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ABSTRACT: In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.
Journal of Biomedical Optics 07/2012; 17(7):077006. · 3.16 Impact Factor
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Chemistry 06/2011; 17(24):6620-4. · 5.93 Impact Factor
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ABSTRACT: Epithelial cell lines were established from the transition and peripheral zones of human prostate by transduction with cdk4 and hTERT. The properties of these lines were investigated using immunocytochemical markers, ability to generate anchorage-independent colonies and by spectral karyotyping (SKY). Cells were exposed to fractionated doses of gamma irradiation to investigate their ability to transform. Cell lines were established from the transition and peripheral zones of human prostate. The expression of CD133, CK5, CK14, CK18, p16, PSCA, p63 and c-myc varied between the lines from the two regions. The line derived from the peripheral zone exhibited properties of a tumour line. A similar pattern was observed in two separate transductions. It was thus unlikely to be an in vitro transformation event, which is very rarely observed with human cells in vitro, and thus more likely to be derived from the immortalisation of a quiescent tumour clone. Fractionated irradiation of the transition zone cell line resulted in forming of transformed colonies. The transformed and tumour line had marked chromosomal rearrangements as demonstrated by SKY analysis. Cell lines have been derived from different zones of human prostate for studies on radiation carcinogenesis. The unirradiated cell line derived from the peripheral zone exhibited chromosomal rearrangements similar to those observed in prostate carcinoma. The cell line derived from the transitional zone exhibited a near diploid karyotype and could be transformed following exposure to fractionated doses of gamma irradiation.
Oncology Reports 01/2011; 25(1):121-8. · 1.84 Impact Factor
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ABSTRACT: Raman spectroscopy permits probing of the molecular and chemical properties of the analyzed sample. However, its applicability has been seriously limited to specific applications by the presence of a strong fluorescence background. In our recent paper [Anal. Chem. 82, 738 (2010)], we reported a new modulation method for separating Raman scattering from fluorescence. By continuously changing the excitation wavelength, we demonstrated that it is possible to continuously shift the Raman peaks while the fluorescence background remains essentially constant. In this way, our method allows separation of the modulated Raman peaks from the static fluorescence background with important advantages when compared to previous work using only two [Appl. Spectrosc. 46, 707 (1992)] or a few shifted excitation wavelengths [Opt. Express 16, 10975 (2008)]. The purpose of the present work is to demonstrate a significant improvement of the efficacy of the modulated method by using different processing algorithms. The merits of each algorithm (Standard Deviation analysis, Fourier Filtering, Least-Squares fitting and Principal Component Analysis) are discussed and the dependence of the modulated Raman signal on several parameters, such as the amplitude and the modulation rate of the Raman excitation wavelength, is analyzed. The results of both simulation and experimental data demonstrate that Principal Component Analysis is the best processing algorithm. It improves the signal-to-noise ratio in the treated Raman spectra, reducing required acquisition times. Additionally, this approach does not require any synchronization procedure, reduces user intervention and renders it suitable for real-time applications.
Optics Express 05/2010; 18(11):11382-95. · 3.59 Impact Factor
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ABSTRACT: The natural tetrapeptide acetyl-ser-asp-lys-pro (AcSDKP) is formed in vivo by enzymatic cleavage of the N terminus of thymosin beta4 by prolyl oligopeptidase (POP). Recently, AcSDKP was shown to promote angiogenesis. Because of the critical role of neovascularization in cancer development, the levels of AcSDKP and POP activity in a number of different malignant tissues were investigated. Our studies revealed that AcSDKP levels were markedly elevated in neoplastic diseases including hematologic malignancies and solid neoplasms. Consistent with this finding, the enhanced activity of POP was also detected in all analyzed specimens of cancer tissues. Both these novel findings are in concert with the previously reported overexpression of thymosin beta4 in a large variety of malignant tumors and with its potential role in cancerogenesis. The physiological relevance of these findings awaits further studies; however, our first results strongly suggest a key role for AcSDKP in the pathogenesis of cancer.
Annals of the New York Academy of Sciences 04/2010; 1194:53-9. · 3.15 Impact Factor
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ABSTRACT: Label-free chemical characterization of single cells is an important aim for biomedical research. Standard Raman spectroscopy provides intrinsic biochemical markers for noninvasive analysis of biological samples but is often hindered by the presence of fluorescence background. In this paper, we present an innovative modulated Raman spectroscopy technique to filter out the Raman spectra from the fluorescence background. The method is based on the principle that the fluorescence background does not change whereas the Raman scattering is shifted by the periodical modulation of the laser wavelength. Exploiting this physical property and importantly the multichannel lock-in detection of the Raman signal, the modulation technique fulfills the requirements of an effective fluorescence subtraction method. Indeed, once the synchronization and calibration procedure is performed, minimal user intervention is required, making the method online and less time-consuming than the other fluorescent suppression methods. We analyze the modulated Raman signal and shifted excitation Raman difference spectroscopy (SERDS) signal of 2 mum-sized polystyrene beads suspended in a solution of fluorescent dye as a function of modulation rate. We show that the signal-to-noise ratio of the modulated Raman spectra at the highest modulation rate is 3 times higher than the SERDS one. To finally evaluate the real benefits of the modulated Raman spectroscopy, we apply our technique to Chinese hamster ovary cells (CHO). Specifically, by analyzing separate spectra from the membrane, cytoplasm, and nucleus of CHO cells, we demonstrate the ability of this method to obtain localized sensitive chemical information from cells, away from the interfering fluorescence background. In particular, statistical analysis of the Raman data and classification using PCA (principal component analysis) indicate that our method allows us to distinguish between different cell locations with higher sensitivity and specificity, avoiding potential misinterpretation of the data obtained using standard background procedures.
