancer. We use a comparison between these “healthy” and “unhealthy” spectra to define forms of variations and corresponding diseases. However, the value of the emitted light varies not only between the patients, but also depending on the position of the tested area inside of one lesion. These variations could be result from two reasons: different degree of damaging and different thickness of the suspicious lesion area. Regarded to the visible image of the lesion, it could be connected with the chroma of colour of the tested area and the lesion homogeneity that corresponds to particular disease. For our investigation, images and spectra of three non-melanoma cutanous malignant tumors are investigated, namely—basal cell carcinoma, squamous cell carcinoma, and keratoacanthoma. The images were processed obtaining the chroma by elimination of the background—healthy tissue, and applying it as a basic signal for transformation from RGB to Lab colorimetric model. The chroma of the areas of emission is compared with the relative value of fluorescence spectra. Specific spectral features are used to develop hybrid diagnostic algorithm (including image and spectral features) for differentiation of these three kinds of malignant cutaneous pathologies.

  7. Melanin-originated carbonaceous dots for triple negative breast cancer diagnosis by fluorescence and photoacoustic dual-mode imaging.

    Science.gov (United States)

    Xiao, Wei; Li, Yuan; Hu, Chuan; Huang, Yuan; He, Qin; Gao, Huile

    2017-07-01

    Carbonaceous dots exhibit increasing applications in diagnosis and drug delivery due to excellent photostability and biocompatibility properties. However, relative short excitation and emission of melanin carbonaceous dots (MCDs) limit the applicability in fluorescence bioimaging. Furthermore, the generally poor spatial resolution of fluorescence imaging limits potential in vivo applications. Due to a variety of beneficial properties, in this study, MCDs were prepared exhibiting great potential in fluorescence and photoacoustic dual-mode bioimaging. The MCDs exhibited a long excitation peak at 615nm and emission peak at 650nm, further highlighting the applicability in fluorescence imaging, while the absorbance peak at 633nm renders MCDs suitable for photoacoustic imaging. In vivo, the photoacoustic signal of MCDs was linearly correlated with the concentration of MCDs. Moreover, the MCDs were shown to be taken up into triple negative breast cancer cell line 4T1 in both a time- and concentration-dependent manner. In vivo fluorescence and photoacoustic imaging of subcutaneous 4T1 tumor demonstrated that MCDs could passively target triple negative breast cancer tissue by enhanced permeability and retention effects and may therefore be used for tumor dual-mode imaging. Furthermore, fluorescence distribution in tissue slices suggested that MCDs may distribute in 4T1 tumor with high efficacy. In conclusion, the MCDs studied offer potential application in fluorescence and photoacoustic dual-mode imaging. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Development of a new detection device using a glass clip emitting infrared fluorescence for laparoscopic surgery of gastric cancer

    International Nuclear Information System (INIS)

    Inada, Shunko Albano; Mori, Kensaku; Fuchi, Shingo; Hasegawa, Junichi; Misawa, Kazunari; Nakanishi, Hayao

    2015-01-01

    In conventional method, to identify location of the tumor intraperitoneally for extirpation of the gastric cancer, charcoal ink is injected around the primary tumor. However, in the time of laparoscopic operation, it is difficult to estimate specific site of primary tumor. In this study we developed a glass phosphors was realized with Yb 3+ , Nd 3+ doped to Bi 2 O 3 -B 2 O 3 based glasses, which have central emission wavelength of 1020 nm and 100 nm of FWHM. Using this glass phosphor, we developed a fluorescent clip and the laparoscopic fluorescent detection system for clip-derived near-infrared light. To evaluated clinical performance of a fluorescent clip and the laparoscopic detection system, we used resected stomach from the patients. Fluorescent clip was fixed on the gastric mucosa, and an excitation light (wavelength: 808nm) was irradiated from outside of stomach for detection of fluorescent through stomach wall. As a result, fluorescent emission from the clip was successfully detected. These results indicate that the glass fluorescent clip in combination with laparoscopic detection system is a very useful method to identify the exact location of the primary gastric cancer. (paper)

  9. Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

    Directory of Open Access Journals (Sweden)

    Zonggao Shi

    2012-01-01

    Full Text Available The noncoding RNA designated as microRNA (miRNA is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH procedures to detect miR-146a with (a different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA incorporated DNA oligonucleotides; (b different reporters for the probes: biotin versus digoxigenin (DIG; (c different visualization: traditional versus tyramide signal amplification (TSA system; (d different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.

  10. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

    Directory of Open Access Journals (Sweden)

    Seung-Ho Shin

    Full Text Available Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5-3, plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5-3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5-3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins

  11. A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers

    Science.gov (United States)

    Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

    2015-01-01

    Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30 minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

  12. Effects of immunosuppressive treatment on protein expression in rat kidney

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  13. On the influence of the instillation time on the results of HAL (Hexvix) fluorescence detection of superficial bladder cancer

    Science.gov (United States)

    Jichlinski, Patrice; Aymon, Daniela; Wagnieres, Georges A.; Marti, Alexandre; Lange, Norbert; Guillou, Louis; Leisinger, Hans-Juerg; van den Bergh, Hubert

    2003-10-01

    Hexyl aminolevulinate (HAL) fluorescence cystoscopy is being investigated as a new diagnostic tool for the detection of flat urothelial malignancies in bladder cancers. However, the influence of the bladder instillation time on the performance of this detection modality has not been addressed up to now. We report our initial experience comparing different instillation schedules of HAL cystoscopy in the diagnosis of superficial bladder cancer. A total of 718 fluorescent positive (433) and fluorescence negative (285) biopsies have been taken in the bladder of 143 patients using the Storz D-light fluorescence imaging system (Karl Storz, Tuttlingen, Germany) which allows both white and blue light (380-450 nm) bladder wall inspection. Following hospitalisation, 50 ml of HAL (8mM) phosphate buffer solution was instilled into the bladder of patients during one hour (1 hour protocol involving 57 patients), or during two hours followed by a two hours resting time after removal of the solution (2+2 hours protocol involving 86 patients). Both instillation subgroups were homogeneous in terms of proportion of high risk disease, previous BCG treatment and/or recurrent disease. This study indicates that the instillation duration does not influence the results of HAL (Hexvix) fluorescence cystoscopy in our conditions. Compared to the standard use of ALA, HAL (Hexvix) fluorescence cystoscopy allows a significant reduction of the instillation time (to less than one hour) without prejudicing the efficacy of the method, what represents a real advantage in daily clinical practice.

  14. Fluorescent imaging of high-grade bladder cancer using a specific antagonist for chemokine receptor CXCR4.

    Science.gov (United States)

    Nishizawa, Koji; Nishiyama, Hiroyuki; Oishi, Shinya; Tanahara, Noriko; Kotani, Hirokazu; Mikami, Yoshiki; Toda, Yoshinobu; Evans, Barry J; Peiper, Stephen C; Saito, Ryoichi; Watanabe, Jun; Fujii, Nobutaka; Ogawa, Osamu

    2010-09-01

    We previously reported that the expression of CXC chemokine receptor-4 (CXCR4) was upregulated in invasive bladder cancers and that the small peptide T140 was a highly sensitive antagonist for CXCR4. In this study, we identified that CXCR4 expression was induced in high-grade superficial bladder tumors, including carcinoma in situ and invasive bladder tumors. To visualize the bladder cancer cells using urinary sediments from the patients and chemically induced mouse bladder cancer model, a novel fluorescent CXCR4 antagonist TY14003 was developed, that is a T140 derivative. TY14003 could label bladder cancer cell lines expressing CXCR4, whereas negative-control fluorescent peptides did not label them. When labeling urinary sediments from patients with invasive bladder cancer, positive-stained cells were identified in all patients with bladder cancer and positive urine cytology but not in controls. Although white blood cells in urine were also labeled with TY14003, they could be easily discriminated from urothelial cells by their shape and size. Finally, intravesical instillation of TY14003 into mouse bladder, using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer model, demonstrated that fluorescent signals were detected in the focal areas of bladder of all mice examined at 12 weeks of BBN drinking by confocal microscopy and fluorescent endoscopy. On the contrary, all the normal bladders were found to be negative for TY14003 staining. In conclusion, these results indicate that TY14003 is a promising diagnostic tool to visualize small or flat high-grade superficial bladder cancer.

  15. Fluorescence-guided surgery of human colon cancer increases complete resection resulting in cures in an orthotopic nude mouse model.

    Science.gov (United States)

    Metildi, Cristina A; Kaushal, Sharmeela; Snyder, Cynthia S; Hoffman, Robert M; Bouvet, Michael

    2013-01-01

    We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer. We established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection. All mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049). Surgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. The effect of near-infrared fluorescence conjugation on the anti-cancer potential of cetuximab.

    Science.gov (United States)

    Yun, Ji Young; Hyun, Byung-Hwa; Nam, Sang Yoon; Yun, Young Won; Lee, Hu-Jang; Lee, Beom-Jun

    2018-03-01

    This study investigated the anti-cancer potential of a near-infrared fluorescence (NIRF) molecule conjugated with Cetuximab (Cetuximab-NIRF) in six-week-old female BALB/c athymic (nu+/nu+) nude mice. A431 cells were cultured and injected into the animals to induce solid tumors. Paclitaxel (30 mg/kg body weight (BW)), Cetuximab (1 mg/kg BW), and Cetuximab-NIRF (0.25, 0.5 and 1.0 mg/kg BW) were intraperitoneally injected twice a week into the A431 cell xenografts of the nude mice. Changes in BW, tumor volume and weight, fat and lean mass, and diameter of the peri-tumoral blood vessel were determined after two weeks. Tumor volumes and weights were significantly decreased in the Cetuximab-NIRF (1 mg/kg BW) group compared with the control group ( P <0.001). Lean mass and total body water content were also conspicuously reduced in the Cetuximab-NIRF (1 mg/kg BW) group compared with the vehicle control group. Peri-tumoral blood vessel diameters were very thin in the Cetuximab-NIRF groups compared with those of the paclitaxel group. These results indicate that the conjugation of Cetuximab with NIRF does not affect the anti-cancer potential of Cetuximab and NIRF can be used for molecular imaging in cancer treatments.

  17. Real-time intraoperative detection of breast cancer using near-infrared fluorescence imaging and Methylene Blue.

    Science.gov (United States)

    Tummers, Q R J G; Verbeek, F P R; Schaafsma, B E; Boonstra, M C; van der Vorst, J R; Liefers, G-J; van de Velde, C J H; Frangioni, J V; Vahrmeijer, A L

    2014-07-01

    Despite recent developments in preoperative breast cancer imaging, intraoperative localization of tumor tissue can be challenging, resulting in tumor-positive resection margins during breast conserving surgery. Based on certain physicochemical similarities between Technetium((99m)Tc)-sestamibi (MIBI), an SPECT radiodiagnostic with a sensitivity of 83-90% to detect breast cancer preoperatively, and the near-infrared (NIR) fluorophore Methylene Blue (MB), we hypothesized that MB might detect breast cancer intraoperatively using NIR fluorescence imaging. Twenty-four patients with breast cancer, planned for surgical resection, were included. Patients were divided in 2 administration groups, which differed with respect to the timing of MB administration. N = 12 patients per group were administered 1.0 mg/kg MB intravenously either immediately or 3 h before surgery. The mini-FLARE imaging system was used to identify the NIR fluorescent signal during surgery and on post-resected specimens transferred to the pathology department. Results were confirmed by NIR fluorescence microscopy. 20/24 (83%) of breast tumors (carcinoma in N = 21 and ductal carcinoma in situ in N = 3) were identified in the resected specimen using NIR fluorescence imaging. Patients with non-detectable tumors were significantly older. No significant relation to receptor status or tumor grade was seen. Overall tumor-to-background ratio (TBR) was 2.4 ± 0.8. There was no significant difference between TBR and background signal between administration groups. In 2/4 patients with positive resection margins, breast cancer tissue identified in the wound bed during surgery would have changed surgical management. Histology confirmed the concordance of fluorescence signal and tumor tissue. This feasibility study demonstrated an overall breast cancer identification rate using MB of 83%, with real-time intraoperative guidance having the potential to alter patient management. Copyright © 2014 Elsevier Ltd. All

  18. Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

    Science.gov (United States)

    Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

    2015-02-10

    Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Design of near-infrared fluorescent bioactive conjugated functional iron oxide nanoparticles for optical detection of colon cancer

    Directory of Open Access Journals (Sweden)

    Corem-Salkmon E

    2012-10-01

    Full Text Available Enav Corem-Salkmon, Benny Perlstein, Shlomo MargelThe Institute of Nanotechnology and Advanced Materials, Department of Chemistry, Bar-Ilan University, Ramat-Gan, IsraelBackground: Colon cancer is one of the major causes of death in the Western world. Early detection significantly improves long-term survival for patients with the disease. Near-infrared (NIR fluorescent nanoparticles hold great promise as contrast agents for tumor detection. NIR offers several advantages for bioimaging compared with fluorescence in the visible spectrum, ie, lower autofluorescence of biological tissues, lower absorbance, and consequently deeper penetration into biomatrices.Methods and results: NIR fluorescent iron oxide nanoparticles with a narrow size distribution were prepared by nucleation, followed by controlled growth of thin iron oxide films onto cyanine NIR dye conjugated gelatin-iron oxide nuclei. For functionalization, and in order to increase the NIR fluorescence intensity, the NIR fluorescent iron oxide nanoparticles obtained were coated with human serum albumin containing cyanine NIR dye. Leakage of the NIR dye from these nanoparticles into phosphate-buffered saline solution containing 4% albumin was not detected. The work presented here is a feasibility study to test the suitability of iron oxide-human serum albumin NIR fluorescent nanoparticles for optical detection of colon cancer. It demonstrates that encapsulation of NIR fluorescent dye within these nanoparticles significantly reduces photobleaching of the dye. Tumor-targeting ligands, peanut agglutinin and anticarcinoembryonic antigen antibodies (αCEA, were covalently conjugated with the NIR fluorescent iron oxide-human serum albumin nanoparticles via a poly(ethylene glycol spacer. Specific colon tumor detection was demonstrated in chicken embryo and mouse models for both nonconjugated and the peanut agglutinin-conjugated or αCEA-conjugated NIR fluorescent iron oxide-human serum albumin

  20. Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti-epidermal growth factor receptor nanobody

    NARCIS (Netherlands)

    Van Driel, P.B.A.A.; Van Der Vorst, J.R.; Verbeek, F.P.R.; Oliveira, S.|info:eu-repo/dai/nl/304841455; Snoeks, T.J.A.; Keereweer, S.; Chan, B.; Boonstra, M.C.; Frangioni, J.V.; Van Bergen En Henegouwen, P.M.P.|info:eu-repo/dai/nl/071919481; Vahrmeijer, A.L.; Lowik, C.W.G.M.

    2014-01-01

    Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for

  1. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  2. A Dual Reporter Iodinated Labeling Reagent for Cancer Positron Emission Tomography Imaging and Fluorescence-Guided Surgery

    Science.gov (United States)

    2018-01-01

    The combination of early diagnosis and complete surgical resection offers the greatest prospect of curative cancer treatment. An iodine-124/fluorescein-based dual-modality labeling reagent, 124I-Green, constitutes a generic tool for one-step installation of a positron emission tomography (PET) and a fluorescent reporter to any cancer-specific antibody. The resulting antibody conjugate would allow both cancer PET imaging and intraoperative fluorescence-guided surgery. 124I-Green was synthesized in excellent radiochemical yields of 92 ± 5% (n = 4) determined by HPLC with an improved one-pot three-component radioiodination reaction. The A5B7 carcinoembryonic antigen (CEA)-specific antibody was conjugated to 124I-Green. High tumor uptake of the dual-labeled A5B7 of 20.21 ± 2.70, 13.31 ± 0.73, and 10.64 ± 1.86%ID/g was observed in CEA-expressing SW1222 xenograft mouse model (n = 3) at 24, 48, and 72 h post intravenous injection, respectively. The xenografts were clearly visualized by both PET/CT and ex vivo fluorescence imaging. These encouraging results warrant the further translational development of 124I-Green for cancer PET imaging and fluorescence-guided surgery. PMID:29388770

  3. Enhancement of early cervical cancer diagnosis with epithelial layer analysis of fluorescence lifetime images.

    Directory of Open Access Journals (Sweden)

    Jun Gu

    Full Text Available This work reports the use of layer analysis to aid the fluorescence lifetime diagnosis of cervical intraepithelial neoplasia (CIN from H&E stained cervical tissue sections. The mean and standard deviation of lifetimes in single region of interest (ROI of cervical epithelium were previously shown to correlate to the gold standard histopathological classification of early cervical cancer. These previously defined single ROIs were evenly divided into layers for analysis. A 10-layer model revealed a steady increase in fluorescence lifetime from the inner to the outer epithelial layers of healthy tissue sections, suggesting a close association with cellular maturity. The shorter lifetime and minimal lifetime increase towards the epithelial surface of CIN-affected regions are in good agreement with the absence of cellular maturation in CIN. Mean layer lifetimes in the top-half cervical epithelium were used as feature vectors for extreme learning machine (ELM classifier discriminations. It was found that the proposed layer analysis technique greatly improves the sensitivity and specificity to 94.6% and 84.3%, respectively, which can better supplement the traditional gold standard cervical histopathological examinations.

  4. Real-time fluorescence target/background (T/B) ratio calculation in multimodal endoscopy for detecting GI tract cancer

    Science.gov (United States)

    Jiang, Yang; Gong, Yuanzheng; Wang, Thomas D.; Seibel, Eric J.

    2017-02-01

    Multimodal endoscopy, with fluorescence-labeled probes binding to overexpressed molecular targets, is a promising technology to visualize early-stage cancer. T/B ratio is the quantitative analysis used to correlate fluorescence regions to cancer. Currently, T/B ratio calculation is post-processing and does not provide real-time feedback to the endoscopist. To achieve real-time computer assisted diagnosis (CAD), we establish image processing protocols for calculating T/B ratio and locating high-risk fluorescence regions for guiding biopsy and therapy in Barrett's esophagus (BE) patients. Methods: Chan-Vese algorithm, an active contour model, is used to segment high-risk regions in fluorescence videos. A semi-implicit gradient descent method was applied to minimize the energy function of this algorithm and evolve the segmentation. The surrounding background was then identified using morphology operation. The average T/B ratio was computed and regions of interest were highlighted based on user-selected thresholding. Evaluation was conducted on 50 fluorescence videos acquired from clinical video recordings using a custom multimodal endoscope. Results: With a processing speed of 2 fps on a laptop computer, we obtained accurate segmentation of high-risk regions examined by experts. For each case, the clinical user could optimize target boundary by changing the penalty on area inside the contour. Conclusion: Automatic and real-time procedure of calculating T/B ratio and identifying high-risk regions of early esophageal cancer was developed. Future work will increase processing speed to <5 fps, refine the clinical interface, and apply to additional GI cancers and fluorescence peptides.

  5. Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer

    Directory of Open Access Journals (Sweden)

    Nakase H

    2017-03-01

    Full Text Available Hiroshi Nakase,1,2 Shinji Sakuma,3 Takumi Fukuchi,4 Takuya Yoshino,1,5 Kohta Mohri,3 Kohei Miyata,3,6 Hironori Kumagai,6 Ken-Ichiro Hiwatari,6 Kazufumi Tsubaki,6 Tetsuya Ikejima,6 Etsuo Tobita,6 Meiying Zhu,7 Kevin J Wilson,7 Kay Washington,8 John C Gore,7,9–13 Wellington Pham7,9–12,14 1Division of Endoscopy, Kyoto University Hospital, Kyoto, Japan; 2Department of Gastroenterology and Hepatology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 3Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, Japan; 4Gastroenterology and Hepatology, Osakafu Saiseikai Nakatsu Hospital, Osaka, Japan; 5Division of Inflammatory Bowel Disease, Digestive Disease Center, Kitano Hospital, Osaka, Japan; 6Advanced Materials R&D Laboratory, ADEKA Corp., Tokyo, Japan; 7Department of Radiology, Vanderbilt University Institute of Imaging Science, 8Division of Gastrointestinal and Hepatic Pathology, Vanderbilt School of Medicine, 9Department of Radiology and Radiological Sciences, 10Department of Biomedical Engineering, 11Vanderbilt Ingram Cancer Center, Vanderbilt School of Medicine, 12Vanderbilt Brain Institute, 13Molecular Physiology and Biophysics, 14Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN, USA Abstract: The Thomsen–Friedenreich (TF antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident. Keywords: TF antigen

  6. General theory for integrated analysis of growth, gene, and protein expression in biofilms.

    Science.gov (United States)

    Zhang, Tianyu; Pabst, Breana; Klapper, Isaac; Stewart, Philip S

    2013-01-01

    A theory for analysis and prediction of spatial and temporal patterns of gene and protein expression within microbial biofilms is derived. The theory integrates phenomena of solute reaction and diffusion, microbial growth, mRNA or protein synthesis, biomass advection, and gene transcript or protein turnover. Case studies illustrate the capacity of the theory to simulate heterogeneous spatial patterns and predict microbial activities in biofilms that are qualitatively different from those of planktonic cells. Specific scenarios analyzed include an inducible GFP or fluorescent protein reporter, a denitrification gene repressed by oxygen, an acid stress response gene, and a quorum sensing circuit. It is shown that the patterns of activity revealed by inducible stable fluorescent proteins or reporter unstable proteins overestimate the region of activity. This is due to advective spreading and finite protein turnover rates. In the cases of a gene induced by either limitation for a metabolic substrate or accumulation of a metabolic product, maximal expression is predicted in an internal stratum of the biofilm. A quorum sensing system that includes an oxygen-responsive negative regulator exhibits behavior that is distinct from any stage of a batch planktonic culture. Though here the analyses have been limited to simultaneous interactions of up to two substrates and two genes, the framework applies to arbitrarily large networks of genes and metabolites. Extension of reaction-diffusion modeling in biofilms to the analysis of individual genes and gene networks is an important advance that dovetails with the growing toolkit of molecular and genetic experimental techniques.

  7. Data of a fluorescent imaging-based analysis of anti-cancer drug effects on three-dimensional cultures of breast cancer cells

    Directory of Open Access Journals (Sweden)

    Junji Itou

    2015-12-01

    Full Text Available Three-dimensional (3D cell culture is a powerful tool to study cell growth under 3D condition. To perform a simple test for anti-cancer drugs in 3D culture, visualization of non-proliferated cells is required. We propose a fluorescent imaging-based assay to analyze cancer cell proliferation in 3D culture. We used a pulse-labeling technique with a photoconvertible fluorescent protein Kaede to identify non-proliferated cells. This assay allows us to observe change in cell proliferation in 3D culture by simple imaging. Using this assay, we obtained the data of the effects of anti-cancer drugs, 5-fluorouracil and PD0332991 in a breast cancer cell line, MCF-7.

  8. Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands.

    Science.gov (United States)

    He, Wei; Kularatne, Sumith A; Kalli, Kimberly R; Prendergast, Franklyn G; Amato, Robert J; Klee, George G; Hartmann, Lynn C; Low, Philip S

    2008-10-15

    Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.

  9. Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells.

    Directory of Open Access Journals (Sweden)

    Valentina Guzmán-Pérez

    Full Text Available Nasturtium (Tropaeolum majus L. contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1. FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i the insulin-signaling pathway, ii the intracellular localization of FOXO1 and, iii the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived-like2 (NRF2 and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1. The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance.

  10. Near-infrared fluorescent aza-BODIPY dye-loaded biodegradable polymeric nanoparticles for optical cancer imaging

    International Nuclear Information System (INIS)

    Hamon, Casey L.; Dorsey, Christopher L.; Özel, Tuğba; Barnes, Eugenia M.; Hudnall, Todd W.; Betancourt, Tania

    2016-01-01

    Nanoparticles are being readily investigated as carriers for the delivery of imaging and therapeutic agents for the detection, monitoring, and treatment of cancer and other diseases. In the present work, the preparation of biodegradable polymeric nanoparticles loaded with a near-infrared fluorescent aza-boron dipyrromethene (NIR-BODIPY) derivative, and their use as contrast agents for optical imaging in cancer are described. Nanoparticles were prepared by nanoprecipitation of amphiphilic block copolymers of poly(lactic acid) and poly(ethylene glycol). The size, morphology, dye loading, spectral properties, quantum yield, cytocompatibility, and in vitro NIR imaging potential of the nanoparticles in breast and ovarian cancer cells were evaluated. Spherical nanoparticles of 30–70 nm in diameter were loaded with 0.73 w/w% BODIPY derivative. At this loading, the dye presented a fluorescence quantum yield in the same order of magnitude as in solution. Nanoparticle suspensions at concentrations up to 580 μg/mL were cytocompatible to breast (MDA-MB-231) and ovarian (SKOV-3 and Caov-3) cancer cells after a four-hour incubation period. Fluorescence microscopy images demonstrated the ability of the nanoparticles to act as imaging agents in all three cell lines in as little as 1 hour. The results shown indicate the potential of these NIR-BODIPY-loaded nanoparticles as contrast agents for near-infrared optical imaging in cancer.Graphical abstract

  11. Near-infrared fluorescent aza-BODIPY dye-loaded biodegradable polymeric nanoparticles for optical cancer imaging

    Energy Technology Data Exchange (ETDEWEB)

    Hamon, Casey L.; Dorsey, Christopher L. [Texas State University, Department of Chemistry and Biochemistry (United States); Özel, Tuğba [Texas State University, Materials Science, Engineering, and Commercialization Program (United States); Barnes, Eugenia M.; Hudnall, Todd W.; Betancourt, Tania, E-mail: tb26@txstate.edu [Texas State University, Department of Chemistry and Biochemistry (United States)

    2016-07-15

    Nanoparticles are being readily investigated as carriers for the delivery of imaging and therapeutic agents for the detection, monitoring, and treatment of cancer and other diseases. In the present work, the preparation of biodegradable polymeric nanoparticles loaded with a near-infrared fluorescent aza-boron dipyrromethene (NIR-BODIPY) derivative, and their use as contrast agents for optical imaging in cancer are described. Nanoparticles were prepared by nanoprecipitation of amphiphilic block copolymers of poly(lactic acid) and poly(ethylene glycol). The size, morphology, dye loading, spectral properties, quantum yield, cytocompatibility, and in vitro NIR imaging potential of the nanoparticles in breast and ovarian cancer cells were evaluated. Spherical nanoparticles of 30–70 nm in diameter were loaded with 0.73 w/w% BODIPY derivative. At this loading, the dye presented a fluorescence quantum yield in the same order of magnitude as in solution. Nanoparticle suspensions at concentrations up to 580 μg/mL were cytocompatible to breast (MDA-MB-231) and ovarian (SKOV-3 and Caov-3) cancer cells after a four-hour incubation period. Fluorescence microscopy images demonstrated the ability of the nanoparticles to act as imaging agents in all three cell lines in as little as 1 hour. The results shown indicate the potential of these NIR-BODIPY-loaded nanoparticles as contrast agents for near-infrared optical imaging in cancer.Graphical abstract.

  12. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

  13. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    Science.gov (United States)

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  14. Photodetection of early cancer by laser-induced fluorescence of a tumor-selective dye: apparatus design and realization

    Science.gov (United States)

    Wagnieres, Georges A.; Depeursinge, Christian D.; Monnier, Philippe; Savary, Jean-Francois; Cornaz, Piet F.; Chatelain, Andre; van den Bergh, Hubert

    1990-07-01

    An apparatus is designed and realized to detect "early" cancer at the surface of the hollow organs in the human body by endoscopic means. The tumor is localized by the laser induced fluorescence of a dye (HPD) which concentrates selectively in the neoplastic tissue after intravenous injection. Fluorescence contrast between the tumor and its normal surroundings is enhanced by subtracting the background autofluorescence which occurs in both types of tissue. This is done by means of 2-color digital images manipulation in real-time. Preliminary clinical tests of the apparatus demonstrated the detection of carcinoma in situ in the esophagus.

  15. Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging.

    Science.gov (United States)

    Bernhem, Kristoffer; Blom, Hans; Brismar, Hjalmar

    2018-01-01

    Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

  16. Scattering features for lung cancer detection in fibered confocal fluorescence microscopy images.

    Science.gov (United States)

    Rakotomamonjy, Alain; Petitjean, Caroline; Salaün, Mathieu; Thiberville, Luc

    2014-06-01

    To assess the feasibility of lung cancer diagnosis using fibered confocal fluorescence microscopy (FCFM) imaging technique and scattering features for pattern recognition. FCFM imaging technique is a new medical imaging technique for which interest has yet to be established for diagnosis. This paper addresses the problem of lung cancer detection using FCFM images and, as a first contribution, assesses the feasibility of computer-aided diagnosis through these images. Towards this aim, we have built a pattern recognition scheme which involves a feature extraction stage and a classification stage. The second contribution relies on the features used for discrimination. Indeed, we have employed the so-called scattering transform for extracting discriminative features, which are robust to small deformations in the images. We have also compared and combined these features with classical yet powerful features like local binary patterns (LBP) and their variants denoted as local quinary patterns (LQP). We show that scattering features yielded to better recognition performances than classical features like LBP and their LQP variants for the FCFM image classification problems. Another finding is that LBP-based and scattering-based features provide complementary discriminative information and, in some situations, we empirically establish that performance can be improved when jointly using LBP, LQP and scattering features. In this work we analyze the joint capability of FCFM images and scattering features for lung cancer diagnosis. The proposed method achieves a good recognition rate for such a diagnosis problem. It also performs well when used in conjunction with other features for other classical medical imaging classification problems. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle as a label for the ultrasensitive detection of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tao Liang [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Song Chaojun; Sun Yuanjie [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China); Li Xiaohua; Li Yunyun [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Jin Boquan [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China); Zhang Zhujun, E-mail: zhangzj@snnu.edu.cn [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Yang Kun, E-mail: yangkunkun@fmmu.edu.cn [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China)

    2013-01-25

    Highlights: Black-Right-Pointing-Pointer Difunctional amino mesoporous silica nanoparticles (FCMSN) were synthesized. Black-Right-Pointing-Pointer The fluorescence and chemiluminescence properties of the FCMSN were studied. Black-Right-Pointing-Pointer The NaIO{sub 4} oxidation method was used for modification of the FCMSN. Black-Right-Pointing-Pointer Liver cancer 7721 cell was detected. Black-Right-Pointing-Pointer The specificity affected by FCMSN's amino groups was studied. - Abstract: A new kind of ultrabright fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle (FCMSN) is reported. A luminescent dye, Rhodamine 6G or tris(2,2 Prime -bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), is doped inside nanochannels of a silica matrix. The hydrophobic groups in the silica matrix avoid the leakage of dye from open channels. The amines groups on the surface of the FCMSN improve the modification performance of the nanoparticle. Because the nanochannels are isolated by a network skeleton of silica, fluorescence quenching based on the inner filter effect of the fluorescent dyes immobilized in nanochannels is weakened effectively. The Quantum Yield of obtained 90 nm silica particles was about 61%. Compared with the fluorescent core-shell nanoparticle, the chemiluminescence reagents can freely enter the nanoparticles to react with fluorescent dyes to create chemiluminescence. The results show that the FCMSN are both fluorescent labels and chemiluminescent labels. In biological applications, the NaIO{sub 4} oxidation method was proven to be superior to the glutaraldehyde method. The amount of amino could affect the specificity of the FCMSN. The fluorescence microscopy imaging demonstrated that the FCMSN is viable for biological applications.

  18. Rapid Chemometric X-Ray Fluorescence approaches for spectral Diagnostics of Cancer utilizing Tissue Trace Metals and Speciation profiles

    International Nuclear Information System (INIS)

    Okonda, J.J.

    2015-01-01

    Energy dispersive X-ray fluorescence (EDXRF) spectroscopy is an analytical method for identification and quantification of elements in materials by measurement of their spectral energy and intensity. EDXRFS spectroscopic technique involves simultaneous non-invasive acquisition of both fluorescence and scatter spectra from samples for quantitative determination of trace elemental content in complex matrix materials. The objective is develop a chemometric-aided EDXRFS method for rapid diagnosis of cancer and its severity (staging) based on analysis of trace elements (Cu, Zn, Fe, Se and Mn), their speciation and multivariate alterations of the elements in cancerous body tissue samples as cancer biomarkers. The quest for early diagnosis of cancer is based on the fact that early intervention translates to higher survival rate and better quality of life. Chemometric aided EDXRFS cancer diagnostic model has been evaluated as a direct and rapid superior alternative for the traditional quantitative methods used in XRF such as FP method. PCA results of cultured samples indicate that it is possible to characterize cancer at early and late stage of development based on trace elemental profiles

  19. Feasibility of Real-Time Near-Infrared Fluorescence Tracer Imaging in Sentinel Node Biopsy for Oral Cavity Cancer Patients

    DEFF Research Database (Denmark)

    Christensen, Anders; Juhl, Karina; Charabi, Birgitte

    2016-01-01

    BACKGROUND: Sentinel node biopsy (SNB) is an established method in oral squamous cell carcinoma (OSCC) for staging the cN0 neck and to select patients who will benefit from a neck dissection. Near-infrared fluorescence (NIRF) imaging has the potential to improve the SNB procedure by facilitating...... intraoperative visual identification of the sentinel lymph node (SN). The purpose of this study was to evaluate the feasibility of fluorescence tracer imaging for SN detection in conjunction with conventional radio-guided technique. METHODS: Prospective study of patients with primary OSCC planned for tumor...... be identified in vivo using NIRF imaging, and the majority of those were located in level 1 close to the primary tumor. CONCLUSIONS: A combined fluorescent and radioactive tracer for SNB is feasible, and the additional use of NIRF imaging may improve the accuracy of SN identification in oral cancer patients...

  20. Intraoperative fluorescence delineation of head and neck cancer with a fluorescent anti-epidermal growth factor receptor nanobody.

    Science.gov (United States)

    van Driel, P B A A; van der Vorst, J R; Verbeek, F P R; Oliveira, S; Snoeks, T J A; Keereweer, S; Chan, B; Boonstra, M C; Frangioni, J V; van Bergen en Henegouwen, P M P; Vahrmeijer, A L; Lowik, C W G M

    2014-06-01

    Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections. © 2013 UICC.

  1. Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

    Science.gov (United States)

    Sakuma, Shinji; Yu, James Y H; Quang, Timothy; Hiwatari, Ken-Ichiro; Kumagai, Hironori; Kao, Stephanie; Holt, Alex; Erskind, Jalysa; McClure, Richard; Siuta, Michael; Kitamura, Tokio; Tobita, Etsuo; Koike, Seiji; Wilson, Kevin; Richards-Kortum, Rebecca; Liu, Eric; Washington, Kay; Omary, Reed; Gore, John C; Pham, Wellington

    2015-03-01

    Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level. © 2014 UICC.

  2. p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

    Science.gov (United States)

    Di Vinci, Angela; Perdelli, Luisa; Banelli, Barbara; Salvi, Sandra; Casciano, Ida; Gelvi, Ilaria; Allemanni, Giorgio; Margallo, Edoardo; Gatteschi, Beatrice; Romani, Massimo

    2005-04-10

    The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma. (c) 2004 Wiley-Liss, Inc.

  3. Efficacy and safety of hexaminolevulinate fluorescence cystoscopy in the diagnosis of bladder cancer.

    Science.gov (United States)

    Lee, Jae Seung; Lee, Seo Yeon; Kim, Woo Jung; Seo, Seong Il; Jeon, Seong Soo; Lee, Hyun Moo; Choi, Han Yong; Jeong, Byong Chang

    2012-12-01

    The aim of this study was to evaluate the efficacy and safety of hexaminolevulinate fluorescence cystoscopy in the diagnosis of bladder cancer. In a prospective design, we included patients who had a bladder lesion suggesting bladder cancer. Patients with massive hematuria, urethral Foley catheter insertion, chronic retention state, or urinary tract infection were excluded. After the bladder was emptied, hexaminolevulinate was gently administered into the bladder. One hour later, cystoscopy under white light and blue light was performed. After marking the lesions confirmed with white light or blue light, transurethral resection of the bladder lesion and pathologic confirmation were done. Transurethral resection of the lesions that were negative in both white and blue light was also performed. From April 2010 to September 2010, 30 patients were enrolled. From the total of 30 patients (25 men and 5 women; mean age, 60.4±9.22 years), 134 specimens were extracted. Among these, 101 specimens showed positive results by blue light cystoscopy (BLC). The sensitivity of BLC and white light cystoscopy (WLC) was 92.3% and 80.8%, respectively (p=0.021). The specificity of BLC and WLC was 48% and 49.1%, respectively (p>0.05). The positive and negative predictive values of BLC were 71.2% and 81.8%, respectively, whereas those of WLC were 72.0% and 68.6%, respectively. With WLC, 48 specimens showed negative findings, but of that group, 15 specimens (31.2%) were revealed to be malignant with BLC. There were no significant side effects in the 24 hours after the instillation of hexaminolevulinate. Photodynamic diagnosis with hexaminolevulinate helps to find tumors that could be missed by use of WLC only. Photodynamic diagnosis might be valuable in complete resection as well as for more accurate diagnosis of bladder tumor.

  4. Fluorescence-guided surgery of a highly-metastatic variant of human triple-negative breast cancer targeted with a cancer-specific GFP adenovirus prevents recurrence

    Science.gov (United States)

    Yano, Shuya; Takehara, Kiyoto; Miwa, Shinji; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Urata, Yasuo; Kagawa, Shunsuke; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M.

    2016-01-01

    We have previously developed a genetically-engineered GFP-expressing telomerase-dependent adenovirus, OBP-401, which can selectively illuminate cancer cells. In the present report, we demonstrate that targeting a triple-negative high-invasive human breast cancer, orthotopically-growing in nude mice, with OBP-401 enables curative fluorescence-guided surgery (FGS). OBP-401 enabled complete resection and prevented local recurrence and greatly inhibited lymph-node metastasis due to the ability of the virus to selectively label and subsequently kill cancer cells. In contrast, residual breast cancer cells become more aggressive after bright (white)-light surgery (BLS). OBP-401-based FGS also improved the overall survival compared with conventional BLS. Thus, metastasis from a highly-aggressive triple-negative breast cancer can be prevented by FGS in a clinically-relevant mouse model. PMID:27689331

  5. A study of MRI-guided diffuse fluorescence molecular tomography for monitoring PDT effects in pancreas cancer

    Science.gov (United States)

    Samkoe, Kimberley S.; Davis, Scott C.; Srinivasan, Subhadra; O'Hara, Julia A.; Hasan, Tayyaba; Pogue, Brian W.

    2009-06-01

    Over the last several decades little progress has been made in the therapy and treatment monitoring of pancreas adenocarcinoma, a devastating and aggressive form of cancer that has a 5-year patient survival rate of 3%. Currently, investigations for the use of interstitial Verteporfin photodynamic therapy (PDT) are being undertaken in both orthotopic xenograft mouse models and in human clinical trials. In the mouse models, magnetic resonance (MR) imaging has been used as a measure of surrogate response to Verteporfin PDT; however, MR imaging alone lacks the molecular information required to assess the metabolic function and growth rates of the tumor immediately after treatment. We propose the implementation of MR-guided fluorescence tomography in conjunction with a fluorescently labeled (IR-Dye 800 CW, LI-COR) epidermal growth factor (EGF) as a molecular measure of surrogate response. To demonstrate the effectiveness of MR-guided diffuse fluorescence tomography for molecular imaging, we have used the AsPC-1 (+EGFR) human pancreatic adenocarcinoma in an orthotopic mouse model. EGF IRDye 800CW was injected 48 hours prior to imaging. MR image sequences were collected simultaneously with the fluorescence data using a MR-coupled diffuse optical tomography system. Image reconstruction was performed multiple times with varying abdominal organ segmentation in order to obtain a optimal tomographic image. It is shown that diffuse fluorescence tomography of the orthotopic pancreas model is feasible, with consideration of confounding fluorescence signals from the multiple organs and tissues surrounding the pancreas. MR-guided diffuse fluorescence tomography will be used to monitor EGF response after photodynamic therapy. Additionally, it provide the opportunity to individualize subsequent therapies based on response to PDT as well as to evaluate the success of combination therapies, such as PDT with chemotherapy, antibody therapy or even radiation.

  6. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    Science.gov (United States)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  7. Virus-resembling nano-structures for near infrared fluorescence imaging of ovarian cancer HER2 receptors

    Science.gov (United States)

    Guerrero, Yadir A.; Bahmani, Baharak; Singh, Sheela P.; Vullev, Valentine I.; Kundra, Vikas; Anvari, Bahman

    2015-10-01

    Ovarian cancer remains the dominant cause of death due to malignancies of the female reproductive system. The capability to identify and remove all tumors during intraoperative procedures may ultimately reduce cancer recurrence, and lead to increased patient survival. The objective of this study is to investigate the effectiveness of an optical nano-structured system for targeted near infrared (NIR) imaging of ovarian cancer cells that over-express the human epidermal growth factor receptor 2 (HER2), an important biomarker associated with ovarian cancer. The nano-structured system is comprised of genome-depleted plant-infecting brome mosaic virus doped with NIR chromophore, indocyanine green, and functionalized at the surface by covalent attachment of monoclonal antibodies against the HER2 receptor. We use absorption and fluorescence spectroscopy, and dynamic light scattering to characterize the physical properties of the constructs. Using fluorescence imaging and flow cytometry, we demonstrate the effectiveness of these nano-structures for targeted NIR imaging of HER2 receptors in vitro. These functionalized nano-materials may provide a platform for NIR imaging of ovarian cancer.

  8. Virus-resembling nano-structures for near infrared fluorescence imaging of ovarian cancer HER2 receptors

    International Nuclear Information System (INIS)

    Guerrero, Yadir A; Bahmani, Baharak; Vullev, Valentine I; Anvari, Bahman; Singh, Sheela P; Kundra, Vikas

    2015-01-01

    Ovarian cancer remains the dominant cause of death due to malignancies of the female reproductive system. The capability to identify and remove all tumors during intraoperative procedures may ultimately reduce cancer recurrence, and lead to increased patient survival. The objective of this study is to investigate the effectiveness of an optical nano-structured system for targeted near infrared (NIR) imaging of ovarian cancer cells that over-express the human epidermal growth factor receptor 2 (HER2), an important biomarker associated with ovarian cancer. The nano-structured system is comprised of genome-depleted plant-infecting brome mosaic virus doped with NIR chromophore, indocyanine green, and functionalized at the surface by covalent attachment of monoclonal antibodies against the HER2 receptor. We use absorption and fluorescence spectroscopy, and dynamic light scattering to characterize the physical properties of the constructs. Using fluorescence imaging and flow cytometry, we demonstrate the effectiveness of these nano-structures for targeted NIR imaging of HER2 receptors in vitro. These functionalized nano-materials may provide a platform for NIR imaging of ovarian cancer. (paper)

  9. Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

    2015-08-27

    Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

  10. Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

    Science.gov (United States)

    Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

    2009-12-01

    The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

  11. Tunable recombinant protein expression with E. coli in a mixed-feed environment.

    Science.gov (United States)

    Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

    2014-04-01

    Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

  12. Differential protein expression in alligator leukocytes in response to bacterial lipopolysaccharide injection.

    Science.gov (United States)

    Merchant, Mark; Kinney, Clint; Sanders, Paige

    2009-12-01

    Blood was collected from three juvenile alligators (Alligator mississippiensis) before, and again 24h after, injection with bacterial lipopolysaccharide (LPS). The leukocytes were collected from both samples, and the proteins were extracted. Each group of proteins was labeled with a different fluorescent dye and the differences in protein expression were analyzed by two dimensional differential in-gel expressions (2D-DIGE). The proteins which appeared to be increased or decreased by treatment with LPS were selected and analyzed by MALDI-TOF to determine mass and LC-MS/MS to acquire the partial protein sequences. The peptide sequences were compared to the NCBI protein sequence database to determine homology with other sequences from other species. Several proteins of interest appeared to be increased upon LPS stimulation. Proteins with homology to human transgelin-2, fish glucose-6-phosphate dehydrogenase, amphibian α-enolase, alligator lactate dehydrogenase, fish ubiquitin-activating enzyme, and fungal β-tubulin were also increased after LPS injection. Proteins with homology to fish vimentin 4, murine heterogeneous nuclear ribonucleoprotein A3, and avian calreticulin were found to be decreased in response to LPS. In addition, five proteins, four of which were up-regulated (827, 560, 512, and 650%) and one that exhibited repressed expression (307%), did not show homology to any protein in the database, and thus may represent newly discovered proteins. We are using this biochemical approach to isolate and characterize alligator proteins with potential relevant immune function.

  13. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  14. Protein expression of saccharomyces cerevisiae in response to uranium exposure

    International Nuclear Information System (INIS)

    Sakamoto, Fuminori; Nankawa, Takuya; Kozai, Naofumi; Ohnuki, Toshihiko; Fujii, Tsutomu; Iefuji, Haruyuki; Francis, A.J.

    2007-01-01

    Protein expression of Saccharomyces cerevisiae grown in the medium containing 238 U (VI) and 233 U (VI) was examined by two-dimensional gel electrophoresis. Saccharomyces cerevisiae of BY4743 was grown in yeast nitrogen base medium containing glucose and glycerol 2-phosphate and 238 U of 0, 2.0, and 5.0 x 10 -4 M or 233 U of 2.5 x 10 -6 M (radioactivity was higher by 350 times than 2.0 x 10 -4 M 238 U) and 5.0 x 10 -6 M for 112 h at 30 degC. The growth of Saccharomyces cerevisiae was monitored by measuring OD 600 at 112 h after the inoculation. Uranium concentrations in the media also were measured by radiometry using a liquid scintillation counter. The growths of the yeast grown in the above media were in the following order: control>2.5 x 10 -6 M 233 U>2.0 x 10 -4 M 238 U>5.0 x 10 -6 M 233 U>5.0 x 10 -4 M 238 U. This result indicated that not only radiological but also chemical effect of U reduced the growth of the yeast. The concentrations of U in the medium containing 238 U or 233 U decreased, suggesting U accumulation by the yeast cells. The 2-D gel electrophoresis analysis showed the appearance of several spots after exposure to 238 U or to 233 U but not in the control containing no uranium. These results show that the yeast cells exposed to U express several specific proteins. (author)

  15. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  16. Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma.

    Directory of Open Access Journals (Sweden)

    Angela B Ortiz

    Full Text Available The oncogenic capacity of cyclin D1 has long been established in breast cancer. CCND1 amplification has been identified in a subset of patients with poor prognosis, but there are conflicting data regarding the predictive value of cyclin D1 protein overexpression. This study was designed to analyze the expression of cyclin D1 and its correlation with CCND1 amplification and their prognostic implications in invasive breast cancer. By using the tissue microarray technique, we performed an immunohistochemical study of ER, PR, HER2, p53, cyclin D1, Ki67 and p16 in 179 invasive breast carcinoma cases. The FISH method was performed to detect HER2/Neu and CCND1 amplification. High cyclin D1 expression was identified in 94/179 (52% of invasive breast cancers. Cyclin D1 overexpression and CCND1 amplification were significantly associated (p = 0.010. Overexpression of cyclin D1 correlated with ER expression, PR expression and Luminal subtypes (p<0.001, with a favorable impact on overall survival in the whole series. However, in the Luminal A group, high expression of cyclin D1 correlated with shorter disease-free survival, suggesting that the prognostic role of cyclin D1 depends on the molecular subtype. CCND1 gene amplification was detected in 17 cases (9% and correlated significantly with high tumor grade (p = 0.038, high Ki-67 protein expression (p = 0.002, and the Luminal B subtype (p = 0.002. Patients with tumors with high amplification of CCND1 had an increased risk of recurrence (HR = 2.5; 95% CI, 1.2-4.9, p = 0.01. These findings suggest that CCND1 amplification could be useful for predicting recurrence in invasive breast cancer.

  17. Fluorescent pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging.

    Science.gov (United States)

    Xue, Yuyuan; Liang, Wanshan; Li, Yuan; Wu, Ying; Peng, Xinwen; Qiu, Xueqing; Liu, Jinbin; Sun, Runcang

    2016-12-28

    A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting spirolactam Rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pK a of 5.35, which indicated that L-SRhB has the potential for pH detection of acidic organelle. In addition, the two probes were internalized successfully by living cells through the endocytosis pathway and could distinguish normal cells from cancer cells by different cell staining rates. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, whereas it was nontoxic to normal cells in the same condition. L-SRhB might have potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH sensor and bioimaging dye for the recognition of cancer cells. The results also provided a new perspective to the high-value utilization of lignin.

  18. Feasibility of Real-Time Near-Infrared Fluorescence Tracer Imaging in Sentinel Node Biopsy for Oral Cavity Cancer Patients.