Analytical Chemistry 12/2009; 82(2):738-45. · 5.86 Impact Factor
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Kristian Unger,
Johannes Wienberg, Andrew Riches,
Ludwig Hieber,
Axel Walch,
Andreas Brown,
Patricia C M O'Brien,
Cäcilia Briscoe,
Lindsey Gray,
Elke Rodriguez,
Gerhard Jackl,
Jeroen Knijnenburg,
Giovanni Tallini,
Malcolm Ferguson-Smith,
Horst Zitzelsberger
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ABSTRACT: Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer, we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1 Mb bacterial artificial chromosome (BAC) arrays. The breakpoints were further narrowed down by fluorescence in situ hybridisation (FISH) with clones from the 32 k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only, it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers.
Endocrine Related Cancer 10/2009; 17(1):87-98. · 4.36 Impact Factor
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ABSTRACT: The cytotoxic effects of strawberry polyphenols were investigated on normal cells and tumour cells derived from the same patient. A human prostate epithelial cell line (P21) and two tumour cell lines (P21 tumour cell line 1 and 2) derived from the same patient, and a normal human breast epithelial cell line (B42) and a tumour line derived from it (B42 clone 16) were used. A polyphenol-rich extract derived from strawberry or anthocyanin or tannin-rich sub-fractions were applied to the cell lines in doses varying from 50 to 1.5 microg/ml. The strawberry extract was cytotoxic with doses of approximately 5 microg/ml causing a 50% reduction in cell survival in both the normal and the tumour lines. The extracts were also cytotoxic to peripheral blood human lymphocytes stimulated with phytohaemagglutinin but higher levels (>20 microg/ml for 50% reduction in cell survival) were required. After fractionation of the strawberry sample, the cytotoxicity was retained in the tannin-rich fraction and this fraction was considerably more toxic to all cells (normal or tumour cell lines or lymphocytes) than the anthocyanin-rich fraction. Established prostate (LNCaP and PC-3) and breast (MCF-7) tumour cell lines were more resistant to the strawberry extract with concentrations of 50 microg/ml required for 50% reduction in cell survival, which is similar to levels in previous studies on the antiproliferative effects of berry extracts. Although these concentrations are much greater than possible physiological levels, they are comparable to those reported in other studies. From these findings, we conclude that there is little evidence to assume that polyphenols from strawberry have a differential cytotoxic effect on tumour cells relative to comparable normal cells from the same tissue derived from the same patient.
International Journal of Oncology 03/2009; 34(3):777-86. · 2.40 Impact Factor
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ABSTRACT: Evidence from clinical and laboratory studies suggests that angiogenesis is important in the progression of solid tumours and hematologic malignancies. We have shown that the naturally occurring tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent angiogenic factor normally present at nanomolar concentrations in the blood. A murine leukemia model was used to assess whether there was a correlation between levels of endogenous AcSDKP and the development of disease. Levels of AcSDKP in the plasma and bone marrow (BM) cells from mice bearing an acute myeloid leukemia (AML) were five- to ten-fold greater than those in non-leukemic mice. Furthermore, a strong correlation between the concentration of endogenous AcSDKP and the progression of AML was demonstrated. These results are consistent with the marked increase in BM vascularity observed in leukemic mice. The physiologic relevance of these findings awaits further studies and the contribution of AcSDKP to the pathogenesis of leukemia is under investigation.
Leukemia and Lymphoma 10/2006; 47(9):1915-20. · 2.58 Impact Factor
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ABSTRACT: Neoplastic transformation induced by ionizing radiation was studied using a human retinal pigment epithelial cell line immortalized by telomerase. Radiation-transformed cell clones were tumorigenic in athymic mice and were analyzed by G-banding and comparative genomic hybridization (CGH). Radiation-transformed cloned cell lines and cell lines derived from tumors produced in athymic nude mice following transplantation exhibited a recurrent karyotype:45,XX,der(10),-13. CGH showed an amplification of 10p11.2 and a deletion of the remaining 10p. Positional cloning of the amplified region by FISH analysis and subsequent sequence analysis of BAC clones showing amplified FISH signals identified the candidate gene PARD3. This gene also was found to be transcriptionally expressed at an increased level. The findings indicate that PARD3 may play an important role in radiation-induced carcinogenesis of RPE cells. This is the first evidence for PARD3 amplification in human cancer cells.
Genes Chromosomes and Cancer 06/2004; 40(1):55-9. · 3.31 Impact Factor
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