    Science.gov (United States)

    Christensen, Anders; Juhl, Karina; Charabi, Birgitte; Mortensen, Jann; Kiss, Katalin; Kjær, Andreas; von Buchwald, Christian

    2016-02-01

    Sentinel node biopsy (SNB) is an established method in oral squamous cell carcinoma (OSCC) for staging the cN0 neck and to select patients who will benefit from a neck dissection. Near-infrared fluorescence (NIRF) imaging has the potential to improve the SNB procedure by facilitating intraoperative visual identification of the sentinel lymph node (SN). The purpose of this study was to evaluate the feasibility of fluorescence tracer imaging for SN detection in conjunction with conventional radio-guided technique. Prospective study of patients with primary OSCC planned for tumor resection and SNB. Thirty patients were injected peritumorally with a bimodal tracer (ICG-99mTc-Nanocoll) followed by lymphoscintigraphy and SPECT/CT to define the SNs and their anatomic allocation preoperatively. SNs were detected intraoperatively with a hand-held gamma-probe and a hand-held NIRF camera. In 29 of 30 subjects (97%), all preoperatively defined SNs could be identified intraoperatively using a combination of radioactive and fluorescence guidance. A total of 94 SNs (mean 3, range 1-5) that were both radioactive and fluorescent ex vivo were harvested. Eleven of 94 SNs (12%) could only be identified in vivo using NIRF imaging, and the majority of those were located in level 1 close to the primary tumor. A combined fluorescent and radioactive tracer for SNB is feasible, and the additional use of NIRF imaging may improve the accuracy of SN identification in oral cancer patients. Intraoperative fluorescence guidance seems of particular value when SNs are located in close proximity to the injection site.

  19. Towards improved bladder cancer diagnosis using fluorescence imaging and Raman spectroscopy

    NARCIS (Netherlands)

    Grimbergen, M.C.M.

    2010-01-01

    Bladder cancer is the fourth most common type of cancer worldwide. Its high recurrence rate makes bladder cancer one of the most prevalent types of cancer in the western world and the most costly type of cancer over the patient’s lifetime. In the Netherlands, each year 5,400 new patients with

  20. Optical design of an optical coherence tomography and multispectral fluorescence imaging endoscope to detect early stage ovarian cancer

    Science.gov (United States)

    Tate, Tyler; Keenan, Molly; Swan, Elizabeth; Black, John; Utzinger, Urs; Barton, Jennifer

    2014-12-01

    The five year survival rate for ovarian cancer is over 90% if early detection occurs, yet no effective early screening method exists. We have designed and are constructing a dual modality Optical Coherence Tomography (OCT) and Multispectral Fluorescence Imaging (MFI) endoscope to optically screen the Fallopian tube and ovary for early stage cancer. The endoscope reaches the ovary via the natural pathway of the vagina, cervix, uterus and Fallopian tube. In order to navigate the Fallopian tube the endoscope must have an outer diameter of 600 μm, be highly flexible, steerable, tracking and nonperforating. The imaging systems consists of six optical subsystems, two from OCT and four from MFI. The optical subsystems have independent and interrelated design criteria. The endoscope will be tested on realistic tissue models and ex vivo tissue to prove feasibility of future human trials. Ultimately the project aims to provide women the first effective ovarian cancer screening technique.

  1. Selective Killing of Breast Cancer Cells by Doxorubicin-Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET.

    Science.gov (United States)

    Chattoraj, Shyamtanu; Amin, Asif; Jana, Batakrishna; Mohapatra, Saswat; Ghosh, Surajit; Bhattacharyya, Kankan

    2016-01-18

    Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin-loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC50 =155 nm). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin-loaded AuNCs is only 40 % (IC50 =4500 nm). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490-515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au10-13 . The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Combination of panoramic and fluorescence endoscopic images to obtain tumor spatial distribution information useful for bladder cancer detection

    Science.gov (United States)

    Olijnyk, S.; Hernández Mier, Y.; Blondel, W. C. P. M.; Daul, C.; Wolf, D.; Bourg-Heckly, G.

    2007-07-01

    Bladder cancer is widely spread. Moreover, carcinoma in situ can be difficult to diagnose as it may be difficult to see, and become invasive in 50 % of case. Non invasive diagnosis methods like photodynamic or autofluorescence endoscopy allow enhancing sensitivity and specificity. Besides, bladder tumors can be multifocal. Multifocality increases the probability of recurrence and infiltration into bladder muscle. Analysis of spatial distribution of tumors could be used to improve diagnosis. We explore the feasibility to combine fluorescence and spatial information on phantoms. We developed a system allowing the acquisition of consecutive images under white light or UV excitation alternatively and automatically along the video sequence. We also developed an automatic image processing algorithm to build a partial panoramic image from a cystoscopic sequence of images. Fluorescence information is extracted from wavelength bandpass filtered images and superimposed over the cartography. Then, spatial distribution measures of fluorescent spots can be computed. This cartography can be positioned on a 3D generic shape of bladder by selecting some reference points. Our first results on phantoms show that it is possible to obtain cartography with fluorescent spots and extract quantitative information of their spatial distribution on a "wide" field of view basis.

  3. Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

    Science.gov (United States)

    Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

    2018-03-01

    Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

  4. Fluorescent CSC models evidence that targeted nanomedicines improve treatment sensitivity of breast and colon cancer stem cells.

    Science.gov (United States)

    Gener, Petra; Gouveia, Luis Pleno; Sabat, Guillem Romero; de Sousa Rafael, Diana Fernandes; Fort, Núria Bergadà; Arranja, Alexandra; Fernández, Yolanda; Prieto, Rafael Miñana; Ortega, Joan Sayos; Arango, Diego; Abasolo, Ibane; Videira, Mafalda; Schwartz, Simo

    2015-11-01

    To be able to study the efficacy of targeted nanomedicines in marginal population of highly aggressive cancer stem cells (CSC), we have developed a novel in vitro fluorescent CSC model that allows us to visualize these cells in heterogeneous population and to monitor CSC biological performance after therapy. In this model tdTomato reporter gene is driven by CSC specific (ALDH1A1) promoter and contrary to other similar models, CSC differentiation and un-differentiation processes are not restrained and longitudinal studies are feasible. We used this model for preclinical validation of poly[(d,l-lactide-co-glycolide)-co-PEG] (PLGA-co-PEG) micelles loaded with paclitaxel. Further, active targeting against CD44 and EGFR receptors was validated in breast and colon cancer cell lines. Accordingly, specific active targeting toward surface receptors enhances the performance of nanomedicines and sensitizes CSC to paclitaxel based chemotherapy. Many current cancer therapies fail because of the failure to target cancer stem cells. This surviving population soon proliferates and differentiates into more cancer cells. In this interesting article, the authors designed an in vitro cancer stem cell model to study the effects of active targeting using antibody-labeled micelles containing chemotherapeutic agent. This new model should allow future testing of various drug/carrier platforms before the clinical phase. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. In vivo detection of oral epithelial cancer using endogenous fluorescence lifetime imaging: a pilot human study (Conference Presentation)

    Science.gov (United States)

    Jo, Javier A.; Hwang, Dae Yon; Palma, Jorge; Cheng, Shuna; Cuenca, Rodrigo; Malik, Bilal; Jabbour, Joey; Cheng, Lisa; Wright, John; Maitland, Kristen

    2016-03-01

    Endogenous fluorescence lifetime imaging (FLIM) provides direct access to the concomitant functional and biochemical changes accompanying tissue transition from benign to precancerous and cancerous. Since FLIM can noninvasively measure different and complementary biomarkers of precancer and cancer, we hypothesize that it will aid in clinically detecting early oral epithelial cancer. Our group has recently demonstrated the detection of benign from premalignant and malignant lesions based on endogenous multispectral FLIM in the hamster cheek-pouch model. Encouraged by these positive preliminary results, we have developed a handheld endoscope capable of acquiring multispectral FLIM images in real time from the oral mucosa. This novel FLIM endoscope is being used for imaging clinically suspicious pre-malignant and malignant lesions from patients before undergoing tissue biopsy for histopathological diagnosis of oral epithelial cancer. Our preliminary results thus far are already suggesting the potential of endogenous FLIM for distinguishing a variety of benign lesions from advanced dysplasia and squamous cell carcinoma (SCC). To the best of out knowledge, this is the first in vivo human study aiming to demonstrate the ability to predict the true malignancy of clinically suspicious lesions using endogenous FLIM. If successful, the resulting clinical tool will allow noninvasive real-time detection of epithelial precancerous and cancerous lesions in the oral mucosa and could potentially be used to assist at every step involved on the clinical management of oral cancer patients, from early screening and diagnosis, to treatment and monitoring of recurrence.

  6. Choline kinase alpha and hexokinase-2 protein expression in hepatocellular carcinoma: association with survival.

    Directory of Open Access Journals (Sweden)

    Sandi A Kwee

    Full Text Available PURPOSE: Hexokinase-2 (HK2 and more recently choline kinase alpha (CKA expression has been correlated with clinical outcomes in several major cancers. This study examines the protein expression of HK2 and CKA in hepatocellular carcinoma (HCC in association with patient survival and other clinicopathologic parameters. METHODS: Immunohistochemical analysis for HK2 and CKA expression was performed on a tissue microarray of 157 HCC tumor samples. Results were analyzed in relation to clinicopathologic data from Surveillance, Epidemiology, and End-Results Program registries. Mortality rates were assessed by Kaplan-Meier estimates and compared using log-rank tests. Predictors of overall survival were assessed using proportional hazards regression. RESULTS: Immunohistochemical expression of HK2 and CKA was detected in 71 (45% and 55 (35% tumor samples, respectively. Differences in tumor HK2 expression were associated with tumor grade (p = 0.008 and cancer stage (p = 0.001, while CKA expression differed significantly only across cancer stage (p = 0.048. Increased mortality was associated with tumor HK2 expression (p = 0.003 as well as CKA expression (p = 0.03 with hazard ratios of 1.86 (95% confidence interval (CI 1.23-2.83 and 1.59 (95% CI 1.04-2.41, respectively. Similar effects on overall survival were noted in a subset analysis of early stage (I and II HCC. Tumor HK2 expression, but not CKA expression, remained a significant predictor of survival in multivariable analyses. CONCLUSION: HK2 and CKA expression may have biologic and prognostic significance in HCC, with tumor HK2 expression being a potential independent predictor of survival.

  7. Fabrication of transferrin functionalized gold nanoclusters/graphene oxide nanocomposite for turn-on near-infrared fluorescent bioimaging of cancer cells and small animals.

    Science.gov (United States)

    Wang, Yong; Chen, Jia-Tong; Yan, Xiu-Ping

    2013-02-19

    Transferrin (Tf)-functionalized gold nanoclusters (Tf-AuNCs)/graphene oxide (GO) nanocomposite (Tf-AuNCs/GO) was fabricated as a turn-on near-infrared (NIR) fluorescent probe for bioimaging cancer cells and small animals. A one-step approach was developed to prepare Tf-AuNCs via a biomineralization process with Tf as the template. Tf acted not only as a stabilizer and a reducer but also as a functional ligand for targeting the transferrin receptor (TfR). The prepared Tf-AuNCs gave intense NIR fluorescence that can avoid interference from biological media such as tissue autofluorescence and scattering light. The assembly of Tf-AuNCs and GO gave the Tf-AuNCs/GO nanocomposite, a turn-on NIR fluorescent probe with negligible background fluorescence due to the super fluorescence quenching property of GO. The NIR fluorescence of the Tf-AuNCs/GO nanocomposite was effectively restored in the presence of TfR, due to the specific interaction between Tf and TfR and the competition of TfR with the GO for the Tf in Tf-AuNCs/GO composite. The developed turn-on NIR fluorescence probe offered excellent water solubility, stability, and biocompatibility, and exhibited high specificity to TfR with negligible cytotoxicity. The probe was successfully applied for turn-on fluorescent bioimaging of cancer cells and small animals.

  8. Twist and YB-1 gene expression in cervical cancer and precancerous tissue and their correlation with cell invasion

    Directory of Open Access Journals (Sweden)

    Qin Tian

    2017-04-01

    Full Text Available Objective: To study the correlation of Twist and YB-1 gene expression in cervical cancer and precancerous tissue with cell invasion. Methods: Cervical cancer tissue, precancerous tissue and normal cervical tissue surgically removed in our hospital between May 2013 and April 2015 were collected; immunohistochemical staining kits were used to detect the positive protein expression rate of Twist and YB-1 gene; fluorescence quantitative PCR kits were used to detect Twist, YB-1 and invasion gene mRNA expression. Results: Twist and YB-1 mRNA expression and positive protein expression rate as well as USP22, Rab11, Rac1 and ANXA5 mRNA expression in cervical cancer tissue and precancerous tissue were significantly higher than those in normal cervical tissue, Twist and YB-1 mRNA expression and positive protein expression rate as well as USP22, Rab11, Rac1 and ANXA5 mRNA expression in cervical cancer tissue were significantly higher than those in precancerous tissue; USP22, Rab11, Rac1 and ANXA5 mRNA expression in cervical cancer tissue and precancerous tissue with positive Twist and YB-1 expression were significantly higher than those in cervical cancer tissue and precancerous tissue with negative Twist and YB-1 expression. Conclusion: Highly expressed Twist and YB-1 in cervical cancer and precancerous tissue can promote cell invasion.

  9. Correlation of histopathologic characteristics to protein expression and function in malignant melanoma.

    Directory of Open Access Journals (Sweden)

    Charlotte Welinder

    Full Text Available Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information.Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease.In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival.In this feasibility

  10. Correlation of histopathologic characteristics to protein expression and function in malignant melanoma

    Science.gov (United States)

    Pawłowski, Krzysztof; Szasz, A. Marcell; Yakovleva, Maria; Sugihara, Yutaka; Malm, Johan; Jönsson, Göran; Ingvar, Christian; Lundgren, Lotta; Baldetorp, Bo; Olsson, Håkan; Rezeli, Melinda; Laurell, Thomas; Wieslander, Elisabet; Marko-Varga, György

    2017-01-01

    Background Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information. Patients and methods Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease. Results In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor

  11. Using In-vivo Fluorescence Imaging in Personalized Cancer Diagnostics and Therapy, an Image and Treat Paradigm

    Science.gov (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Capala, Jacek; Hassan, Moinuddin; Zielinsky, Rafal; Griffiths, Gary; Achilefu, Samuel; Smith, Paul; Gandjbakhckhe, Amir

    2013-01-01

    The major goal in developing drugs targeting specific tumor receptors, such as Monoclonal AntiBodies (MAB), is to make a drug compound that targets selectively the cancer-causing biomarkers, inhibits their functionality, and/or delivers the toxin specifically to the malignant cells. Recent advances in MABs show that their efficacy depends strongly on characterization of tumor biomarkers. Therefore, one of the main tasks in cancer diagnostics and treatment is to develop non-invasive in-vivo imaging techniques for detection of cancer biomarkers and monitoring their down regulation during the treatment. Such methods can potentially result in a new imaging and treatment paradigm for cancer therapy. In this article we have reviewed fluorescence imaging approaches, including those developed in our group, to detect and monitor Human Epidermal Growth Factor 2 (HER2) receptors before and during therapy. Transition of these techniques from the bench to bedside is the ultimate goal of our project. Similar approaches can be used potentially for characterization of other cancer related cell biomarkers. PMID:22066595

  12. Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

    International Nuclear Information System (INIS)

    Li, Chao; Ruan, Jing; Yang, Meng; Pan, Fei; Gao, Guo; Qu, Su; Shen, You-Lan; Dang, Yong-Jun; Wang, Kan; Jin, Wei-Lin; Cui, Da-Xiang

    2015-01-01

    Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer

  13. Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

    Science.gov (United States)

    Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

    2016-07-01

    The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

  14. Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe.

    Science.gov (United States)

    Shi, Rongguang; Huang, Lu; Duan, Xiaoxue; Sun, Guohao; Yin, Gui; Wang, Ruiyong; Zhu, Jun-Jie

    2017-10-02

    Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Study on discrimination of oral cancer from normal using blood plasma based on fluorescence steady and excited state at excitation wavelength 280 nm

    Science.gov (United States)

    Rekha, Pachaiappan; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2016-03-01

    Many research works based on fluorescence spectroscopy have proven its potential in the diagnosis of various diseases using the spectral signatures of the native key fluorophores such as tryptophan, tyrosine, collagen, NADH, FAD and porphyrin. These fluorophores distribution, concentration and their conformation may be changed depending upon the pathological and metabolic conditions of cells and tissues. In this study, we have made an attempt to characterize the blood plasma of normal subject and oral cancer patients by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed by employing the multivariate statistical method - linear discriminant analyses (LDA) using leaves one out cross validation method. The results illustrate the potential of fluorescence spectroscopy technique in the diagnosis of oral cancer using blood plasma.

  16. In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment

    Science.gov (United States)

    Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

    2015-01-01

    Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

  17. In Vivo Imaging of Prostate Cancer Tumors and Metastasis Using Non-Specific Fluorescent Nanoparticles in Mice

    Directory of Open Access Journals (Sweden)

    Coralie Genevois

    2017-12-01

    Full Text Available With the growing interest in the use of nanoparticles (NPs in nanomedicine, there is a crucial need for imaging and targeted therapies to determine NP distribution in the body after systemic administration, and to achieve strong accumulation in tumors with low background in other tissues. Accumulation of NPs in tumors results from different mechanisms, and appears extremely heterogeneous in mice models and rather limited in humans. Developing new tumor models in mice, with their low spontaneous NP accumulation, is thus necessary for screening imaging probes and for testing new targeting strategies. In the present work, accumulation of LipImageTM 815, a non-specific nanosized fluorescent imaging agent, was compared in subcutaneous, orthotopic and metastatic tumors of RM1 cells (murine prostate cancer cell line by in vivo and ex vivo fluorescence imaging techniques. LipImageTM 815 mainly accumulated in liver at 24 h but also in orthotopic tumors. Limited accumulation occurred in subcutaneous tumors, and very low fluorescence was detected in metastasis. Altogether, these different tumor models in mice offered a wide range of NP accumulation levels, and a panel of in vivo models that may be useful to further challenge NP targeting properties.

  18. Optimizing the diagnosis and treatment of bladder cancer using fluorescence cystoscopy and Raman spectroscopy

    NARCIS (Netherlands)

    Draga, R.O.P.

    2013-01-01

    The gold standard for the diagnosis and treatment of bladder cancer is transurethral resection of bladder tumors (TURBT). A relative high recurrence rate and the need for repeated treatments make bladder cancer one the most expensive cancers from diagnosis till death of the patient. The TURBT

  19. In vitro protein expression: an emerging alternative to cell-based approaches.

    Science.gov (United States)

    He, Mingyue

    2011-04-30

    Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. A hybrid mathematical modeling approach of the metabolic fate of a fluorescent sphingolipid analogue to predict cancer chemosensitivity.

    Science.gov (United States)

    Molina-Mora, J A; Kop-Montero, M; Quirós-Fernández, I; Quiros, S; Crespo-Mariño, J L; Mora-Rodríguez, R A

    2018-04-13

    Sphingolipid (SL) metabolism is a complex biological system that produces and transforms ceramides and other molecules able to modulate other cellular processes, including survival or death pathways key to cell fate decisions. This signaling pathway integrates several types of stress signals, including chemotherapy, into changes in the activity of its metabolic enzymes, altering thereby the cellular composition of bioactive SLs. Therefore, the SL pathway is a promising sensor of chemosensitivity in cancer and a target hub to overcome resistance. However, there is still a gap in our understanding of how chemotherapeutic drugs can disturb the SL pathway in order to control cellular fate. We propose to bridge this gap by a systems biology approach to integrate i) a dynamic model of SL analogue (BODIPY-FL fluorescent-sphingomyelin analogue, SM-BOD) metabolism, ii) a Gaussian mixture model (GMM) of the fluorescence features to identify how the SL pathway senses the effect of chemotherapy and iii) a fuzzy logic model (FLM) to associate SL composition with cell viability by semi-quantitative rules. Altogether, this hybrid model approach was able to predict the cell viability of double experimental perturbations with chemotherapy, indicating that the SL pathway is a promising sensor to design strategies to overcome drug resistance in cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

    2011-07-01

    Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

  2. Dual-Color Fluorescence Imaging of EpCAM and EGFR in Breast Cancer Cells with a Bull's Eye-Type Plasmonic Chip.

    Science.gov (United States)

    Izumi, Shota; Yamamura, Shohei; Hayashi, Naoko; Toma, Mana; Tawa, Keiko

    2017-12-19

    Surface plasmon field-enhanced fluorescence microscopic observation of a live breast cancer cell was performed with a plasmonic chip. Two cell lines, MDA-MB-231 and Michigan Cancer Foundation-7 (MCF-7), were selected as breast cancer cells, with two kinds of membrane protein, epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR), observed in both cells. The membrane proteins are surface markers used to differentiate and classify breast cancer cells. EGFR and EpCAM were detected with Alexa Fluor ® 488-labeled anti-EGFR antibody (488-EGFR) and allophycocyanin (APC)-labeled anti-EpCAM antibody (APC-EpCAM), respectively. In MDA-MB231 cells, three-fold plus or minus one and seven-fold plus or minus two brighter fluorescence of 488-EGFR were observed on the 480-nm pitch and the 400-nm pitch compared with that on a glass slide. Results show the 400-nm pitch is useful. Dual-color fluorescence of 488-EGFR and APC-EpCAM in MDA-MB231 was clearly observed with seven-fold plus or minus two and nine-fold plus or minus three, respectively, on the 400-nm pitch pattern of a plasmonic chip. Therefore, the 400-nm pitch contributed to the dual-color fluorescence enhancement for these wavelengths. An optimal grating pitch of a plasmonic chip improved a fluorescence image of membrane proteins with the help of the surface plasmon-enhanced field.

  3. Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

    Science.gov (United States)

    Pauletti, G; Godolphin, W; Press, M F; Slamon, D J

    1996-07-04

    Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

  4. Analysis of APC allelic imbalance/loss of heterozygosity and APC protein expression in cutaneous squamous cell carcinomas.

    LENUS (Irish Health Repository)

    Gray, Sarah E

    2012-02-01

    BACKGROUND: The adenomatous polyposis coli (APC) gene is a tumor suppressor gene which is mutated in the hereditary disease, familial adenomatous polyposis (FAP). Somatic mutations of the APC gene have also been identified in the majority of sporadic colorectal carcinomas, and mutation of the APC gene appears to be an early step in the initiation of colon cancer. Loss of heterozygosity (LOH) of APC has been described in a variety of other cancer types, including renal cell carcinoma, gastric cancer, non-small cell lung cancer, endometrial cancer and oral squamous cell carcinomas (SCC). AIM: To determine the role played by APC gene in the genesis of cutaneous SCC. MATERIALS AND METHODS: Allelic imbalance\\/loss of heterozygosity (AI\\/LOH) was examined in twenty-two histologically confirmed cutaneous squamous cell carcinomas (SCC) using microsatellite markers, proximal to the APC gene. Immunohistochemical analysis of APC protein expression was also examined in the cutaneous SCC. RESULTS: AI\\/LOH was detected in 60% of the SCC samples using D5S346 marker (proximal to the APC gene). Ninty-five percent of the SCC samples showed positive reduced APC expression, however the localization of the APC protein was abnormal. CONCLUSION: The abnormal expression of APC suggests that APC gene may play a role in cutaneous SCC development.

  5. Antibody-Based Detection of ERG Rearrangement-Positive Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Kyung Park

    2010-07-01

    Full Text Available TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancertissues using a combined immunohistochemistry(IHC and fluorescence in situ hybridization (FISH analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial reoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role. Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5% of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combired pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. Ir conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Giver the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtypirg prostate cancer based or ERG rearrangement status and suggests clinical utility it prostate needle biopsy evaluation.

  6. Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    João Vitor Dutra Molino

    Full Text Available Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.

  7. [A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

    Science.gov (United States)

    Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

    2012-06-01

    To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

  8. Descriptive data on cancerous lung lesions detected by auto-fluorescence bronchoscope: A five-year study

    Directory of Open Access Journals (Sweden)

    Asmitananda Thakur

    2012-01-01

    Full Text Available Background: Auto-fluorescence bronchoscopy (AFB has been used for the identification and localization of intra-epithelial pre-neoplastic and neoplastic lesions within the bronchus. Objectives: To determine the applicability of AFB for the detection and localization of precancerous and cancerous lesions, in addition to analyzing the morphologic presentation, their association to histological type and the variation between genders. Methods: A five-year study involving 4983 patients, who underwent routine bronchoscopy [B] examination in a local tertiary teaching hospital, was done. The B examination was performed under intratracheal lidocaine, and samples were obtained using suitable approach. One thousand four hundred and eighty-five pathologically confirmed lung cancer patients were included in the study. The following parameters were studied: Morphological presentation, biopsy sites, histology. Differences between the groups were analyzed using Chi square test. Result: One thousand four hundred and eighty-five patients who had hyperplasia or neoplastic lesions were further confirmed as lung cancer pathologically. Lung cancer was more commonly found in the right lung (51.58% vs. 42.82%. The lesion occurred more frequently in the upper lobe than the lower lobe (44.17% vs. 22.42%. Male patients with squamous cell carcinoma showed upper lobe involvement more commonly, while the left main bronchus was more commonly involved in female patients. Adenocarcinoma mostly involved lesion of the upper lobe. Squamous cell carcinoma and small cell carcinoma were the major proliferative types (80.15% and 76.16% respectively. Conclusion: AFB is efficient in the detection of pre-invasive and invasive lung lesions. The morphological presentation is associated to the histological type. There is variation in the presentation and histology of cancerous lung lesions between genders.

  9. Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

    Science.gov (United States)

    Winnard, Paul T; Bharti, Santosh K; Penet, Marie-France; Marik, Radharani; Mironchik, Yelena; Wildes, Flonne; Maitra, Anirban; Bhujwalla, Zaver M

    2016-03-15

    The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development. ©2015 American Association for Cancer Research.

  10. The Impact of Hypergravity and Vibration on Gene and Protein Expression of Thyroid Cells

    Science.gov (United States)

    Wehland, Markus; Warnke, Elisabeth; Frett, Timo; Hemmersbach, Ruth; Hauslage, Jens; Ma, Xiao; Aleshcheva, Ganna; Pietsch, Jessica; Bauer, Johann; Grimm, Daniela

    2016-06-01

    Experiments in space either on orbital missions on-board the ISS, or in suborbital missions using sounding rockets, like TEXUS as well as parabolic flight campaigns are still the gold standard to achieve real microgravity conditions in the field of gravitational biology and medicine. However, during launch, and in flight, hypergravity and vibrations occur which might interfere with the effects of microgravity. It is therefore important to know these effects and discriminate them from the microgravity effects. This can be achieved by ground-based facilities like centrifuges or vibration platforms. Recently, we have conducted several experiments with different thyroid cancer cell lines. This study, as part of the ESA-CORA-GBF 2010-203 project, focused on the influence of vibration and hypergravity on benign human thyroid follicular epithelial cells (Nthy-ori 3-1 cell line). Gene and in part protein expression regulation under both conditions were analyzed for VCAN, ITGA10, ITGB1, OPN, ADAM19, ANXA1, TNFA, ABL2, ACTB, PFN2, TLN1, EZR, RDX, MSN, CTGF, PRKCA, and PRKAA1 using quantitative real-time PCR and Western Blot. We found that hypergravity and vibration affected genes and proteins involved in the extracellular matrix, the cytoskeleton, apoptosis, cell growth and signaling. Vibration always led to a down-regulation, whereas hypergravity resulted in a more heterogeneous expression pattern. Overall we conclude that both conditions can influence gene regulation and production of various genes and proteins. As a consequence, it is important to perform control experiments on hypergravity and vibration facilities in parallel to flight experiments.

  11. Evaluation of intratumoral HER-2 heterogeneity by fluorescence in situ hybridization in invasive breast cancer: a single institution study.

    Science.gov (United States)

    Lee, Sarah; Jung, Woohee; Hong, Soon-Won; Koo, Ja Seung

    2011-08-01

    This study aimed to determine the incidence and characteristics of HER-2 gene heterogeneity in invasive breast cancer in a single institution. Included were 971 cases of primary invasive breast cancer diagnosed between 2008 and 2010. Fluorescence in situ hybridization (FISH) image files were retrospectively reviewed and HER-2 gene heterogeneity was defined as more than 5% but less than 50% of analyzed invasive tumor cells with a HER-2/Chr17 ratio higher than 2.2, according to the College of American Pathologists guidelines. HER-2 gene heterogeneity was identified in 24 (2.5%) cases. The mean proportion of invasive tumor cells with a HER-2/chromosome 17 ratio higher than 2.2 was 11.6% (range: 5%-25%). Of 24 cases, HER-2 gene status was not amplified in 8, showed borderline amplification in 2, and amplification in 14. All HER-2 amplification cases were low-grade. In conclusion, HER-2 gene heterogeneity of invasive breast cancer is identified in routine FISH examination. This may affect the results of HER-2 gene amplification status in FISH studies.

  12. Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

    Science.gov (United States)

    Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

    2014-04-01

    The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

  13. uPAR-targeted optical near-infrared (NIR) fluorescence imaging and PET for image-guided surgery in head and neck cancer

    DEFF Research Database (Denmark)

    Christensen, Anders; Juhl, Karina; Persson, Morten

    2017-01-01

    . Histological analysis showed co-localization of the fluorescent signal, uPAR expression and tumor deposits. In addition, the feasibility of uPARguided robotic cancer surgery was demonstrated. Also, uPAR-PET imaging showed a clear and localized signal in the tongue tumors. Conclusions: This study demonstrated...

  14. Heat shock protein expression in canine malignant mammary tumours

    International Nuclear Information System (INIS)

    Romanucci, Mariarita; Marinelli, Alessia; Sarli, Giuseppe; Salda, Leonardo Della

    2006-01-01

    Abnormal levels of Heat Shock Proteins (HSPs) have been observed in many human neoplasms including breast cancer and it has been demonstrated that they have both prognostic and therapeutic implications. In this study, we evaluated immunohistochemical expression of HSPs in normal and neoplastic canine mammary glands and confronted these results with overall survival (OS), in order to understand the role of HSPs in carcinogenesis and to establish their potential prognostic and/or therapeutic value. Immunohistochemical expression of Hsp27, Hsp72, Hsp73 and Hsp90 was evaluated in 3 normal canine mammary glands and 30 malignant mammary tumours (10 in situ carcinomas, 10 invasive carcinomas limited to local structures without identifiable invasion of blood or lymphatic vessels, 10 carcinomas with invasion of blood or lymphatic vessels and/or metastases to regional lymph nodes). A semi-quantitative method was used for the analysis of the results. Widespread constitutive expression of Hsp73 and Hsp90 was detected in normal tissue, Hsp72 appeared to be focally distributed and Hsp27 showed a negative to rare weak immunostaining. In mammary tumours, a significant increase in Hsp27 (P < 0.01), Hsp72 (P < 0.05) and Hsp90 (P < 0.01) expression was observed as well as a significant reduction in Hsp73 (P < 0.01) immunoreactivity compared to normal mammary gland tissue. Hsp27 demonstrated a strong positivity in infiltrating tumour cells and metaplastic squamous elements of invasive groups. High Hsp27 expression also appeared to be significantly correlated to a shorter OS (P = 0.00087). Intense immunolabelling of Hsp72 and Hsp73 was frequently detected in infiltrative or inflammatory tumour areas. Hsp90 expression was high in all tumours and, like Hsp73, it also showed an intense positivity in lymphatic emboli. These results suggest that Hsp27, Hsp72 and Hsp90 are involved in canine mammary gland carcinogenesis. In addition, Hsp27 appears to be implicated in tumour invasiveness and

  15. Characterizing the Utility and Limitations of Repurposing an Open-Field Optical Imaging Device for Fluorescence-Guided Surgery in Head and Neck Cancer Patients.

    Science.gov (United States)

    Moore, Lindsay S; Rosenthal, Eben L; Chung, Thomas K; de Boer, Esther; Patel, Neel; Prince, Andrew C; Korb, Melissa L; Walsh, Erika M; Young, E Scott; Stevens, Todd M; Withrow, Kirk P; Morlandt, Anthony B; Richman, Joshua S; Carroll, William R; Zinn, Kurt R; Warram, Jason M

    2017-02-01

    The purpose of this study was to assess the potential of U.S. Food and Drug Administration-cleared devices designed for indocyanine green-based perfusion imaging to identify cancer-specific bioconjugates with overlapping excitation and emission wavelengths. Recent clinical trials have demonstrated potential for fluorescence-guided surgery, but the time and cost of the approval process may impede clinical translation. To expedite this translation, we explored the feasibility of repurposing existing optical imaging devices for fluorescence-guided surgery. Consenting patients (n = 15) scheduled for curative resection were enrolled in a clinical trial evaluating the safety and specificity of cetuximab-IRDye800 (NCT01987375). Open-field fluorescence imaging was performed preoperatively and during the surgical resection. Fluorescence intensity was quantified using integrated instrument software, and the tumor-to-background ratio characterized fluorescence contrast. In the preoperative clinic, the open-field device demonstrated potential to guide preoperative mapping of tumor borders, optimize the day of surgery, and identify occult lesions. Intraoperatively, the device demonstrated robust potential to guide surgical resections, as all peak tumor-to-background ratios were greater than 2 (range, 2.2-14.1). Postresection wound bed fluorescence was significantly less than preresection tumor fluorescence (P open-field imaging device was successfully repurposed to distinguish cancer from normal tissue in the preoperative clinic and throughout surgical resection. This study illuminated the potential for existing open-field optical imaging devices with overlapping excitation and emission spectra to be used for fluorescence-guided surgery. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

  16. Fluorescence and Diffuse Reflectance Spectroscopy for Breast Cancer Diagnosis During Core Needle Biopsy

    National Research Council Canada - National Science Library

    Zhu, Changfang

    2007-01-01

    .... Both empirically based and model based approaches have been explored for the extraction of diagnostically useful information from the tissue spectra, as well as the diagnosis of breast cancer based...

  17. A Comprehensive Evaluation Of HER-2/NEU In Human Breast Cancer By Immunohistochemistry And Fluorescence In Situ Hybridization

    International Nuclear Information System (INIS)

    Abdel Fattah, H.I.; Seif, A.A.; El Hadidi, E.S.; Mohamed, A.A.; El Shinawi, M.E.; Shabaan, M.A.M.

    2012-01-01

    Background: Accurate diagnostic assessment of human epidermal growth factor receptor-2 (HER2/neu) is essential and a prerequisite for appropriate application of the humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) to the treatment of patients with breast cancer. The food drug administration (FDA) approved immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) for HER2/neu evaluation. IHC is the most widely applicable diagnostic modality in studying HER-2 status. FISH is used for HER2/neu assessment in cases with an equivocal IHC status (score 2+). Objectives: The aim of this study was to detect the amplification and/or expression of HER2/neu in breast cancer using FISH and IHC techniques and to evaluate these applied techniques for their potential and clinical application, with special consideration of equivocal cases by IHC (2+). Subjects and Methods: Assessment of HER2/neu gene expression was made by FISH analysis using the HER2/CEP dual color probe (Vysis) in paraffin-embedded tissue sections of 32 breast cancer patients who were grouped into stages 1+, 2+ or 3+ based on IHC (CBII monoclonal antibody), 4 were classified as IHC 0, 4 were classified as IHC 1 +, 22 were classified as the borderline group; IHC 2+, and 2 were classified as IHC 3+. ER, PR, CEA and CA15-3 were performed to all cases. Survival data was obtained from 25 patients only. Results: The cut-off suggested for HER2/neu amplification by FISH ratio was > 1.3. No statistical significance was found between HER2/neu -by either FISH or IHC- and the different prognostic parameters. The overall survival for the studied patients -in average 3 years- was 16/25 (64%). A significant statistical association was revealed between breast cancer patients’ survival outcome and HER2/neu amplification (p < 0.05) using chi square test. Conclusion: All breast cancer patients should be assessed for HER2/neu status. IHC is a well established method for assessing HER2/neu status in

  18. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    Science.gov (United States)

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

  19. Multi-site and multi-depth in vivo cancer localization enhancement after auto-fluorescence removal

    International Nuclear Information System (INIS)

    Montcuquet, A.S.; Herve, L.; Navarro, F.; Dinten, J.M.; Mars, J.I.

    2011-01-01

    Fluorescence imaging in diffusive media locates tumors tagged by injected fluorescent markers in NIR wavelengths. For deep embedded markers, natural auto-fluorescence of tissues comes to be a limiting factor to tumor detection and accurate FDOT reconstructions. A spectroscopic approach coupled with Non-negative Matrix Factorization source separation method is explored to discriminate fluorescence sources according to their fluorescence spectra and remove unwanted auto-fluorescence. We successfully removed auto-fluorescence from acquisitions on living mice with a single subcutaneous tumor or two capillary tubes inserted at different depths. (authors)

  20. Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience.

    Science.gov (United States)

    Bogdanovska-Todorovska, Magdalena; Petrushevska, Gordana; Janevska, Vesna; Spasevska, Liljana; Kostadinova-Kunovska, Slavica

    2018-05-20

    Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

  1. NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring.

    Science.gov (United States)

    Komljenovic, Dorde; Wiessler, Manfred; Waldeck, Waldemar; Ehemann, Volker; Pipkorn, Ruediger; Schrenk, Hans-Hermann; Debus, Jürgen; Braun, Klaus

    2016-01-01

    Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

  2. Imaging lysosomal highly reactive oxygen species and lighting up cancer cells and tumors enabled by a Si-rhodamine-based near-infrared fluorescent probe.

    Science.gov (United States)

    Zhang, Hongxing; Liu, Jing; Liu, Chenlu; Yu, Pengcheng; Sun, Minjia; Yan, Xiaohan; Guo, Jian-Ping; Guo, Wei

    2017-07-01

    Lysosomes have recently been regarded as the attractive pharmacological targets for selectively killing of cancer cells via lysosomal cell death (LCD) pathway that is closely associated with reactive oxygen species (ROS). However, the details on the ROS-induced LCD of cancer cells are still poorly understood, partially due to the absence of a lysosome-targetable, robust, and biocompatible imaging tool for ROS. In this work, we brought forward a Si-rhodamine-based fluorescent probe, named PSiR, which could selectively and sensitively image the pathologically more relavent highly reactive oxygen species (hROS: HClO, HO, and ONOO - ) in lysosomes of cancer cells. Compared with many of the existing hROS fluorescent probes, its superiorities are mainly embodied in the high stability against autoxidation and photoxidation, near-infrared exitation and emission, fast fluorescence off-on response, and specific lysosomal localization. Its practicality has been demonstrated by the real-time imaging of hROS generation in lysosomes of human non-small-cell lung cancer cells stimulated by anticancer drug β-lapachone. Moreover, the probe was sensitive enough for basal hROS in cancer cells, allowing its further imaging applications to discriminate not only cancer cells from normal cells, but also tumors from healthy tissues. Overall, our results strongly indicated that PSiR is a very promising imaging tool for the studies of ROS-related LCD of cancer cells, screening of new anticancer drugs, and early diagnosis of cancers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. A feasibility study of NIR fluorescent image-guided surgery in head and neck cancer based on the assessment of optimum surgical time as revealed through dynamic imaging

    Directory of Open Access Journals (Sweden)

    Yokoyama J

    2013-04-01

    Full Text Available Junkichi Yokoyama,* Mitsuhisa Fujimaki,* Shinichi Ohba, Takashi Anzai, Ryota Yoshii, Shin Ito, Masataka Kojima, Katsuhisa IkedaDepartment of Otolaryngology-Head and Neck Surgery, Juntendo University School of Medicine, Tokyo, Japan *These authors contributed equally to this study Background: In order to minimize surgical stress and preserve organs, endoscopic or robotic surgery is often performed when conducting head and neck surgery. However, it is impossible to physically touch tumors or to observe diffusely invaded deep organs through the procedure of endoscopic or robotic surgery. In order to visualize and safely resect tumors even in these cases, we propose using an indocyanine green (ICG fluorescence method for navigation surgery in head and neck cancer. Objective: To determine the optimum surgical time for tumor resection after the administration of ICG based on the investigation of dynamic ICG fluorescence imaging. Methods: Nine patients underwent dynamic ICG fluorescence imaging for 360 minutes, assessing tumor visibility at 10, 30, 60, 120, 180, and 360 minutes. All cases were scored according to near-infrared (NIR fluorescence imaging visibility scored from 0 to 5. Results: Dynamic NIR fluorescence imaging under the HyperEye Medical System indicated that the greatest contrast in fluorescent images between tumor and normal tissue could be observed from 30 minutes to 1 hour after the administration of ICG. The optimum surgical time was determined to be between 30 minutes to 2 hours after ICG injection. These findings are particularly useful for detection and safe resection of tumors invading the parapharyngeal space. Conclusion: ICG fluorescence imaging is effective for the detection of head and neck cancer. Preliminary findings suggest that the optimum timing for surgery is from 30 minutes to 2 hours after the ICG injection. Keywords: indocyanine green (ICG, navigation surgery, robotic surgery, endoscopic surgery, minimally invasive

  4. Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer

    NARCIS (Netherlands)

    van Brussel, Aram S A; Adams, Arthur; Oliveira, Sabrina; Dorresteijn, Bram; El Khattabi, Mohamed; Vermeulen, J. F.; van der Wall, Elsken; Mali, Willem P Th M; Derksen, Patrick W B; van Diest, Paul J; van Bergen En Henegouwen, Paul M P

    PURPOSE: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. PROCEDURES: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated

  5. Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer

    NARCIS (Netherlands)

    van Brussel, Aram S A; Adams, Arthur; Oliveira, Sabrina; Dorresteijn, Bram; El Khattabi, Mohamed; Vermeulen, Jeroen F.; van der Wall, Elsken; Mali, W.P.T.M.; Derksen, Patrick W B; van Diest, Paul J.; van Bergen En Henegouwen, Paul M P

    Purpose: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. Procedures: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated

  6. Lycopene Inhibits Metastasis of Human Liver Adenocarcinoma SK-Hep-1 Cells by Downregulation of NADPH Oxidase 4 Protein Expression.

    Science.gov (United States)

    Jhou, Bo-Yi; Song, Tuzz-Ying; Lee, Inn; Hu, Miao-Lin; Yang, Nae-Cherng

    2017-08-16

    NADPH oxidase 4 (NOX4), with the sole function to produce reactive oxygen species (ROS), can be a molecular target for disrupting cancer metastasis. Several studies have indicated that lycopene exhibited anti-metastatic actions in vitro and in vivo. However, the role of NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first confirmed the anti-metastatic effect of lycopene (0.1-5 μM) on human liver adenocarcinoma SK-Hep-1 cells. We showed that lycopene significantly inhibited NOX4 protein expression, with the strongest inhibition of 64.3 ± 10.2% (P lycopene. Lycopene also significantly inhibited NOX4 mRNA expression, NOX activity, and intracellular ROS levels in SK-Hep-1 cells. We then determined the effects of lycopene on transforming growth factor β (TGF-β)-induced metastasis. We found that TGF-β (5 ng/mL) significantly increased migration, invasion, and adhesion activity, the intracellular ROS level, matrix metalloproteinase 9 (MMP-9) and MMP-2 activities, the level of NOX4 protein expression, and NOX activity. All these TGF-β-induced effects were antagonized by the incubation of SK-Hep-1 cells with lycopene (2.5 μM). Using transient transfection of siRNA against NOX4, we found that the downregulation of NOX4 could mimic lycopene by inhibiting cell migration and the activities of MMP-9 and MMP-2 during the incubation with or without TGF-β on SK-Hep-1 cells. The results demonstrate that the downregulation of NOX4 plays a crucial role in the anti-metastatic action of lycopene in SK-Hep-1 cells.

  7. Urothelial dysfunction and sensory protein expressions in patients with urological or systemic diseases and hypersensitive bladder

    Directory of Open Access Journals (Sweden)

    Hueih-Ling Ong

    2017-09-01

    Conclusion: Patients with OAB or HSB showed increased urothelial inflammation and lower barrier protein expression. Increased M3/β3-AR or M2/β3-AR in the urothelium was associated with OAB, whereas decreased M3/β3-AR or M2/β3-AR was associated with poor voiding efficiency and large PVR in LUTD.

  8. Glycoprofiling of N-linked glycans of erythropoietin therapeutic protein expressed in Yarrowia lipolytica

    CSIR Research Space (South Africa)

    Kahari, D

    2008-11-01

    Full Text Available profiling techniques. The gene encoding Lip2 was cloned as a C-terminally His-tagged protein, expressed in Yarrowia lipolytica (Madzak, C et al;2004) and the glycan composition of the purified protein was analysed by HPLC and MALDITOF. The HPLC techniques...

  9. C-fos protein expression in central nervous system. Effects of acute whole-body irradiation

    International Nuclear Information System (INIS)

    Martin, C.; Chollat, S.; Mahfoudi, H.; Lambert, F.; Baille Le Crom, V.; Fatome, M.

    1995-01-01

    Study of c-Fos protein expression in the rat striatum after gamma or (neutron-gamma) irradiation was carried on. c-Fos protein is expressed one hour after gamma exposure at the dose of 15 Gy but specificity of the response must be verified. (author)

  10. Evolutionary Divergence of Gene and Protein Expression in the Brains of Humans and Chimpanzees.

    Science.gov (United States)

    Bauernfeind, Amy L; Soderblom, Erik J; Turner, Meredith E; Moseley, M Arthur; Ely, John J; Hof, Patrick R; Sherwood, Chet C; Wray, Gregory A; Babbitt, Courtney C

    2015-07-10

    Although transcriptomic profiling has become the standard approach for exploring molecular differences in the primate brain, very little is known about how the expression levels of gene transcripts relate to downstream protein abundance. Moreover, it is unknown whether the relationship changes depending on the brain region or species under investigation. We performed high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses on two regions of the human and chimpanzee brain: The anterior cingulate cortex and caudate nucleus. In both brain regions, we found a lower correlation between mRNA and protein expression levels in humans and chimpanzees than has been reported for other tissues and cell types, suggesting that the brain may engage extensive tissue-specific regulation affecting protein abundance. In both species, only a few categories of biological function exhibited strong correlations between mRNA and protein expression levels. These categories included oxidative metabolism and protein synthesis and modification, indicating that the expression levels of mRNA transcripts supporting these biological functions are more predictive of protein expression compared with other functional categories. More generally, however, the two measures of molecular expression provided strikingly divergent perspectives into differential expression between human and chimpanzee brains: mRNA comparisons revealed significant differences in neuronal communication, ion transport, and regulatory processes, whereas protein comparisons indicated differences in perception and cognition, metabolic processes, and organization of the cytoskeleton. Our results highlight the importance of examining protein expression in evolutionary analyses and call for a more thorough understanding of tissue-specific protein expression levels. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular

  11. Thick tissue diffusion model with binding to optimize topical staining in fluorescence breast cancer margin imaging

    Science.gov (United States)

    Xu, Xiaochun; Kang, Soyoung; Navarro-Comes, Eric; Wang, Yu; Liu, Jonathan T. C.; Tichauer, Kenneth M.

    2018-03-01

    Intraoperative tumor/surgical margin assessment is required to achieve higher tumor resection rate in breast-conserving surgery. Though current histology provides incomparable accuracy in margin assessment, thin tissue sectioning and the limited field of view of microscopy makes histology too time-consuming for intraoperative applications. If thick tissue, wide-field imaging can provide an acceptable assessment of tumor cells at the surface of resected tissues, an intraoperative protocol can be developed to guide the surgery and provide immediate feedback for surgeons. Topical staining of margins with cancer-targeted molecular imaging agents has the potential to provide the sensitivity needed to see microscopic cancer on a wide-field image; however, diffusion and nonspecific retention of imaging agents in thick tissue can significantly diminish tumor contrast with conventional methods. Here, we present a mathematical model to accurately simulate nonspecific retention, binding, and diffusion of imaging agents in thick tissue topical staining to guide and optimize future thick tissue staining and imaging protocol. In order to verify the accuracy and applicability of the model, diffusion profiles of cancer targeted and untargeted (control) nanoparticles at different staining times in A431 tumor xenografts were acquired for model comparison and tuning. The initial findings suggest the existence of nonspecific retention in the tissue, especially at the tissue surface. The simulator can be used to compare the effect of nonspecific retention, receptor binding and diffusion under various conditions (tissue type, imaging agent) and provides optimal staining and imaging protocols for targeted and control imaging agent.

  12. Effects of salinomycin and 17-AAG on proliferation of human gastric cancer cells in vitro

    Science.gov (United States)

    Zhang, Zuwen; Zhao, Jumei; Mi, Zhikuan; Pang, Qiuxia; Wang, Aihong; Chen, Meini; Liu, Xiaobin; Wei, Xiaoli; Liu, Tao

    2017-01-01

    The aim of the present study was to investigate the effects and mechanisms of 17-AAG combined with salinomycin treatment on proliferation and apoptosis of the SGC-7901 gastric cancer cell line. An MTT assay was used to detect the proliferation of SGC-7901 cells. Morphological alterations of cells were observed under inverted phase-contrast and fluorescence microscopes. Cell cycle and apoptosis were assessed by flow cytometry analysis. The protein expression of nuclear factor (NF)-κB p65 and Fas-ligand (L) were evaluated by immunocytochemistry. Salinomycin with a concentration range of 1–32 µmol/l was demonstrated to inhibit growth of SGC-7901 cells effectively, affect the morphology and apoptosis rate of cells, and arrest SGC-7901 cells in S phase. Furthermore, salinomycin significantly increased the protein expression of Fas-L and decreased the protein expression of NF-κB p65. The alterations in SGC-7901 cells co-treated with salinomycin and 17-AAG were more significant compared with cells treated with one drug only. In conclusion, the individual use of salinomycin and combined use with 17-AAG may significantly inhibit SGC-7901 gastric cancer cell proliferation and induce cell apoptosis. The potential mechanisms may be associated with upregulation of Fas-L and downregulation of NF-κB. These results provide a basis for the potential use of salinomycin in gastric cancer treatment. PMID:28627587

  13. An Optimized Set of Fluorescence In Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples.

    Science.gov (United States)

    Barr Fritcher, Emily G; Voss, Jesse S; Brankley, Shannon M; Campion, Michael B; Jenkins, Sarah M; Keeney, Matthew E; Henry, Michael R; Kerr, Sarah M; Chaiteerakij, Roongruedee; Pestova, Ekaterina V; Clayton, Amy C; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C; Kipp, Benjamin R

    2015-12-01

    Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis. We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH. The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of

  14. Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Takashi Jin

    2009-11-01

    Full Text Available The early detection of HER2 (human epidermal growth factor receptor 2 status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC. As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs with fluorescence quantum yields of 0.23~0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission on the fluorescence image of KPL-4 cells.

  15. Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

    Science.gov (United States)

    Adinolfi, Barbara; Pellegrino, Mario; Giannetti, Ambra; Tombelli, Sara; Trono, Cosimo; Sotgiu, Giovanna; Varchi, Greta; Ballestri, Marco; Posati, Tamara; Carpi, Sara; Nieri, Paola; Baldini, Francesco

    2017-02-15

    One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm 2 ), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm 2 ). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

    Science.gov (United States)

    Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

    2017-03-01

    Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

  17. Standardization and optimization of fluorescence in situ hybridization (FISH for HER-2 assessment in breast cancer: A single center experience

    Directory of Open Access Journals (Sweden)

    Magdalena Bogdanovska-Todorovska

    2018-05-01

    Full Text Available Accurate assessment of human epidermal growth factor receptor 2 (HER-2 is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC and fluorescence in situ hybridization (FISH. Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples and DAKO Histology FISH Accessory Kit (30 samples. The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70; the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001. The greatest discordance (82% between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

  18. Indicadores de Prognóstico em Câncer de Mama com Axila Negativa: Receptor de Estrógeno e Expressão de P53 e de c-erbB-2 Prognostic Indicators In Lymph Node-Negative Breast Cancer: Estrogen Receptor and P53 and c-erbB-2 Protein Expression

    Directory of Open Access Journals (Sweden)

    Adriana Harter Teixeira Bolaséll

    2000-08-01

    dos linfonodos axilares tem pior prognóstico, em relação ao intervalo livre de doença, quando apresentam associação de tumor pouco diferenciado com RE negativo, p53 positivo e c-erbB-2 positivo.Purpose: to evaluate the prognostic value of estrogen receptor and p53 and c-erbB-2 proteins in lymph node-negative breast cancer. Methods: an immunohistochemical study was made in paraffin-embedded tissues from the file of the Instituto de Pesquisas Cito-Oncológicas of the Fundação Faculdade Federal de Ciências Médicas de Porto Alegre of fifty cases of postmenopausal women, who were treated at the Irmandade da Santa Casa de Misericórdia de Porto Alegre and at the Santa Rita Hospital from 1990 to 1994. For statistical analysis c² with Yates correction, as well as exact Fisher tests were used and Kaplan Meier curves compared with log-rank test. The mean follow-up of the patients was 3.6 years (3.1-4.5. Of the 50 cases, 14 showed recurrence during the period of follow-up. Results: the mean age was 61 years (46-78. Modified radical mastectomy was performed in 35 patients (70% and 15 (30% were submitted to lumpectomy/axillary dissection and postoperative radiation therapy. Fifty percent of the patients who showed recurrence did it in the first three years after the diagnosis. The mean size of the tumor was 2.8 cm (1.98-3.13 and the most frequent histological type was invasive ductal carcinoma of no special type (92%, according to the Bloom and Richardson graduation, 3 being stage I (6.6%, 35 stage II (76% and 8 stage III (17.4%. In the tumors with recurrence, there was no grade I, 9 stage II (25.7% and 3 stage III (37.5%. In relation to the prognosis, the disease-free interval was less when there was association of a poorly differentiated tumor with negative estrogen receptor (p = 0.006, positive p53 (p = 0.006 and positive c-erbB-2 (p = 0.001. Conclusion: postmenopausal women with lymph node-negative breast cancer showed worse prognosis in relation to disease

  19. Automated Image Analysis of HER2 Fluorescence In Situ Hybridization to Refine Definitions of Genetic Heterogeneity in Breast Cancer Tissue.

    Science.gov (United States)

    Radziuviene, Gedmante; Rasmusson, Allan; Augulis, Renaldas; Lesciute-Krilaviciene, Daiva; Laurinaviciene, Aida; Clim, Eduard; Laurinavicius, Arvydas

    2017-01-01

    Human epidermal growth factor receptor 2 gene- (HER2-) targeted therapy for breast cancer relies primarily on HER2 overexpression established by immunohistochemistry (IHC) with borderline cases being further tested for amplification by fluorescence in situ hybridization (FISH). Manual interpretation of HER2 FISH is based on a limited number of cells and rather complex definitions of equivocal, polysomic, and genetically heterogeneous (GH) cases. Image analysis (IA) can extract high-capacity data and potentially improve HER2 testing in borderline cases. We investigated statistically derived indicators of HER2 heterogeneity in HER2 FISH data obtained by automated IA of 50 IHC borderline (2+) cases of invasive ductal breast carcinoma. Overall, IA significantly underestimated the conventional HER2, CEP17 counts, and HER2/CEP17 ratio; however, it collected more amplified cells in some cases below the lower limit of GH definition by manual procedure. Indicators for amplification, polysomy, and bimodality were extracted by factor analysis and allowed clustering of the tumors into amplified, nonamplified, and equivocal/polysomy categories. The bimodality indicator provided independent cell diversity characteristics for all clusters. Tumors classified as bimodal only partially coincided with the conventional GH heterogeneity category. We conclude that automated high-capacity nonselective tumor cell assay can generate evidence-based HER2 intratumor heterogeneity indicators to refine GH definitions.

  20. Magnetic Enrichment of Dendritic Cell Vaccine in Lymph Node with Fluorescent-Magnetic Nanoparticles Enhanced Cancer Immunotherapy

    Science.gov (United States)

    Jin, Honglin; Qian, Yuan; Dai, Yanfeng; Qiao, Sha; Huang, Chuan; Lu, Lisen; Luo, Qingming; Chen, Jing; Zhang, Zhihong

    2016-01-01

    Dendritic cell (DC) migration to the lymph node is a key component of DC-based immunotherapy. However, the DC homing rate to the lymphoid tissues is poor, thus hindering the DC-mediated activation of antigen-specific T cells. Here, we developed a system using fluorescent magnetic nanoparticles (α-AP-fmNPs; loaded with antigen peptide, iron oxide nanoparticles, and indocyanine green) in combination with magnetic pull force (MPF) to successfully manipulate DC migration in vitro and in vivo. α-AP-fmNPs endowed DCs with MPF-responsiveness, antigen presentation, and simultaneous optical and magnetic resonance imaging detectability. We showed for the first time that α-AP-fmNP-loaded DCs were sensitive to MPF, and their migration efficiency could be dramatically improved both in vitro and in vivo through MPF treatment. Due to the enhanced migration of DCs, MPF treatment significantly augmented antitumor efficacy of the nanoparticle-loaded DCs. Therefore, we have developed a biocompatible approach with which to improve the homing efficiency of DCs and subsequent anti-tumor efficacy, and track their migration by multi-modality imaging, with great potential applications for DC-based cancer immunotherapy. PMID:27698936

  1. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera

    Directory of Open Access Journals (Sweden)

    Nagata Tatsuya

    2011-05-01

    Full Text Available Abstract Background Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE containing the envelope gene (env of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Results Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. Conclusions The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine.

  2. Low birthweight is associated with specific changes in muscle insulin-signalling protein expression

    DEFF Research Database (Denmark)

    Ozanne, SE; Jensen, CB; Tingey, KJ

    2005-01-01

    muscle in a human cohort and a rat model. METHODS: We recruited 20 young men with low birthweight (mean birthweight 2702+/-202 g) and 20 age-matched control subjects (mean birthweight 3801+/-99 g). Biopsies were obtained from the vastus lateralis muscle and protein expression of selected insulin......-signalling proteins was determined. Rats used for this study were male offspring born to dams fed a standard (20%) protein diet or a low (8%) protein diet during pregnancy and lactation. Protein expression was determined in soleus muscle from adult offspring. RESULTS: Low-birthweight subjects showed reduced muscle...... expression of protein kinase C (PKC)zeta, p85alpha, p110beta and GLUT4. PKCzeta, GLUT4 and p85 were also reduced in the muscle of rats fed a low-protein diet. Other proteins studied were unchanged in low-birthweight humans and in rats fed a low-protein diet when compared with control groups. CONCLUSIONS...

  3. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    Science.gov (United States)

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...... to control conditions. We therefore hypothesize that cysteine residues increase TRPC6 channel protein expression in humans....

  5. Increase of bcl-2 Protein Expression in Aggressive Basal Cell Carcinoma of Head and Neck

    OpenAIRE

    Cláudia CAZAL; Mariana Roesch ELY; Ana Paula Veras SOBRAL; Wilton Wilney Nascimento PADILHA

    2006-01-01

    Objective: The aim of this study was to verify the bcl-2 protein expression in 22 cutaneous basal cell carcinomas (BCC) of the head and neck, and to compare it with its aggressive behavior. Method: Tumors were histologically classified in non-aggressive (BCC 1) and aggressive (BCC 2) and then submitted to the immunohistochemistry technique with the streptavidin-biotin peroxidase method using the anti-bcl-2 antibody. Results: After proceeding to morphological analysis, sixteen tumors (72.7%) w...

  6. WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome

    International Nuclear Information System (INIS)

    Nunez, María I; Mills, Gordon B; Aldaz, C Marcelo; Rosen, Daniel G; Ludes-Meyers, John H; Abba, Martín C; Kil, Hyunsuk; Page, Robert; Klein-Szanto, Andres JP; Godwin, Andrew K; Liu, Jinsong

    2005-01-01

    The putative tumor suppressor WWOX gene spans the common chromosomal fragile site 16D (FRA16D) at chromosome area 16q23.3-24.1. This region is a frequent target for loss of heterozygosity and chromosomal rearrangement in ovarian, breast, hepatocellular, prostate carcinomas and other neoplasias. The goal of these studies was to evaluate WWOX protein expression levels in ovarian carcinomas to determine if they correlated with clinico-pathological parameters, thus providing additional support for WWOX functioning as a tumor suppressor. We performed WWOX protein expression analyses by means of immunobloting and immunohistochemistry on normal ovaries and specific human ovarian carcinoma Tissue Microarrays (n = 444). Univariate analysis of clinical-pathological parameters based on WWOX staining was determined by χ 2 test with Yates' correction. The basic significance level was fixed at p < 0.05. Immunoblotting analysis from normal ovarian samples demonstrated consistently strong WWOX expression while 37% ovarian carcinomas showed reduced or undetectable WWOX protein expression levels. The immunohistochemistry of normal human ovarian tissue sections confirmed strong WWOX expression in ovarian surface epithelial cells and in epithelial inclusion cysts within the cortex. Out of 444 ovarian carcinoma samples analyzed 30% of tumors showed lack of or barely detectable WWOX expression. The remaining ovarian carcinomas (70%) stained moderately to strongly positive for this protein. The two histotypes showing significant loss of WWOX expression were of the Mucinous (70%) and Clear Cell (42%) types. Reduced WWOX expression demonstrated a significant association with clinical Stage IV (FIGO) (p = 0.007), negative Progesterone Receptor (PR) status (p = 0.008) and shorter overall survival (p = 0.03). These data indicate that WWOX protein expression is highly variable among ovarian carcinoma histotypes. It was also observed that subsets of ovarian tumors demonstrated loss of

  7. Changes in protein expression in p53 deleted spontaneous thymic lymphomas

    DEFF Research Database (Denmark)

    Honoré, Bent; Vorum, Henrik; Pedersen, Anders Elm

    2004-01-01

    with the protein expression in p53+/+ and p53-/- thymocytes. Only a minority (13 proteins) of the quantitatively changed proteins were common for the two thymic lymphoma cell lines, suggesting that the p53 deficiency mainly results in genetic dysfunctions which are individual for a given tumor. Two of the detected...... structure containing motifs of the glyoxalase-bleomycin resistance protein family (MDR) as deduced from the cDNA....

  8. Role of KCNMA1 in breast cancer.

    Directory of Open Access Journals (Sweden)

    Martin Oeggerli

    Full Text Available KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+-activated (BK potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%. We performed an extensive analysis on breast cancer tissue microarrays (TMA of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

  9. Exercise alters myostatin protein expression in sedentary and exercised streptozotocin-diabetic rats.

    Science.gov (United States)

    Bassi, Daniela; Bueno, Patricia de Godoy; Nonaka, Keico Okino; Selistre-Araujo, Heloisa Sobreiro; Leal, Angela Merice de Oliveira

    2015-04-01

    The aim of this study was to analyze the effect of exercise on the pattern of muscle myostatin (MSTN) protein expression in two important metabolic disorders, i.e., obesity and diabetes mellitus. MSTN, is a negative regulator of skeletal muscle mass. We evaluated the effect of exercise on MSTN protein expression in diabetes mellitus and high fat diet-induced obesity. MSTN protein expression in gastrocnemius muscle was analyzed by Western Blot. P sedentary or exercised obese animals. Diabetes reduced gastrocnemius muscle weight in sedentary animals. However, gastrocnemius muscle weight increased in diabetic exercised animals. Both the precursor and processed forms of muscle MSTN protein were significantly higher in sedentary diabetic rats than in control rats. The precursor form was significantly lower in diabetic exercised animals than in diabetic sedentary animals. However, the processed form did not change. These results demonstrate that exercise can modulate the muscle expression of MSTN protein in diabetic rats and suggest that MSTN may be involved in energy homeostasis.

  10. Actual Proliferating Index and p53 protein expression as prognostic marker in odontogenic cysts.

    Science.gov (United States)

    Gadbail, A R; Chaudhary, M; Patil, S; Gawande, M

    2009-10-01

    The purpose of this study was to evaluate the biological aggressiveness of odontogenic keratocyst/keratocystic odontogenic tumour (KCOT), radicular cyst (RC) and dentigerous cyst (DC) by observing the actual proliferative activity of epithelium, and p53 protein expression. The actual proliferative activity was measured by Ki-67 Labelling Index and argyrophilic nucleolar organizing regions (AgNOR) count per nucleus. The p53 protein expression was also evaluated. Ki-67 positive cells were observed higher in suprabasal cell layers of KCOT with uniform distribution, a few of them were predominantly observed in basal cell layer in RC and DC. The AgNOR count was significantly higher in suprabasal cell layers of KCOT. The actual proliferative activity was noted to be higher in suprabasal cell layers of KCOT. The p53 immunolabelling was dense and scattered in basal and suprabasal cell layers in KCOT. The weakly stained p53 positive cells were observed diffusely distributed in KCOT, whereas they were mainly seen in basal cell layer of RC and DC. The quantitative and qualitative differences of the proliferative activity and the p53 protein expression in sporadic KCOT may be associated with intrinsic growth potential that could play a role in its development and explain locally aggressive biological behaviour. AgNOR count and p53 protein detection in odontogenic lesions can be of great consequence to predict the biological behaviour and prognosis.

  11. Protein expression in the nucleus accumbens of rats exposed to developmental vitamin D deficiency.

    Directory of Open Access Journals (Sweden)

    John McGrath

    Full Text Available INTRODUCTION: Developmental vitamin D (DVD deficiency is a candidate risk factor for schizophrenia. Animal models have confirmed that DVD deficiency is associated with a range of altered genomic, proteomic, structural and behavioural outcomes in the rat. Because the nucleus accumbens has been implicated in neuropsychiatric disorders, in the current study we examined protein expression in this region in adult rats exposed to DVD deficiency METHODS: Female Sprague Dawley rats were maintained on a vitamin D deficient diet for 6 weeks, mated and allowed to give birth, after which a diet containing vitamin D was reintroduced. Male adult offspring (n = 8 were compared to control male (n = 8. 2-D gel electrophoresis-based proteomics and mass spectroscopy were used to investigate differential protein expression. RESULTS: There were 35 spots, mapped to 33 unique proteins, which were significantly different between the two groups. Of these, 22 were down-regulated and 13 up-regulated. The fold changes were uniformly small, with the largest FC being -1.67. Within the significantly different spots, three calcium binding proteins (calbindin1, calbindin2 and hippocalcin were altered. Other proteins associated with DVD deficiency related to mitochondrial function, and the dynamin-like proteins. CONCLUSIONS: Developmental vitamin D deficiency was associated with subtle changes in protein expression in the nucleus accumbens. Disruptions in pathways related to calcium-binding proteins and mitochondrial function may underlie some of the behavioural features associated with animal models of developmental vitamin D deficiency.

  12. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  13. Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

    Science.gov (United States)

    He, Ding; Pengtao, Gong; Ju, Yang; Jianhua, Li; He, Li; Guocai, Zhang; Xichen, Zhang

    2017-04-01

    Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis , we detected 2 strains of T. vaginalis ; the virus-infected (V + ) and uninfected (V - ) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V + compared with V - isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V + isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V + and V - isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

  14. A comparative study of cell cycle mediator protein expression patterns in anaplastic and papillary thyroid carcinoma.

    Science.gov (United States)

    Evans, Juanita J; Crist, Henry S; Durvesh, Saima; Bruggeman, Richard D; Goldenberg, David

    2012-07-01

    Anaplastic thyroid carcinoma (ATC) is an extremely aggressive and rapidly fatal neoplasm. The aim of this study was to identify a limited cell cycle associated protein expression pattern unique to ATC and to correlate that pattern with clinical outcome. This represents one of the largest tissue micro-array projects comparing the cell cycle protein expression data of ATC to other well-differentiated tumors in the literature. Tissue microarrays were created from 21 patients with ATC and an age and gender matched cohort of patients with papillary thyroid carcinoma (PTC). Expression of epidermal growth factor receptor, cyclin D1, cyclin E, p53, p21, p16, aurora kinase A, opioid growth factor (OGF), OGF-receptor, thyroglobulin and Ki-67 was evaluated in a semi-quantitative fashion. Differences in protein expression between the cohorts were evaluated using chi-square tests with Bonferroni adjustments. Survival time and presence of metastasis at presentation were collected. The ATC cohort showed a statistically significant decrease (p cycle with aberrant expression of multiple protein markers suggesting increased proliferative activity and loss of control of cell cycle progression to G₁ phase. These findings support the assertion that ATC may represent the furthest end of a continuum of thyroid carcinoma dedifferentiation.

  15. Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

    Science.gov (United States)

    Suguro, Hisashi; Mikami, Yoshikazu; Koshi, Rieko; Ogiso, Bunnai; Watanabe, Eri; Watanabe, Nobukazu; Honda, Masaki J; Asano, Masatake; Komiyama, Kazuo

    2011-08-01

    Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  17. Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis with out Helicobacter pylori infection

    International Nuclear Information System (INIS)

    Zhang, Lin; Hou, Yanhong; Wu, Kai; Li, Dan

    2012-01-01

    Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules

  18. Comparative proteomics analysis of chronic atrophic gastritis: changes of protein expression in chronic atrophic gastritis with out Helicobacter pylori infection

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lin; Hou, Yanhong; Wu, Kai; Li, Dan [Department of Gastroenterology and Hepatology, The 309 Hospital of People' s Liberation Army, Beijing (China)

    2012-03-02

    Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.

  19. Evolutionary tuning of protein expression levels of a positively autoregulated two-component system.

    Directory of Open Access Journals (Sweden)

    Rong Gao

    2013-10-01

    Full Text Available Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is 'appropriate' for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the E. coli PhoB/PhoR two-component system (TCS. In response to phosphate (Pi-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural

  20. Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients

    Science.gov (United States)

    Martinez, Pablo; Hernández-Losa, Javier; Cedrés, Susana; Castellví, Josep; Martinez-Marti, Alex; Tallada, Natalia; Murtra-Garrell, Nuria; Navarro-Mendivill, Alejandro; Rodriguez-Freixinos, Victor; Canela, Mercedes; Ramon y Cajal, Santiago; Felip, Enriqueta

    2013-01-01

    Background Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients. Methods NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored. Results ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative. Conclusions ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients. PMID:23359795

  1. Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

    International Nuclear Information System (INIS)

    Lim, Yong Taik; Cho, Mi Young; Noh, Young-Woock; Chung, Bong Hyun; Chung, Jin Woong

    2009-01-01

    This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

  2. Real-time navigation system for sentinel lymph node biopsy in breast cancer patients using projection mapping with indocyanine green fluorescence.

    Science.gov (United States)

    Takada, Masahiro; Takeuchi, Megumi; Suzuki, Eiji; Sato, Fumiaki; Matsumoto, Yoshiaki; Torii, Masae; Kawaguchi-Sakita, Nobuko; Nishino, Hiroto; Seo, Satoru; Hatano, Etsuro; Toi, Masakazu

    2018-05-09

    Inability to visualize indocyanine green fluorescence images in the surgical field limits the application of current near-infrared fluorescence imaging (NIR) systems for real-time navigation during sentinel lymph node (SLN) biopsy in breast cancer patients. The aim of this study was to evaluate the usefulness of the Medical Imaging Projection System (MIPS), which uses active projection mapping, for SLN biopsy. A total of 56 patients (59 procedures) underwent SLN biopsy using the MIPS between March 2016 and November 2017. After SLN biopsy using the MIPS, residual SLNs were removed using a conventional NIR camera and/or radioisotope method. The primary endpoint of this study was identification rate of SLNs using the MIPS. In all procedures, at least one SLN was detected by the MIPS, giving an SLN identification rate of 100% [95% confidence interval (CI) 94-100%]. SLN biopsy was successfully performed without operating lights in all procedures. In total, 3 positive SLNs were excised using MIPS, but were not included in the additional SLNs excised by other methods. The median number of SLNs excised using the MIPS was 3 (range 1-7). Of procedures performed after preoperative systemic therapy, the median number of SLNs excised using the MIPS was 3 (range 2-6). The MIPS is effective in detecting SLNs in patients with breast cancer, providing continuous and accurate projection of fluorescence signals in the surgical field, without need for operating lights, and could be useful in real-time navigation surgery for SLN biopsy.

  3. Frequency of polymorphisms and protein expression of cyclin-dependent kinase inhibitor 1A (CDKN1A in central nervous system tumors

    Directory of Open Access Journals (Sweden)

    Mev Dominguez Valentin

    Full Text Available CONTEXT AND OBJECTIVE: Genetic investigation of central nervous system (CNS tumors provides valuable information about the genes regulating proliferation, differentiation, angiogenesis, migration and apoptosis in the CNS. The aim of our study was to determine the prevalence of genetic polymorphisms (codon 31 and 3' untranslated region, 3'UTR and protein expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A gene in patients with and without CNS tumors. DESIGN AND SETTING: Analytical cross-sectional study with a control group, at the Molecular Biology Laboratory, Pediatric Oncology Department, Hospital das Clínicas de Ribeirão Preto. METHODS: 41 patients with CNS tumors and a control group of 161 subjects without cancer and paires for sex, age and ethnicity were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Protein analysis was performed on 36 patients with CNS tumors, using the Western Blotting technique. RESULTS: The frequencies of the heterozygote (Ser/Arg and polymorphic homozygote (Arg/Arg genotypes of codon 31 in the control subjects were 28.0% and 1.2%, respectively. However, the 3'UTR site presented frequencies of 24.2% (C/T and 0.6% (T/T. These frequencies were not statistically different (P > 0.05 from those seen in the patients with CNS tumors (19.4% and 0.0%, codon 31; 15.8% and 2.6%, 3'UTR site. Regarding the protein expression in ependymomas, 66.67% did not express the protein CDKN1A. The results for medulloblastomas and astrocytomas were similar: neither of them expressed the protein (57.14% and 61.54%, respectively. CONCLUSION: No significant differences in protein expression patterns or polymorphisms of CDKN1A in relation to the three types of CNS tumors were observed among Brazilian subjects.

  4. Automated image analysis of cyclin D1 protein expression in invasive lobular breast carcinoma provides independent prognostic information.

    Science.gov (United States)

    Tobin, Nicholas P; Lundgren, Katja L; Conway, Catherine; Anagnostaki, Lola; Costello, Sean; Landberg, Göran

    2012-11-01

    The emergence of automated image analysis algorithms has aided the enumeration, quantification, and immunohistochemical analyses of tumor cells in both whole section and tissue microarray samples. To date, the focus of such algorithms in the breast cancer setting has been on traditional markers in the common invasive ductal carcinoma subtype. Here, we aimed to optimize and validate an automated analysis of the cell cycle regulator cyclin D1 in a large collection of invasive lobular carcinoma and relate its expression to clinicopathologic data. The image analysis algorithm was trained to optimally match manual scoring of cyclin D1 protein expression in a subset of invasive lobular carcinoma tissue microarray cores. The algorithm was capable of distinguishing cyclin D1-positive cells and illustrated high correlation with traditional manual scoring (κ=0.63). It was then applied to our entire cohort of 483 patients, with subsequent statistical comparisons to clinical data. We found no correlation between cyclin D1 expression and tumor size, grade, and lymph node status. However, overexpression of the protein was associated with reduced recurrence-free survival (P=.029), as was positive nodal status (Pinvasive lobular carcinoma. Finally, high cyclin D1 expression was associated with increased hazard ratio in multivariate analysis (hazard ratio, 1.75; 95% confidence interval, 1.05-2.89). In conclusion, we describe an image analysis algorithm capable of reliably analyzing cyclin D1 staining in invasive lobular carcinoma and have linked overexpression of the protein to increased recurrence risk. Our findings support the use of cyclin D1 as a clinically informative biomarker for invasive lobular breast cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

    Directory of Open Access Journals (Sweden)

    Katherine D. Shives

    2016-10-01

    Full Text Available West Nile virus (WNV is a (+ sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1 for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K and eukaryotic translation initiation factor 4E-binding protein (4EBP pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E interaction and eukaryotic initiation factor 4F (eIF4F complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6 and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  6. PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

    Directory of Open Access Journals (Sweden)

    Mao-lin HUANG

    2014-09-01

    Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

  7. Effects of Diabetes on Salivary Gland Protein Expression of Tetrahydrobiopterin and Nitric Oxide Synthesis and Function.

    Science.gov (United States)

    Stewart, Cassandra R; Obi, Nneka; Epane, Elodie C; Akbari, Alexander A; Halpern, Leslie; Southerland, Janet H; Gangula, Pandu R

    2016-06-01

    Xerostomia is defined as dry mouth resulting from a change in the amount or composition of saliva and is often a major oral health complication associated with diabetes mellitus (DM). Studies have shown that xerostomia is more common in females at the onset of DM. Evidence suggests that nitric oxide (NO) plays a critical role in healthy salivary gland function. However, the specific mechanisms by which NO regulates salivary gland function at the onset of DM have yet to be determined. This study has two aims: 1) to determine whether protein expression or dimerization of NO synthase enzymes (neuronal [nNOS] and endothelial [eNOS]) are altered in the onset of diabetic xerostomia; and 2) to determine whether the changes in nNOS/eNOS protein expression or dimerization are correlated with changes in NO cofactor tetrahydrobiopterin (BH4) biosynthetic enzymes (guanosine triphosphate cyclohydrolase-1 and dihydrofolate reductase). Functional and Western blot studies were performed in streptozotocin-induced and control Sprague-Dawley female rats with DM (type 1 [t1DM]) using standardized protocols. Confirmation of xerostomia was determined by increased water intake and decreased salivary flow rate. The results showed that in female rats with DM, salivary hypofunction is correlated with decreased submandibular and parotid gland sizes. The results also show a decrease in NOS and BH4 biosynthetic enzyme in submandibular glands. These results indicate that a decrease in submandibular NO-BH4 protein expression may provide insight pertaining to mechanisms for the development of hyposalivation in DM-induced xerostomia. Furthermore, understanding the role of the NO-BH4 pathway may give insight into possible treatment options for the patient with DM experiencing xerostomia.

  8. 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

    Science.gov (United States)

    Shives, Katherine D; Massey, Aaron R; May, Nicholas A; Morrison, Thomas E; Beckham, J David

    2016-10-18

    West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7 GpppN m 5' cap with 2'- O -methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

  9. NM23 protein expression in colorectal carcinoma using TMA (tissue microarray: association with metastases and survival

    Directory of Open Access Journals (Sweden)

    Levindo Alves de Oliveira

    2010-12-01

    Full Text Available CONTEXT: NM23, a metastasis suppressor gene, may be associated with prognosis in patients with colorectal carcinoma. OBJECTIVE: To analyze NM23 expression and its association with the presence of lymph node and liver metastases and survival in patients operated on for colorectal carcinoma. METHODS: One hundred thirty patients operated on for colorectal carcinoma were investigated. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic mucosa were obtained and analyzed by immunohistochemical staining using a monoclonal anti-NM23 antibody. Immunohistochemical expression was assessed using a semiquantitative scoring method, counting the percentage of stained cells. The results were compared regarding morphological and histological characteristics of the colorectal carcinoma, presence of lymph node and liver metastases, tumor staging, and patient survival. Statistical analysis was performed using the Mann-Whitney test, the Kruskal-Wallis test and Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. RESULTS: NM23 expression was higher in colorectal carcinoma tissue than in adjacent non-neoplastic mucosa (P<0.0001. NM23 protein expression did not correlate with degree of cell differentiation (P = 0.57, vascular invasion (P = 0.85, lymphatic invasion (P = 0.41, perineural infiltration (P = 0.46, staging (P = 0.19, lymph node metastases (P = 0.08, or liver metastases (P = 0.59. Disease-free survival showed significant association (P = 0.01 with the intensity of NM23 protein immunohistochemical expression in colorectal carcinoma tissue, whereas overall survival showed no association with NM23 protein expression (P = 0.13. CONCLUSIONS: NM23 protein expression was higher in neoplastic colorectal carcinoma tissue than in adjacent non-neoplastic mucosa, showing no correlation with morphological aspects, presence of lymph node or liver metastases, colorectal carcinoma

  10. Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications.

    Science.gov (United States)

    Juturu, Veeresh; Wu, Jin Chuan

    2018-01-04

    Pichia pastoris is a well-known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent-free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site-specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α-factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co-expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high-quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol-II, and hyaluronic acid. The cell-surface display of enzymes has obviously increased enzyme stability, and high-level intracellular expression of acyl-CoA and ethanol O-acyltransferase, lipase and d-amino acid oxidase has opened up applications in whole-cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High-level expression of various food-grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Determination of the activity of telomerase in cancer cells by using BSA-protected gold nanoclusters as a fluorescent probe.

    Science.gov (United States)

    Xu, Yujuan; Zhang, Peng; Wang, Zhen; Lv, Shaoping; Ding, Caifeng

    2018-02-27

    Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650 nm) on photoexcitation with ultraviolet light (365 nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity. Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.

  12. Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris

    International Nuclear Information System (INIS)

    Clark, Lindsay; Zahm, Jacob A.; Ali, Rustam; Kukula, Maciej; Bian, Liangqiao; Patrie, Steven M.; Gardner, Kevin H.; Rosen, Michael K.; Rosenbaum, Daniel M.

    2015-01-01

    13 C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13 C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13 C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets

  13. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    International Nuclear Information System (INIS)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João

    2013-01-01

    Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag + presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag + (10 μg L −1 ) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag + . Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag + , with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one protein involved in

  14. Machine learning in computational biology to accelerate high-throughput protein expression

    DEFF Research Database (Denmark)

    Sastry, Anand; Monk, Jonathan M.; Tegel, Hanna

    2017-01-01

    and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide...... the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. Availability and implementation: We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template...

  15. Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Tânia; Pereira, Catarina G.; Cardoso, Cátia; Bebianno, Maria João, E-mail: mbebian@ualg.pt

    2013-07-15

    Highlights: •Different protein expression profiles between tissues and Ag forms. •Ag NPs and Ag{sup +} presented different mechanisms of toxic action. •Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades. •New biomarkers for Ag NPs were proposed, i.e. MVP, ras partial and precol-P. -- Abstract: Ag NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag{sup +} (10 μg L{sup −1}) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two–dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag{sup +}. Fifteen of these proteins were subsequently identified by MALDI–TOF–TOF and database search. Ag NPs affected similar cellular pathways as Ag{sup +}, with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one

  16. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    Science.gov (United States)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  17. Effect of polysaccharides from Angelica sinensis on Bcl-2 and Bax protein expression of irradiated liver cells

    International Nuclear Information System (INIS)

    Sun Yuanlin; Tang Jian; Gu Xiaohong; Li Deyuan

    2009-01-01

    Objective: To investigate the effect of polysaccharides from Angelica sinensis (ASP3) on Bcl-2 and Bax protein expression of irradiated liver cells from mice. Methods: Bcl-2 and Bax protein expression of liver cells in vitro exposed to 2.0 Gy rays were examined by using immunohistochemistry method. Results: The expression of apoptosis-accelerating protein Bax in the irradiation group was enhanced obviously (70.83%), while apoptosis inhibiting protein Bcl-2 tended to decline (55.60%), with the statistically significant difference (P <0.01) compared with that of the control. ASP3 pretreatment could regulate Bcl-2 and Bax protein expression of liver cells, inhibiting Bax protein expression(64.14/58.37%) and increasing Bcl-2 protein expression(59.21%/ 67.45%). The differences between the high dosage (100 mg/L of ASP3) and the irradiation group were statistically significant (P<0.05). Conclusions: ASP3 pretreatment could prohibit the apoptosis of radiation- damaged liver cells due to abnormal expression of Bcl-2 and Bax, and reduce the cell apoptosis by increasing Bcl-2/Bax protein expression so as to enhance the radiation endurance of liver cells. (authors)

  18. Homogeneous immunoassay for the cancer marker alpha-fetoprotein using single wavelength excitation fluorescence cross-correlation spectroscopy and CdSe/ZnS quantum dots and fluorescent dyes as labels

    International Nuclear Information System (INIS)

    Wang, Jinjie; Liu, Heng; Huang, Xiangyi; Ren, Jicun

    2016-01-01

    The article describes sensitive and selective homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP) in human serum by using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). Both competitive and sandwich immunoassay modes were applied, and AFP served as a model analyte. Fluorescent CdSe/ZnS quantum dots (with a 655 nm emission peak) and the fluorophore Alexa Fluor 488 (520 nm emission) were chosen to label the antibodies in the sandwich mode, and the antibody and the antigen in the competitive mode. Under optimized conditions, the sandwich assay has a linear dynamic range that covers the 20 pM to 5.0 nM concentration range. The competitive assay, in turn, extends from 180 pM to 15.0 nM. The respective detection limits are 20 pM and 180 pM. The method was successfully applied to directly determine AFP in (spiked) clinical samples, and results were in good agreement with data obtained via ELISAs. (author)

  19. Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

    Science.gov (United States)

    Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Reddy, P Vijay Anand

    2012-03-01

    Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value

  20. Parasitization by Scleroderma guani influences protein expression in Tenebrio molitor pupae.

    Science.gov (United States)

    Zhu, Jia-Ying; Wu, Guo-Xing; Ze, Sang-Zi; Stanley, David W; Yang, Bin

    2014-07-01

    Ectoparasitoid wasps deposit their eggs onto the surface and inject venom into their hosts. Venoms are chemically complex and they exert substantial impact on hosts, including permanent or temporary paralysis and developmental arrest. These visible venom effects are due to changes in expression of genes encoding physiologically relevant proteins. While the influence of parasitization on gene expression in several lepidopterans has been reported, the molecular details of parasitoid/beetle relationships remain mostly unknown. This shortcoming led us to pose the hypothesis that envenomation by the ectoparasitic ant-like bethylid wasp Scleroderma guani leads to changes in protein expression in the yellow mealworm beetle Tenebrio molitor. We tested our hypothesis by comparing the proteomes of non-parasitized and parasitized host pupae using iTRAQ-based proteomics. We identified 41 proteins that were differentially expressed (32↑- and 9↓-regulated) in parasitized pupae. We assigned these proteins to functional categories, including immunity, stress and detoxification, energy metabolism, development, cytoskeleton, signaling and others. We recorded parallel changes in mRNA levels and protein abundance in 14 selected proteins following parasitization. Our findings support our hypothesis by documenting changes in protein expression in parasitized hosts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

    Science.gov (United States)

    Ambrosio, Javier R; Ostoa-Saloma, Pedro; Palacios-Arreola, M Isabel; Ruíz-Rosado, Azucena; Sánchez-Orellana, Pedro L; Reynoso-Ducoing, Olivia; Nava-Castro, Karen E; Martínez-Velázquez, Nancy; Escobedo, Galileo; Ibarra-Coronado, Elizabeth G; Valverde-Islas, Laura; Morales-Montor, Jorge

    2014-09-01

    We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  2. iQuantitator: A tool for protein expression inference using iTRAQ

    Directory of Open Access Journals (Sweden)

    Comte-Walters Susana

    2009-10-01

    Full Text Available Abstract Background Isobaric Tags for Relative and Absolute Quantitation (iTRAQ™ [Applied Biosystems] have seen increased application in differential protein expression analysis. To facilitate the growing need to analyze iTRAQ data, especially for cases involving multiple iTRAQ experiments, we have developed a modeling approach, statistical methods, and tools for estimating the relative changes in protein expression under various treatments and experimental conditions. Results This modeling approach provides a unified analysis of data from multiple iTRAQ experiments and links the observed quantity (reporter ion peak area to the experiment design and the calculated quantity of interest (treatment-dependent protein and peptide fold change through an additive model under log transformation. Others have demonstrated, through a case study, this modeling approach and noted the computational challenges of parameter inference in the unbalanced data set typical of multiple iTRAQ experiments. Here we present the development of an inference approach, based on hierarchical regression with batching of regression coefficients and Markov Chain Monte Carlo (MCMC methods that overcomes some of these challenges. In addition to our discussion of the underlying method, we also present our implementation of the software, simulation results, experimental results, and sample output from the resulting analysis report. Conclusion iQuantitator's process-based modeling approach overcomes limitations in current methods and allows for application in a variety of experimental designs. Additionally, hypertext-linked documents produced by the tool aid in the interpretation and exploration of results.

  3. Biological activity of Bt proteins expressed in different structures of transgenic corn against Spodoptera frugiperda

    Directory of Open Access Journals (Sweden)

    Daniel Bernardi

    2016-06-01

    Full Text Available ABSTRACT: Spodoptera frugiperda (J. E. Smith is the main target pest of Bt corn technologies, such as YieldGard VT PRO(tm (Cry1A.105/Cry2Ab2 and PowerCore(tm (Cry1A.105/Cry2Ab2/Cry1F. In this study, it was evaluated the biological activity of Bt proteins expressed in different plant structures of YieldGard VT PRO(tm and PowerCore(tm corn against S. frugiperda . Complete mortality of S. frugiperda neonates was observed on leaf-disc of both Bt corn technologies. However, the mortality in silks and grains was lower than 50 and 6%, respectively. In addition, more than 49% of the surviving larvae in silks and grains completed the biological cycle. However, all life table parameters were negatively affected in insects that developed in silks and grains of both Bt corn events. In summary, the low biological activity of Bt proteins expressed on silks and grains of YieldGard VT PRO(tm and PowerCore(tm corn can contribute to the resistance evolution in S. frugiperda populations.

  4. Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

    Directory of Open Access Journals (Sweden)

    Yawei Gao

    2017-12-01

    Full Text Available Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

  5. Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells

    Directory of Open Access Journals (Sweden)

    Hyun Kim

    2012-05-01

    Full Text Available Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL. Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

  6. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    International Nuclear Information System (INIS)

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-01-01

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export

  7. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  8. Clinical Significance of "Double-hit" and "Double-protein" expression in Primary Gastric B-cell Lymphomas.

    Science.gov (United States)

    He, Miaoxia; Chen, Keting; Li, Suhong; Zhang, Shimin; Zheng, Jianming; Hu, Xiaoxia; Gao, Lei; Chen, Jie; Song, Xianmin; Zhang, Weiping; Wang, Jianmin; Yang, Jianmin

    2016-01-01

    Primary gastric B-cell lymphoma is the second most common malignancy of the stomach. There are many controversial issues about its diagnosis, treatment and clinical management. "Double-hit" and "double-protein" involving gene rearrangement and protein expression of c-Myc and bcl2/bcl6 are the most used terms to describe DLBCL poor prognostic factors in recent years. However, very little is known about the role of these prognostic factors in primary gastric B-cell lymphomas. This study aims to obtain a molecular pathology prognostic model of gastric B-cell lymphoma for clinical stratified management by evaluating how the "double-hit" and "double-protein" in tumor cells as well as microenvironmental reaction of tumor stromal tissue affect clinical outcome in primary gastric B-cell lymphomas. Data and tissues of 188 cases diagnosed with gastric B-cell lymphomas were used in this study. Tumor tissue microarray (TMA) of formalin fixed and paraffin embedded (FFPE) tissues was constructed for fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analysis with a serial of biomarkers containing MYC, BCL2, BCL6, CD31, SPARC, CD10, MUM1 and Ki-67. Modeled period analysis was used to estimate 3-year and 5-year overall survival (OS) and disease-free survival (DFS) distributions. There was no definite "double-hit" case though the gene rearrangement of c-Myc (5.9%), bcl2 (0.1%) and bcl6 (7.4%) was found in gastric B-cell lymphomas. The gene amplification or copy gains of c-Myc (10.1%), bcl-2 (17.0%) and bcl-6 (0.9%) were present in these lymphomas. There were 12 cases of the lymphomas with the "double-protein" expression of MYC and BCL2/BCL6. All patients with "double-protein" gastric B-cell lymphomas had poor outcome compared with those without. More importantly, "MYC-BCL2-BCL6" negative group of gastric B-cell lymphoma patients had favorable clinical outcome regardless clinical stage, pathological types and therapeutic modalities. And the similar better

  9. Fiscal 1999 achievement report on research and development project on intellectual infrastructure creation and utilization technologies. Development of efficient protein expression system (Development of efficient protein expression system utilizing protein folding mechanism of hyperthermophilic bacteria); 1999 nendo kokoritsu tanpakushitsu hatsugen system no kaihatsu seika hokokusho. Chokonetsukin no tanpakushitsu oritatami kiko wo riyoshita kokoritsu tanpakushitsu hatsugen system no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    Efforts were exerted to achieve efficient expression of proteins of hyperthermophilic bacteria, hyperthermophilic archaeabacteria in particular, using a heterogene expression system in which Escherichia coli was the host. In an effort to search for genes related to protein folding and to elucidate the mechanism of folding, chaperonin and prefoldin subunit genes, out of various factors participating in protein folding in hyperthermophilic archaeabacteria, were cloned, and expressed in Escherichia coli. As a system for analyzing protein folding reaction, an experimental system was established on a substrate comprising isopropyl malate dehydrogenase, citrate synthase, glucose dehydrogenase, and a green fluorescent protein. Studies were further conducted to elucidate the mechanism of expression of enzyme genes in Escherichia coli for the establishment of a mass production method for useful enzymes. Also carried out was the research and development of an element technology evaluation system involving protein expression. (NEDO)

  10. In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

    Science.gov (United States)

    Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

    2014-09-01

    In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

  11. Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

    Science.gov (United States)

    Sarkar, Mitul; Koland, John G

    2016-01-01

    The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

  12. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundati......Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

  13. Protein expression changes induced in a malignant melanoma cell line by the curcumin analogue compound D6

    International Nuclear Information System (INIS)

    Pisano, Marina; Palomba, Antonio; Tanca, Alessandro; Pagnozzi, Daniela; Uzzau, Sergio; Addis, Maria Filippa; Dettori, Maria Antonietta; Fabbri, Davide; Palmieri, Giuseppe; Rozzo, Carla

    2016-01-01

    We have previously demonstrated that the hydroxylated biphenyl compound D6 (3E,3′E)-4,4′-(5,5′,6,6′-tetramethoxy-[1,1′-biphenyl]-3,3′-diyl)bis (but-3-en-2-one), a structural analogue of curcumin, exerts a strong antitumor activity on melanoma cells both in vitro and in vivo. Although the mechanism of action of D6 is yet to be clarified, this compound is thought to inhibit cancer cell growth by arresting the cell cycle in G2/M phase, and to induce apoptosis through the mitochondrial intrinsic pathway. To investigate the changes in protein expression induced by exposure of melanoma cells to D6, a differential proteomic study was carried out on D6-treated and untreated primary melanoma LB24Dagi cells. Proteins were fractionated by SDS-PAGE and subjected to in gel digestion. The peptide mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry. Proteins were identified and quantified using database search and spectral counting. Proteomic data were finally uploaded into the Ingenuity Pathway Analysis software to find significantly modulated networks and pathways. Analysis of the differentially expressed protein profiles revealed the activation of a strong cellular stress response, with overexpression of several HSPs and stimulation of ubiquitin-proteasome pathways. These were accompanied by a decrease of protein synthesis, evidenced by downregulation of proteins involved in mRNA processing and translation. These findings are consistent with our previous results on gene expression profiling in melanoma cells treated with D6. Our findings confirm that the curcumin analogue D6 triggers a strong stress response in melanoma cells, turning down majority of cell functions and finally driving cells to apoptosis. The online version of this article (doi:10.1186/s12885-016-2362-6) contains supplementary material, which is available to authorized users

  14. The protein expression landscape of mitosis and meiosis in diploid budding yeast.

    Science.gov (United States)

    Becker, Emmanuelle; Com, Emmanuelle; Lavigne, Régis; Guilleux, Marie-Hélène; Evrard, Bertrand; Pineau, Charles; Primig, Michael

    2017-03-06

    Saccharomyces cerevisiae is an established model organism for the molecular analysis of fundamental biological processes. The genomes of numerous strains have been sequenced, and the transcriptome and proteome ofmajor phases during the haploid and diploid yeast life cycle have been determined. However, much less is known about dynamic changes of the proteome when cells switch from mitotic growth to meiotic development. We report a quantitative protein profiling analysis of yeast cell division and differentiation based on mass spectrometry. Information about protein levels was integrated with strand-specific tiling array expression data. We identified a total of 2366 proteins in at least one condition, including 175 proteins showing a statistically significant>5-fold change across the sample set, and 136 proteins detectable in sporulating but not respiring cells. We correlate protein expression patterns with biological processes and molecular function by Gene Ontology term enrichment, chemoprofiling, transcription interference and the formation of double stranded RNAs by overlapping sense/antisense transcripts. Our work provides initial quantitative insight into protein expression in diploid respiring and differentiating yeast cells. Critically, it associates developmentally regulated induction of antisense long noncoding RNAs and double stranded RNAs with fluctuating protein concentrations during growth and development. This integrated genomics analysis helps better understand how the transcriptome and the proteome correlate in diploid yeast cells undergoing mitotic growth in the presence of acetate (respiration) versus meiotic differentiation (Meiosis I and II). The study (i) provides quantitative expression data for 2366 proteins and their cognate mRNAs in at least one sample, (ii) shows strongly fluctuating protein levels during growth and differentiation for 175 cases, and (iii) identifies 136 proteins absent in mitotic but present in meiotic yeast cells. We

  15. Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

    Science.gov (United States)

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

  16. Quantitative Time-Resolved Fluorescence Imaging of Androgen Receptor and Prostate-Specific Antigen in Prostate Tissue Sections.

    Science.gov (United States)

    Krzyzanowska, Agnieszka; Lippolis, Giuseppe; Helczynski, Leszek; Anand, Aseem; Peltola, Mari; Pettersson, Kim; Lilja, Hans; Bjartell, Anders

    2016-05-01

    Androgen receptor (AR) and prostate-specific antigen (PSA) are expressed in the prostate and are involved in prostate cancer (PCa). The aim of this study was to develop reliable protocols for reproducible quantification of AR and PSA in benign and malignant prostate tissue using time-resolved fluorescence (TRF) imaging techniques. AR and PSA were detected with TRF in tissue microarrays from 91 PCa patients. p63/ alpha-methylacyl-CoA racemase (AMACR) staining on consecutive sections was used to categorize tissue areas as benign or cancerous. Automated image analysis was used to quantify staining intensity. AR intensity was significantly higher in AMACR+ and lower in AMACR- cancer areas as compared with benign epithelium. The PSA intensity was significantly lower in cancer areas, particularly in AMACR- glands. The AR/PSA ratio varied significantly in the AMACR+ tumor cells as compared with benign glands. There was a trend of more rapid disease progression in patients with higher AR/PSA ratios in the AMACR- areas. This study demonstrates the feasibility of developing reproducible protocols for TRF imaging and automated image analysis to study the expression of AR and PSA in benign and malignant prostate. It also highlighted the differences in AR and PSA protein expression within AMACR- and AMACR+ cancer regions. © 2016 The Histochemical Society.

  17. Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

    Science.gov (United States)

    Gamboa, Maribet; Tsuchiya, Maria Claret; Matsumoto, Suguru; Iwata, Hisato; Watanabe, Kozo

    2017-11-01

    Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies. © 2017 Wiley Periodicals, Inc.

  18. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    Science.gov (United States)

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

  19. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    International Nuclear Information System (INIS)

    Manno, D; Filippo, E; Fiore, R; Serra, A; Urso, E; Rizzello, A; Maffia, M

    2010-01-01

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP C . In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP C at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  20. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Calmon, Marilia Freitas [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil); Sichero, Laura [Molecular Biology Laboratory, Centre for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo (Brazil); Boccardo, Enrique [Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo., São Paulo (Brazil); Villa, Luisa Lina [Department of Radiology and Oncology, Faculdade de Medicina, Universidade de São Paulo, São Paulo (Brazil); Rahal, Paula, E-mail: rahalp@yahoo.com.br [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil)

    2016-09-15

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  1. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    International Nuclear Information System (INIS)

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

    2016-01-01

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  2. Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

    Science.gov (United States)

    Wang, Dongdong; Tosevska, Anela; Heiß, Elke H; Ladurner, Angela; Mölzer, Christine; Wallner, Marlies; Bulmer, Andrew; Wagner, Karl-Heinz; Dirsch, Verena M; Atanasov, Atanas G

    2017-04-28

    Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 μmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease. © 2017 The Authors. Published on

  3. Changes in HSP gene and protein expression in natural scrapie with brain damage

    Science.gov (United States)

    2011-01-01

    Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease. PMID:21314976

  4. Changes in HSP gene and protein expression in natural scrapie with brain damage

    Directory of Open Access Journals (Sweden)

    Serrano Carmen

    2011-01-01

    Full Text Available Abstract Heat shock proteins (Hsp perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

  5. Oxidant production and SOD1 protein expression in single skeletal myofibers from Down syndrome mice

    Directory of Open Access Journals (Sweden)

    Patrick M. Cowley

    2017-10-01

    Full Text Available Down syndrome (DS is a genetic condition caused by the triplication of chromosome 21. Persons with DS exhibit pronounced muscle weakness, which also occurs in the Ts65Dn mouse model of DS. Oxidative stress is thought to be an underlying factor in the development of DS-related pathologies including muscle dysfunction. High-levels of oxidative stress have been attributed to triplication and elevated expression of superoxide dismutase 1 (SOD1; a gene located on chromosome 21. The elevated expression of SOD1 is postulated to increase production of hydrogen peroxide and cause oxidative injury and cell death. However, it is unknown whether SOD1 protein expression is associated with greater oxidant production in skeletal muscle from Ts65Dn mice. Thus, our objective was to assess levels of SOD1 expression and oxidant production in skeletal myofibers from the flexor digitorum brevis obtained from Ts65Dn and control mice. Measurements of oxidant production were obtained from myofibers loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA in the basal state and following 15 min of stimulated unloaded contraction. Ts65Dn myofibers exhibited a significant decrease in basal DCF emissions (p 0.05. Myofibers from Ts65Dn mice tended to be smaller and myonuclear domain was lower (p < 0.05. In summary, myofibers from Ts65Dn mice exhibited decreased basal DCF emissions that were coupled with elevated protein expression of SOD1. Stimulated contraction in isolated myofibers did not affect DCF emissions in either group. These findings suggest the skeletal muscle dysfunction in the adult Ts65Dn mouse is not associated with skeletal muscle oxidative stress.

  6. [HER-2 oncogene amplification assessment in invasive breast cancer by dual-color in situ hybridization (dc-CISH): a comparative study with fluorescent in situ hybridization (FISH)].

    Science.gov (United States)

    Akhdar, Abbas; Bronsard, Marc; Lemieux, Renald; Geha, Sameh

    2011-12-01

    The amplification of the gene encoding for the human epidermal growth factor receptor 2 (HER-2 oncogene), located on chromosome 17 (17q21-q22), or the overexpression of this receptor have prognostic and therapeutic implications in invasive breast cancer. An evaluation of the HER-2 status by immunohistochemistry (IHC) is performed on all invasive breast cancer cases. Fluorescent in situ hybridization (FISH) is considered as the gold standard for the detection of HER-2 gene amplification for IHC equivocal cases (score 2+). A more recent in situ hybridization technique, the dual-color chromogenic in situ hybridization (dc-CISH), has been proposed as an alternative to FISH. The aim of this study was to measure the correlation between dc-CISH and FISH for HER-2 oncogene amplification assessment in invasive breast cancer. We built four tissue micro-array (TMA) blocs with 100 breast invasive cancer cases that had been previously tested by IHC for HER-2 detection: 10 score 0 cases, 10 score 3+cases, 39 score 1+and 41 score 2+cases. Both FISH and dc-CISH techniques were applied on all TMA cases as well as on two additional slides serving as controls. Interpretation of dc-CISH was carried out by a pathologist using an optical microscope. For FISH, the interpretation was done by a professional from the medical genetics department using a fluorescent microscope linked to a computer system for image capturing and analysis. The interpretation of the HER-2/CEN-17 ratio for both tests was in accordance with the values of the updated recommendations from the Canadian National Consensus Meeting on HER-2/neu testing in breast cancer and from the ASCO/CAP. Among the 100 cases initially included in the study, eight were excluded from the analysis due to sampling or technical flaws. From the 92 remaining cases, we obtained a concordance of 97.8% (90/92 cases) between the two techniques (Kappa coefficient 0.97, 95% confidence interval). The correlation coefficient (rho) between ratios

  7. Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

    Directory of Open Access Journals (Sweden)

    Engelbrecht Christoph

    2009-12-01

    Full Text Available Abstract Background In the past decade, an enormous number of new bioprocesses have evolved in the biotechnology industry. These bioprocesses have to be developed fast and at a maximum productivity. Up to now, only few microbioreactors were developed to fulfill these demands and to facilitate sample processing. One predominant reaction platform is the shaken microtiter plate (MTP, which provides high-throughput at minimal expenses in time, money and work effort. By taking advantage of this simple and efficient microbioreactor array, a new online monitoring technique for biomass and fluorescence, called BioLector, has been recently developed. The combination of high-throughput and high information content makes the BioLector a very powerful tool in bioprocess development. Nevertheless, the scalabilty of results from the micro-scale to laboratory or even larger scales is very important for short development times. Therefore, engineering parameters regarding the reactor design and its operation conditions play an important role even on a micro-scale. In order to evaluate the scale-up from a microtiter plate scale (200 μL to a stirred tank fermenter scale (1.4 L, two standard microbial expression systems, Escherichia coli and Hansenula polymorpha, were fermented in parallel at both scales and compared with regard to the biomass and protein formation. Results Volumetric mass transfer coefficients (kLa ranging from 100 to 350 1/h were obtained in 96-well microtiter plates. Even with a suboptimal mass transfer condition in the microtiter plate compared to the stirred tank fermenter (kLa = 370-600 1/h, identical growth and protein expression kinetics were attained in bacteria and yeast fermentations. The bioprocess kinetics were evaluated by optical online measurements of biomass and protein concentrations exhibiting the same fermentation times and maximum signal deviations below 10% between the scales. In the experiments, the widely applied green

  8. Enhanced fatty acid oxidation and FATP4 protein expression after endurance exercise training in human skeletal muscle

    DEFF Research Database (Denmark)

    Jeppesen, Jacob; Jordy, Andreas B; Sjøberg, Kim A

    2012-01-01

    ; however, it is not known whether this involves up-regulation of FATP1 and FATP4 protein. Therefore, the aim of this project was to investigate FATP1 and FATP4 protein expression in the vastus lateralis muscle from healthy human individuals and to what extent FATP1 and FATP4 protein expression were......FATP1 and FATP4 appear to be important for the cellular uptake and handling of long chain fatty acids (LCFA). These findings were obtained from loss- or gain of function models. However, reports on FATP1 and FATP4 in human skeletal muscle are limited. Aerobic training enhances lipid oxidation...

  9. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

    Science.gov (United States)

    Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2013-12-01

    The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.

  10. Effective delivery of hydrophobic drugs to breast (MCF-7) and Liver (HepG2) cancer cells: A detailed investigation using Cytotoxicity assays, fluorescence imaging and flow cytometry.

    Science.gov (United States)

    Manatunga, Danushika C; de Silva, Rohini M; Nalin de Silva, K M; Neelika Malavige, Gathsaurie; Wijeratne, Dulharie T; Williams, Gareth R; Jayasinghe, Chanika D; Udagama, Preethi V

    2018-04-03

    This study aimed to develop a drug carrier system consisting of a polymer containing hydroxyapatite (HAp) shell and a magnetic core of iron oxide nanoparticles. Doxorubicin and/or curcumin were loaded into the carrier via a simple diffusion deposition approach, with encapsulation efficiencies (EE) for curcumin and doxorubicin of 93.03 ± 0.3% and 97.37 ± 0.12% respectively. The co-loading of curcumin and doxorubicin led to a total EE of 76.02 ± 0.48%. Release studies were carried out at pH 7.4 and 5.3, and revealed higher release was at pH 5.3 expressing the potential application in tumor microenvironments. Cytotoxicity assays, fluorescence imaging and flow cytometry showed the formulations could effectively inhibit the growth of MCF-7 and HEpG2 cancer cells, being more potent than the free drug molecules both in dose and time dependent manner. Additionally, hemolysis tests and cytotoxicity evaluations determined the drug-loaded carriers to be non-toxic towards non-cancerous cells. These formulations thus have great potential in the development of new cancer therapeutics. Copyright © 2018. Published by Elsevier B.V.

  11. A fluorescence-coupled assay for gamma aminobutyric acid (GABA reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    Directory of Open Access Journals (Sweden)

    Joseph E Ippolito

    Full Text Available Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA have been implicated in the pathogenesis of high grade neuroendocrine (NE neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1, was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies.

  12. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  13. BTG2 Antiproliferative Gene and Prostate Cancer

    National Research Council Canada - National Science Library

    Walden, Paul D

    2008-01-01

    .... During this study we showed that BTG2 protein expression is lost as an early event in prostate carcinogenesis and that prostate cancer cells degrade BTG2 at a greater rate than noncancerous prostate cells...

  14. C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

    Science.gov (United States)

    Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

    2012-01-01

    The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

  15. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  16. Correlation among genetic variations of c-MET in Chinese patients with non-small cell lung cancer.

    Science.gov (United States)

    Duan, Jianchun; Yang, Xiaodan; Zhao, Jun; Zhuo, Minglei; Wang, Zhijie; An, Tongtong; Bai, Hua; Wang, Jie

    2018-01-05

    The purpose of our research was to determine the correlation of amplification, protein expression and somatic mutation of c-MET in IIIb-IV stage NSCLC (Non-small cell lung cancer). We also explored correlation of c-MET variation with clinical outcome. c-MET expression was observed in 28.6% (56/196) cases, and among those 13.8% (27/196) were shown to be FISH positive. Only 2.67% patients in this study carried the c-MET mutation. Cases with c-MET FISH positive were all IHC positive ,but in IHC positive cases, only half were FISH positive. Among patients with IHC 2+ staining, 35.5% was FISH positive, while cases with IHC 3+ staining,64% was FISH positive. Both protein expression and copy number of c-MET did not significantly correlate with clinical prognosis in these patients treated with EGFR-TKIs. IHC could be used as a preliminary screening method for c-MET copy number amplification and should be confirmed by FISH only in IHC positive case which facilitate selection of ALK or MET inhibitor therapy. c-MET gene copy number, protein expression and somatic mutation for exon 14 were detected by fluorescent- In-Situ -Hybridization (FISH), Immunohistochemistry (IHC), and Denaturing-High-Performance-Liquid-Chromatography (DHPLC), respectively, in 196 NSCLC patients. The relationship between c-MET abnormalities and clinical outcome of targeted therapy was analyzed by McNemar's test.

  17. Evaluation of outer dense fiber-1 and -2 protein expression in asthenozoospermic infertile men

    Directory of Open Access Journals (Sweden)

    Silvia W. Lestari

    2015-07-01

    Full Text Available Background: Most of male infertility are caused by defect in sperm motility (asthenozoospermia. The molecular mechanism of low sperm motility in asthenozoospermic patients has not been fully understood. Sperm motility is strongly related to the axoneme structure which is composed of microtubules and supported by outer dense fiber (ODF and fibrous sheath (FS protein. The objective of this study was to characterize the ODF (ODF1 and ODF2 expression in asthenozoospermic infertile male and control normozoospermic fertile male.Methods: Asthenozoospermic samples (n=18 were collected from infertile patients at Andrology Lab, Cipto Mangunkusumo Hospital Jakarta and control were taken from normozoospermic fertile donor (n=18. After motility analyses by computer-assisted sperm analysis (CASA, semen were divided into two parts, for Western blot and for immunocytochemistry analysis. Antibody against ODF1 and ODF2 protein were used in both analyses.Results: Analysis of ODF1 protein expression showed bands with molecular weight of ~30 kDa and ODF2 ~85 kDa. The mean band intensity of ODF1 and ODF2 protein were lower in the asthenozoospermic group (AG compared to normozoospermic group (NG. Moreover, both ODF proteins were less intense and less localized in the AG than NG. Sperm motility was lower in AG, compared to control NG, i.e. average path velocity (VAP = 32.07 ± 7.03 vs 37.58 ± 8.73 µm/s, p = 0.455; straight line velocity (VSL = 24.17 ± 6.90 vs 27.61 ± 4.50 µm/s, p = 0.317 and curvilinear velocity (VCL = 45.68 ± 7.91 vs 55.55 ± 16.40 µm/s, p = 0.099.Conclusion: There is down-regulation of ODF1 and ODF2 protein expression and less-compact localization in AG sperm compared to the NG. These changes might have caused disturbances in the sperm motility as observed in this study.

  18. Calcium affecting protein expression in longan under simulated acid rain stress.

    Science.gov (United States)

    Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

    2015-08-01

    Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

  19. A novel approach to sequence validating protein expression clones with automated decision making

    Directory of Open Access Journals (Sweden)

    Mohr Stephanie E

    2007-06-01

    Full Text Available Abstract Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence

  20. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    International Nuclear Information System (INIS)

    Cavalcanti, M.B.; Fernandes, T.S.; Melo, J.A.; Neves, M.A.B.; Machado, C.G.F

    2005-01-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10 6 /mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO 2 at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results obtained

  1. Study of p53 protein expression levels from irradiated peripheral blood lymphocytes for biodosimetry

    Energy Technology Data Exchange (ETDEWEB)

    Cavalcanti, M.B.; Fernandes, T.S. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear; Amaral, A. [Universite Paris XII (UPXII) (France); Melo, J.A. [Centro de Radioterapia de Pernambuco (CERAPE), PE (Brazil); Neves, M.A.B.; Machado, C.G.F, E-mail: maribrayner@yahoo.com.br [Fundacao de Hematologia e Hemoterapia de Pernambuco, PE (Brazil)

    2005-07-01

    Biodosimetry can be defined as the investigation of radioinduced biological effects in order to correlate them with the absorbed dose. Scoring of unstable chromosomal aberrations and micronuclei, from in vitro irradiated peripheral blood lymphocytes, is commonly used for biodosimetry based on cytogenetic analysis. However, this method of analysis is time-consuming, which may represent a pitfall when fast investigation of a possible exposure to ionizing radiation (IR) is needed. The interaction of IR with the living cell can cause injuries in the DNA molecules. However, normal cells possess mechanisms of repair that are capable to correct those damages. During the repair process of the DNA various proteins are expressed. Among these proteins, p53 plays an important role. This protein is a transcription factor that helps in the maintenance of the genomic integrity. p53 protein is found into the cytoplasm in reduced concentrations and has a short average life. However, expression of p53 protein can be induced by DNA harmful radioinduced, which increases the concentration and the average life of this protein, making possible its detection. Thus, the correlation between the increasing of p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the objective of this research was to evaluate the p53 protein expression levels from lymphocytes of the human peripheral blood after in vitro irradiation. For this, samples of peripheral blood from healthy individuals were irradiated with known doses. Lymphocytes were separated on ficoll gradient by centrifugation and re-suspended at 1x 10{sub 6}/mL in RPMI medium enriched with fetal calf serum. Hence, lymphocytes were incubated in 5% CO{sub 2} at 37 deg C prior to the methodology of flow cytometry, using intranuclear antigens for the quantification of p53. In this report, the methodology performed and the results

  2. Loss of PTEN expression is associated with aggressive behavior and poor prognosis in Middle Eastern triple-negative breast cancer.

    Science.gov (United States)

    Beg, Shaham; Siraj, Abdul K; Prabhakaran, Sarita; Jehan, Zeenath; Ajarim, Dahish; Al-Dayel, Fouad; Tulbah, Asma; Al-Kuraya, Khawla S

    2015-06-01

    PTEN is a tumor suppressor that negatively regulates the PI3 K-AKT signaling pathway which is involved in the pathogenesis of many different tumor types and serves as a prognostic marker in breast cancer. However, the significance of the role of PTEN in Middle Eastern ethnic breast cancer has not been explored especially with the fact that breast cancer originating from this ethnic population tend to behave more aggressively than breast cancer in the west. In this study, we analyzed PTEN alteration in a tissue microarray format containing more than 1000 primary breast cancers with clinical follow-up data. Tissue Microarray sections were analyzed for protein expression and copy number change using immunohistochemistry and fluorescence in situ hybridization. Loss of PTEN immunostaining was observed in 77 % of the cases. PTEN loss was significantly associated with large tumor size (p = 0.0030), high grade (p = 0.0281), tumor recurrence (p = 0.0333), and triple-negative breast cancers (p = 0.0086). PTEN loss in triple-negative breast cancers was significantly associated with rapid tumor cell proliferation (p = 0.0396) and poor prognosis (p = 0.0408). PTEN deletion was found only in 60 cases (6.4 %). Loss of PTEN protein expression occurs at high frequency in Middle Eastern breast cancer. PTEN inactivation may potentially lead to an aggressive behavior of tumor cells through stimulation of tumor cell proliferation. Furthermore, PTEN signaling pathway might be used as potential therapeutic target in triple-negative breast cancers since loss of its expression is shown to be significantly associated with this aggressive subtype of breast cancer.

  3. Integrin-Targeted Hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography for Imaging Tumor Progression and Early Response in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xiaopeng Ma

    2017-01-01

    Full Text Available Integrins play an important role in tumor progression, invasion and metastasis. Therefore we aimed to evaluate a preclinical imaging approach applying ανβ3 integrin targeted hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT for monitoring tumor progression as well as early therapy response in a syngeneic murine Non-Small Cell Lung Cancer (NSCLC model. Lewis Lung Carcinomas were grown orthotopically in C57BL/6 J mice and imaged in-vivo using a ανβ3 targeted near-infrared fluorescence (NIRF probe. ανβ3-targeted FMT-XCT was able to track tumor progression. Cilengitide was able to substantially block the binding of the NIRF probe and suppress the imaging signal. Additionally mice were treated with an established chemotherapy regimen of Cisplatin and Bevacizumab or with a novel MEK inhibitor (Refametinib for 2 weeks. While μCT revealed only a moderate slowdown of tumor growth, ανβ3 dependent signal decreased significantly compared to non-treated mice already at one week post treatment. ανβ3 targeted imaging might therefore become a promising tool for assessment of early therapy response in the future.

  4. Overexpression of pucC improves the heterologous protein expression level in a Rhodobacter sphaeroides expression system.

    Science.gov (United States)

    Cheng, L; Chen, G; Ding, G; Zhao, Z; Dong, T; Hu, Z

    2015-04-27

    The Rhodobacter sphaeroides system has been used to express membrane proteins. However, its low yield has substantially limited its application. In order to promote the protein expression capability of this system, the pucC gene, which plays a crucial role in assembling the R. sphaeroides light-harvesting 2 complex (LH2), was overexpressed. To build a pucC overexpression strain, a pucC overexpression vector was constructed and transformed into R. sphaeroides CQU68. The overexpression efficiency was evaluated by quantitative real-time polymerase chain reaction. A well-used reporter β-glucuronidase (GUS) was fusion-expressed with LH2 to evaluate the heterologous protein expression level. As a result, the cell culture and protein in the pucC overexpression strain showed much higher typical spectral absorption peaks at 800 and 850 nm compared with the non-overexpression strain, suggesting a higher expression level of LH2-GUS fusion protein in the pucC overexpression strain. This result was further confirmed by Western blot, which also showed a much higher level of heterologous protein expression in the pucC overexpression strain. We further compared GUS activity in pucC overexpression and non-overexpression strains, the results of which showed that GUS activity in the pucC overexpression strain was approximately ten-fold that in the non-overexpression strain. These results demonstrate that overexpressed pucC can promote heterologous protein expression levels in R. sphaeroides.

  5. Comparison of Gene and Protein Expressions in Rats Residing in Standard Cages with Those Having Access to an Exercise Wheel

    Directory of Open Access Journals (Sweden)

    Helaine M. Alessio

    2014-01-01

    Full Text Available Lifelong physical inactivity is associated with morbidity in adulthood, possibly influenced by changes in gene and protein expressions occurring earlier in life. mRNA (Affymetrix gene array and proteomic (2D-DIGE MALDI-TOF/MS analyses were determined in cardiac tissue of young (3 months and old (16 months Sprague-Dawley rats housed with no access to physical activity (SED versus an exercise wheel (EX. Unfavorable phenotypes for body weight, dyslipidemia, and tumorogenesis appeared more often in adult SED versus EX. No differentially expressed genes (DEGs occurred between groups at 3 or 16 months. Within groups, SED and EX shared 215 age-associated DEGs. In SED, ten unique DEGs occurred with age; three had cell adhesion functions (fn1, lgals3, ncam2. In EX, five unique DEGs occurred with age; two involved hypothalamic, pituitary, and gonadal hormone axis (nrob2, xpnpep2. Protein expression involved in binding, sugar metabolic processes, and vascular regulation declined with age in SED (KNT1, ALBU, GPX1, PYGB, LDHB, G3P, PYGM, PGM1, ENOB. Protein expression increased with age in EX for ATP metabolic processes (MYH6, MYH7, ATP5J, ATPA and vascular function (KNT1, ALBU, GPX1. Differences in select gene and protein expressions within sedentary and active animals occurred with age and contributed to distinct health-related phenotypes in adulthood.

  6. Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series

    NARCIS (Netherlands)

    Roelofsen, Johan; Alvarez Llamas, Gloria; Dijkstra, Martijn; Breitling, Rainer; Havenga, Klaas; Bijzet, Johannes; Zandbergen, Wouter; de Vries, Marcel; Ploeg, Rutger J.; Vonk, Roel J.

    Analyses of intricate kinetics of the serum proteome during and after colon surgery by protein expression time series.Roelofsen H, Alvarez-Llamas G, Dijkstra M, Breitling R, Havenga K, Bijzet J, Zandbergen W, de Vries MP, Ploeg RJ, Vonk RJ. Centre for Medical Biomics, University Medical Centre

  7. An estrogen-responsive plasma protein expression signature in Atlantic cod (Gadus morhua) revealed by SELDI-TOF MS

    DEFF Research Database (Denmark)

    Nielsen, Mari Mæland; Meyer, Sonnich; Larsen, Bodil Katrine

    2011-01-01

    Compound-specific protein expression signatures( PESs) can be revealed by proteomic techniques. The SELDI-TOF MS approach is advantageous due to its simplicity and high-throughput capacity,however, there are concerns regarding the reproducibility of this method. The aim of this study was to define...

  8. Mechanistic target of rapamycin (MTOR) protein expression in the tumor and its microenvironment correlates with more aggressive pathology at cystectomy

    NARCIS (Netherlands)

    Winters, B.R. (Brian R.); Vakar-Lopez, F. (Funda); Brown, L. (Lisha); Montgomery, B. (Bruce); Seiler, R. (Roland); P.C. Black (Peter C.); J.L. Boormans (Joost); Dall′Era, M. (Marc); Davincioni, E. (Elai); Douglas, J. (James); Gibb, E.A. (Ewan A.); B.W. van Rhijn (Bas); M.S. van der Heijden (Michiel); Hsieh, A.C. (Andrew C.); Wright, J.L. (Jonathan L.); Lam, H.-M. (Hung-Ming)

    2018-01-01

    textabstractBackground: The mechanistic target of rapamycin (mTOR) has been implicated in driving tumor biology in multiple malignancies, including urothelial carcinoma (UC). We investigate how mTOR and phosphorylated mTOR (pmTOR) protein expression correlate with chemoresponsiveness in the tumor

  9. Concordant association validates MGMT methylation and protein expression as favorable prognostic factors in glioma patients on alkylating chemotherapy (Temozolomide).

    Science.gov (United States)

    Pandith, Arshad A; Qasim, Iqbal; Zahoor, Wani; Shah, Parveen; Bhat, Abdul R; Sanadhya, Dheera; Shah, Zafar A; Naikoo, Niyaz A

    2018-04-30

    O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation and its subsequent loss of protein expression has been identified to have a variable impact on clinical outcome of glioma patients indicated for chemotherapy with alkylating agents (Temozolomide). This study investigated methylation status of MGMT gene along with in situ protein expression in malignant glioma patients of different histological types to evaluate the associated clinical outcome vis-a-vis use of alkylating drugs and radiotherapy. Sixty three cases of glioma were evaluated for MGMT promoter methylation by methylation-specific PCR (MS-PCR) and protein expression by immunostaining (IHC). Methylation status of MGMT and loss of protein expression showed a very high concordant association with better survival and progression free survival (PFS) (p < 0.0001). Multivariate Cox regression analysis showed both MGMT methylation and loss of protein as significant independent prognostic factors in glioma patients with respect to lower Hazard Ratio (HR) for better OS and PFS) [p < 0.05]. Interestingly concordant MGMT methylation and lack of protein showed better response in TMZ therapy treated patient subgroups with HR of 2.02 and 0.76 (p < 0.05). We found the merits of prognostication of MGMT parameters, methylation as well as loss of its protein as predictive factors for favorable outcome in terms of better survival for TMZ therapy.

  10. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

  11. α-Lipoic acid stabilized DTX/IR780 micelles for photoacoustic/fluorescence imaging guided photothermal therapy/chemotherapy of breast cancer.

    Science.gov (United States)

    Li, WenTing; Peng, JinRong; Yang, Qian; Chen, LiJuan; Zhang, Lan; Chen, XiaoXin; Qian, ZhiYong

    2018-05-01

    Micellar nanoparticles have unique advantages as carriers for therapeutic or imaging agents, owing to their smaller size and better penetration of tumors. However, some agents, due to their physical or chemical properties, are difficult to load into micelles. IR780 is one of these agents, and is also a promising near-infrared dye for fluorescence imaging (FI)/photoacoustic imaging (PAI) and cancer photothermal therapy (PTT). Its hydrophobic and high crystallization structure results in limited bioavailability in vivo. It is difficult to load into micelles constructed from an amphiphilic block polymer with relatively low molecular weight. In this study, we use computer simulation and introduce another small biomolecule, α-lipoic acid, into the micelles constructed from a mPEG-PCL copolymer, to lower the energy of molecular interaction between MPEG-PCL and IR780, and expect to enhance the loading capacity of the micelles to IR780. The introduction of α-lipoic acid decreases the energy of molecular interaction between MEPG-PCL and IR780 from -46.18 kJ mol-1 to -196.52 kJ mol-1 and increases the loading capacity and stability of the mPEG-PCL micelles to IR780, which also maintains the loading capacity to DTX. We further construct DTX/IR780 co-loaded mPEG-PCL micelles for FI/PAI dual modal imaging guided PTT/chemotherapy of cancer. By FI and PAI evaluation in vitro and in vivo, we demonstrate that the DTX/IR780 co-loaded micelles can be used as FI and PAI probes. By further evaluating the therapeutic outcome of PTT/chemotherapy co-therapy of breast cancer, we demonstrate that the DTX/IR780 co-loaded mPEG-PCL micelles can serve as promising candidates for FI and PAI guided PTT/chemotherapy of breast cancer.

  12. The use of fed batch approaches to maximise yields in bacterial fermentation and protein expression

    International Nuclear Information System (INIS)

    McLean, A.

    2001-01-01

    A fermentation facility for the scale up of bacterial and yeast fermentations has been set up at the University of Queensland under the auspices of the ARC Special Research Centre for Functional and Applied Genomics. A major application is the production of recombinant proteins for determination of tertiary structures by X-ray crystallography or nuclear magnetic resonance. For this purpose, large amounts of protein arc needed and the yield from a single fermentation run is crucial to success within constrained laboratory budgets. To achieve maximal yields we are optimising fed batch approaches in bacterial fermentation. Fed batch offers many advantages over batch cultures. Coupled with the ability to monitor online the internal conditions of the fermentation including pH and dissolved oxygen and stirrer cascading functions it is possible to ensure that the nutritional environment of the microorganism is optimised for its growth and or for optimal protein expression. The poster will describe some of our experience in setting up fed batch fermentations and successful applications of fed batches to increasing protein yield. It will also outline services that are available to academic groups outside the University of Queensland For structure determination and functional studies, the production of radiolabelled proteins can also be an advantage. We will describe initial experiments aimed at coupling the principles of fed batch fermentation to the introduction of carbon or nitrogen isotopes into the recombinant protein

  13. Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing

    Energy Technology Data Exchange (ETDEWEB)

    Yan Chunlan; Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Li Haiyan [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Huzhou Maternity and Child Care Hospital, Huzhou, Zhejiang 313000 (China); Zhang Guanglin [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Zhejiang-California International Nanosystems Institute, Hangzhou, Zhejiang 310029 (China)

    2010-04-01

    Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage.

  14. Lipopolysaccharide regulated protein expression is only partly impaired in monocytes from patients with type I diabetes

    Directory of Open Access Journals (Sweden)

    Abke Sabine

    2006-03-01

    Full Text Available Abstract Background Monocytes play an important role in innate immunity and atherosclerosis. A disturbed secretion of cytokines in lipopolysaccharide (LPS activated monocytes from type 1 diabetes (T1D patients has been described and may contribute to the impaired inflammatory response in these individuals. In the present study the influence of LPS on five different proteins with a function in immunity and atherosclerosis was analyzed in monocytes from controls and T1D patients. Methods Monocytes were isolated from controls and T1D patients and the LPS-stimulated increase of IL-6, CXCL8, monocyte chemotactic protein 1 (CCL2, MCP-1 and superoxide dismutase (SOD 2, as well as the LPS-mediated decrease of apolipoprotein E (Apo E in primary human monocytes from controls and T1D patients was determined. Results CCL2 and IL-6 secretion in response to LPS was found significantly reduced in monocytes from T1D patients when compared to controls whereas basal CCL2 release was similar in control and T1D cells. In contrast, CXCL8 and apolipoprotein E secretion and SOD 2 expression upon LPS stimulation is similar from T1D and control monocytes. Conclusion These data indicate that LPS-mediated protein expression is only partly disturbed in monocytes from T1D patients. Reduced secretion of IL-6 and CCL2 in activated monocytes of these patients may contribute to an impaired inflammatory response and vascular disease.

  15. Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications

    Energy Technology Data Exchange (ETDEWEB)

    Manno, D; Filippo, E; Fiore, R; Serra, A [Dipartimento di Scienza dei Materiali, Universita del Salento, Lecce (Italy); Urso, E; Rizzello, A; Maffia, M [Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita del Salento, Lecce (Italy)

    2010-04-23

    Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP{sup C}. In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP{sup C} at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

  16. Machine learning in computational biology to accelerate high-throughput protein expression.

    Science.gov (United States)

    Sastry, Anand; Monk, Jonathan; Tegel, Hanna; Uhlen, Mathias; Palsson, Bernhard O; Rockberg, Johan; Brunk, Elizabeth

    2017-08-15

    The Human Protein Atlas (HPA) enables the simultaneous characterization of thousands of proteins across various tissues to pinpoint their spatial location in the human body. This has been achieved through transcriptomics and high-throughput immunohistochemistry-based approaches, where over 40 000 unique human protein fragments have been expressed in E. coli. These datasets enable quantitative tracking of entire cellular proteomes and present new avenues for understanding molecular-level properties influencing expression and solubility. Combining computational biology and machine learning identifies protein properties that hinder the HPA high-throughput antibody production pipeline. We predict protein expression and solubility with accuracies of 70% and 80%, respectively, based on a subset of key properties (aromaticity, hydropathy and isoelectric point). We guide the selection of protein fragments based on these characteristics to optimize high-throughput experimentation. We present the machine learning workflow as a series of IPython notebooks hosted on GitHub (https://github.com/SBRG/Protein_ML). The workflow can be used as a template for analysis of further expression and solubility datasets. ebrunk@ucsd.edu or johanr@biotech.kth.se. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  17. Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii.

    Science.gov (United States)

    Pasquevich, M Y; Dreon, M S; Gutierrez Rivera, J N; Vázquez Boucard, C; Heras, H

    2013-05-01

    Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Singh Upinder

    2009-02-01

    Full Text Available Abstract Background Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. Results An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Conclusion Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  19. Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.

    Science.gov (United States)

    Linford, Alicia S; Moreno, Heriberto; Good, Katelyn R; Zhang, Hanbang; Singh, Upinder; Petri, William A

    2009-02-17

    Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  20. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Fabian Machens

    2017-10-01

    Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

  1. Time-dependent changes in protein expression in rainbow trout muscle following hypoxia.

    Science.gov (United States)

    Wulff, Tune; Jokumsen, Alfred; Højrup, Peter; Jessen, Flemming

    2012-04-18

    Adaptation to hypoxia is a complex process, and individual proteins will be up- or down-regulated in order to address the main challenges at any given time. To investigate the dynamics of the adaptation, rainbow trout (Oncorhynchus mykiss) was exposed to 30% of normal oxygen tension for 1, 2, 5 and 24 h respectively, after which muscle samples were taken. The successful investigation of numerous proteins in a single study was achieved by selectively separating the sarcoplasmic proteins using 2-DE. In total 46 protein spots were identified as changing in abundance in response to hypoxia using one-way ANOVA and multivariate data analysis. Proteins of interest were subsequently identified by MS/MS following tryptic digestion. The observed regulation following hypoxia in skeletal muscle was determined to be time specific, as only a limited number of proteins were regulated in response to more than one time point. The cellular response to hypoxia included regulation of proteins involved in maintaining iron homeostasis, energy levels and muscle structure. In conclusion, this proteome-based study presents a comprehensive investigation of the expression profiles of numerous proteins at four different time points. This increases our understanding of timed changes in protein expression in rainbow trout muscle following hypoxia. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. p53 protein expression in corneal squamous cell carcinomas of dogs

    Directory of Open Access Journals (Sweden)

    Lucas Bahdour Cossi

    2015-06-01

    Full Text Available Ocular tumors play an increasing concern in veterinary ophthalmology. Corneal squamous cell carcinoma is unfrequent in dogs, and by this way it has little studies. Studies that investigated the carcinogenesis mechanisms wich could help to the development of ocular squamous cell carcinoma (SCC in dog are rare. The aim of this work was to identify by immunohistochemical techniques, the p53 protein expression in the spontaneous dog corneal SCC. For this work, were used five cases of corneal SCC and one case of actinic keratitis. The sections were obtained from paraffin-wax blocks and submitted to histopathological and immunohistochemical analysis. All the six samples showed immunolabeling to cytokeratin and p53 protein. These results support the conclusions that the immunoreactivity of p53 protein by immunohistochemistry is present in canine corneal SCC suppporting its role in carcinogenesis of this tumor, but not provides prognostic indicators in cases of SCC corneal in dog; and can be a association of exposure to solar radiation with the possible mutation of the TP53 gene.

  3. Altered hypothalamic protein expression in a rat model of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Wei-na Cong

    Full Text Available Huntington's disease (HD is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP, heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4, glycogen synthase1 (Gys1 and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1. In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.

  4. Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy.

    Science.gov (United States)

    Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

    2006-06-01

    To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

  5. Altered Osteocyte-Specific Protein Expression in Bone after Childhood Solid Organ Transplantation.

    Science.gov (United States)

    Pereira, Renata C; Valta, Helena; Tumber, Navdeep; Salusky, Isidro B; Jalanko, Hannu; Mäkitie, Outi; Wesseling Perry, Katherine

    2015-01-01

    Bone fragility is common post solid organ transplantation but little is known about bone pathology on a tissue level. Abnormal osteocytic protein expression has been linked to compromised bone health in chronic kidney disease (CKD) and immunosuppressant medications may impact osteocyte function. Transiliac bone biopsies were obtained from 22 pediatric solid organ allograft recipients (average age 15.6 years) an average of 6.3 ± 1.2 years after transplantation and from 12 pediatric pre-dialysis CKD patients (average age 13.2 years). Histomorphometry and immunohistochemistry for FGF23, DMP1, sclerostin, and osteopontin were performed on all biopsies. FGF23 and sclerostin were increased in transplant recipients relative to non-transplant CKD, regardless of the type of allograft received and despite, in the case of liver and heart recipients, a higher GFR. Bone DMP1 expression was higher in liver or heart than in kidney recipients, concomitant with higher serum phosphate values. Osteopontin expression was higher in CKD than in transplant recipients (pBone FGF23 and sclerostin correlated directly (r = 0.38, pbone FGF23 expression and osteoid thickness correlated inversely (r = - 0.46, ptransplantation is associated with increased FGF23 and sclerostin expression. The contribution of these findings to compromised bone health post transplantation warrants further evaluation.

  6. Fragile X mental retardation protein expression in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Abigail J Renoux

    2014-10-01

    Full Text Available The FMR1 protein product, FMRP, is an mRNA binding protein associated with translational inhibition of target transcripts. One FMRP target is the amyloid precursor protein (APP mRNA, and APP levels are elevated in Fmr1 KO mice. Given that elevated APP protein expression can elicit Alzheimer’s disease (AD in patients and model systems, we evaluated whether FMRP expression might be altered in Alzheimer’s autopsy brain samples and mouse models compared to controls. In a double transgenic mouse model of AD (APP/PS1, we found no difference in FMRP expression in aged AD model mice compared to littermate controls. FMRP expression was also similar in AD and control patient frontal cortex and cerebellum samples. Fragile X-associated tremor/ataxia syndrome (FXTAS is an age related neurodegenerative disorder caused by expanded CGG repeats in the 5’UTR of the FMR1 gene. Patients experience cognitive impairment and dementia in addition to motor symptoms. In parallel studies, we measured FMRP expression in cortex and cerebellum from three FXTAS patients and found reduced expression compared to both controls and Alzheimer’s patient brains, consistent with animal models. We also find increased APP levels in cerebellar, but not cortical, samples of FXTAS patients compared to controls. Taken together, these data suggest that a decrease in FMRP expression is unlikely to be a primary contributor to Alzheimer’s disease pathogenesis.

  7. 17β-estradiol-induced ACSL4 protein expression promotes an invasive phenotype in estrogen receptor positive mammary carcinoma cells.

    Science.gov (United States)

    Belkaid, Anissa; Ouellette, Rodney J; Surette, Marc E

    2017-04-01

    Long chain acyl-CoA synthase-4 (ACSL4) expression has been associated with an aggressive phenotype in breast carcinoma cells, whereas its role in ERα-positive breast cancer has not been studied. ACSL4 prefers 20-carbon polyunsaturated fatty acid (PUFA) substrates, and along with other ACSLs has been associated with cellular uptake of exogenous fatty acids. 17β-estradiol induces proliferation and invasive capacities in ERα+ve breast carcinoma that is associated with modifications of cellular lipid metabolism. In this study, treatment of steroid-starved ERα-positive MCF-7 and T47D mammary carcinoma cells with 17β-estradiol resulted in increased cellular uptake of the PUFA arachidonic acid (AA) and eicosapentaenoic acid (EPA), important building blocks for cellular membranes, and increased ACSL4 protein levels. There was no change in the expression of the ACSL1, ACSL3 and ACSL6 protein isotypes. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life compared to untreated cells. ERα silencing reversed the impact of 17β-estradiol on ACSL4 protein levels and half-life. Silencing of ACSL4 eliminated the 17β-estradiol-induced increase in AA and EPA uptake, as well as the 17β-estradiol-induced cell migration, proliferation and invasion capacities. ASCL4 silencing also prevented the 17β-estradiol induced increases in p-Akt and p-GSK3β, and decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. Taken together, these results demonstrate that ACSL4 is a target of 17β-estradiol-stimulated ERα and is required for the cellular uptake of exogenous PUFA and the manifestation of a more malignant phenotype in ERα+ve breast carcinoma cells. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Alteration of protein expression pattern of vascular endothelial growth factor (VEGF) from soluble to cell-associated isoform during tumourigenesis

    International Nuclear Information System (INIS)

    Cressey, Ratchada; Wattananupong, Onusa; Lertprasertsuke, Nirush; Vinitketkumnuen, Usanee

    2005-01-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells, and its expression has been correlated with increased tumour angiogenesis. Although numerous publications dealing with the measurement of circulating VEGF for diagnostic and therapeutic monitoring have been published, the relationship between the production of tissue VEGF and its concentration in blood is still unclear. The aims of this study were to determine: 1) The expression pattern of VEGF isoforms at the protein level in colorectal and lung adenocarcinoma in comparison to the pattern in corresponding adjacent normal tissues 2) The relationship between the expression pattern of VEGF and total level of circulating VEGF in the blood to clarify whether the results of measuring circulating VEGF can be used to predict VEGF expression in tumour tissues. Ninety-four tissue samples were obtained from patients, 76 colorectal tumour tissues and 18 lung tumour tissues. VEGF protein expression pattern and total circulating VEGF were examined using western blot and capture ELISA, respectively. Three major protein bands were predominately detected in tumour samples with an apparent molecular mass under reducing conditions of 18, 23 and 26 kDa. The 18 kDa VEGF protein was expressed equally in both normal and colorectal tumour tissues and predominately expressed in normal tissues of lung, whereas the 23 and 26 kDa protein was only detected at higher levels in tumour tissues. The 18, 23 and 26 kDa proteins are believed to represent the VEGF 121 , the VEGF 165 and the VEGF 189 , respectively. There was a significant correlation of the expression of VEGF 165 with a smaller tumour size maximum diameter <5 cm (p < 0.05), and there was a significant correlation of VEGF 189 with advanced clinical stage of colorectal tumours. The measurement of total circulating VEGF in serum revealed that cancer patients significantly (p < 0.001) possessed a higher level of circulating VEGF (1081 ± 652 pg/ml in

  9. Combination of Bcl-2 and MYC protein expression improves high-risk stratification in diffuse large B-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Wang J

    2015-09-01

    Full Text Available Jing Wang,* Min Zhou,* Jing-Yan Xu,* Bing Chen, Jian OuyangDepartment of Hematology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, People’s Republic of China*These authors contributed equally to this work and should be considered as cofirst authorsPurpose: To evaluate whether the addition of two biological markers (MYC and BCL-2 protein overexpression improves the stratification of high-risk patients with diffuse large B-cell lymphoma (DLBCL.Method: Seven risk factors were identified at diagnosis, and a maximum of 7 points were assigned to each patient. The patients were classified according to four risk groups: low (0–1, low-intermediate (2–3, high-intermediate (4, and high (5–7. Only high-risk patients with DLBCL were included in this analysis. We retrospectively examined 20 cases from 2008 to 2013 at the Nanjing Drum Tower Hospital.Results: The median expression of MYC protein was 60%, and 17 of 20 (65% evaluable cases overexpressed MYC. The median expression of BCL-2 protein was also 60%. Eighteen of 20 (90% evaluable cases showed BCL-2 overexpression. Additionally, 12 out of 20 cases (60% demonstrated coexpression of MYC and BCL-2 proteins. The percentages of overall survival and progression-free survival at the median follow-up time (36 months were 33.3%±16.1% and 16.9%±13.5%, respectively. By comparison, nine, four, and 20 patients were classified as high risk based on the International Prognostic Index (IPI, National Comprehensive Cancer Network(NCCN-IPI, and revised IPI criteria, respectively. According to the IPI and NCCN-IPI stratification, the risk groups demonstrated closely overlapping survival curves. In addition, four out of 20 cases were identified as low-intermediate risk according to the NCCN-IPI criteria.Conclusion: The addition of MYC and BCL-2 protein expression to the IPI could identify a subset of DLBCL patients with high-risk clinicopathological characteristics and

  10. Role of Light and Dark Cycle and Different Temperatures in the Regulation of Growth and Protein Expression in Oscillatoria agardhii Strain

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    Gajendra Kumar Dautania

    2014-12-01

    Full Text Available The cyanobacterium Oscillatoria agardhii was isolated from the fresh water Mawatha lake, Jaipur and was grown in Zarrouk's medium at 25 ± 2°C, illuminated with white fluorescent light at the intensity of 2 500 lux with 12:12 h light and dark photoperiod. The effects of photoperiod and temperature on the growth and protein expression by Oscillatoria agardhii were studied under different controlled culture conditions (ALR, ALC, CLR, CLC, and NDL, measuring optical density, cell count and dry weight. Protein content was measured quantitatively by Bradford assay and qualitatively by SDS-PAGE. The densitometric analysis was also carried out for the measurement of the expression level of different proteins/peptides under different culture conditions. Maximum growth and protein content were observed in ALR condition while minimum was in the CLC. Alternate light and dark periods proved efficient as contrasting banding patterns were observed with many new unique polypeptides such as 32, 36.3, 47.9, 60.8, and 67.0 kDa, whereas, expressions of three polypeptides of 57.2, 110.1, and 117.3 kDa were inhibited in constant light cultures.

  11. An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

    Science.gov (United States)

    Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

    2013-04-15

    NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

    Directory of Open Access Journals (Sweden)

    Lobstein Julie

    2012-05-01

    Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

  13. Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region

    DEFF Research Database (Denmark)

    Goltermann, Lise; Borch Jensen, Martin; Bentin, Thomas

    2011-01-01

    intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding...

  14. Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Kiyose, Shinichiro; Igarashi, Hisaki; Nagura, Kiyoko; Kamo, Takaharu; Kawane, Kazunori; Mori, Hiroki; Ozawa, Takachika; Maeda, Matsuyoshi; Konno, Keisuke; Hoshino, Hideaki; Konno, Hiroyuki; Ogura, Hiroyuki; Shinmura, Kazuya; Hattori, Naohiko; Sugimura, Haruhiko

    2012-11-01

    The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

  15. ICG-fluorescence imaging for detection of peritoneal metastases and residual tumoral scars in locally advanced ovarian cancer: A pilot study.

    Science.gov (United States)

    Veys, Isabelle; Pop, Florin-Catalin; Vankerckhove, Sophie; Barbieux, Romain; Chintinne, Marie; Moreau, Michel; Nogaret, Jean-Marie; Larsimont, Denis; Donckier, Vincent; Bourgeois, Pierre; Liberale, Gabriel

    2018-02-01

    No intraoperative imaging techniques exist for detecting tumor nodules or tumor scar tissues in patients treated with upfront or interval cytoreductive surgery (CS) after neoadjuvant chemotherapy (NAC). The aims of this study were to evaluate the role of indocyanine green (ICG) fluorescence imaging (FI) for the detection of peritoneal metastases (PM) and evaluate whether it can be used to detect remnant tumor cells in scar tissue. Patients with PM from ovarian cancer admitted for CS were included. ICG, at 0.25 mg per kg of patient weight, was injected intraoperatively after explorative laparotomy before CS. A total of 108 peritoneal lesions, including 25 scars, were imaged in 20 patients. Seventy-three were malignant (67.6%) and 35 benign (32.4%). The mean Tumor to Background Ratio (ex vivo) was 1.8 (SD 1.3) in malig