Dielectrophoresis-Based SERS Sensors for the Detection of Cancer Cells in Microfluidic Chips.

The detection of freely circulating cancer cells (CTCs) is one of the greatest challenges of modern medical diagnostics. For several years, there has been increased attention on the use of surface-enhanced Raman spectroscopy (SERS) for the detection of CTCs. SERS is a non-destructive, accurate and precise technique, and the use of special SERS platforms even enables the amplification of weak signals from biological objects. In the current study, we demonstrate the unique arrangement of the SERS technique combined with the deposition of CTCs cells on the surface of the SERS platform via a dielectrophoretic effect. The appropriate frequencies of an alternating electric field and a selected shape of the electric field can result in the efficient deposition of CTCs on the SERS platform. The geometry of the microfluidic chip, the type of the cancer cells and the positive dielectrophoretic phenomenon resulted in the trapping of CTCs on the surface of the SERS platform. We presented results for two type of breast cancer cells, MCF-7 and MDA-MB-231, deposited from the 0.1 PBS solution. The limit of detection (LOD) is 20 cells/mL, which reflects the clinical potential and usefulness of the developed approach. We also provide a proof-of-concept for these CTCs deposited on the SERS platform from blood plasma.

Effect of Varying Expression of EpCAM on the Efficiency of CTCs Detection by SERS-Based Immunomagnetic Optofluidic Device.

The circulating tumor cells (CTCs) isolation and characterization has a great potential for non-invasive biopsy. In the present research, the surface-enhanced Raman spectroscopy (SERS)-based assay utilizing magnetic nanoparticles and solid SERS-active support integrated in the external field assisted microfluidic device was designed for efficient isolation of CTCs from blood samples. Magnetic nanospheres (Fe2O3) were coated with SERS-active metal and then modified with p-mercaptobenzoic acid (p-MBA) which works simultaneously as a Raman reporter and linker to an antiepithelial-cell-adhesion-molecule (anti-EpCAM) antibodies. The newly developed laser-induced SERS-active silicon substrate with a very strong enhancement factor (up to 108) and high stability and reproducibility provide the additional extra-enhancement in the sandwich plasmonic configuration of immune assay which finally leads to increase the efficiency of detection. The sensitive immune recognition of cancer cells is assisted by the introducing of the controllable external magnetic field into the microfluidic chip. Moreover, the integration of the SERS-active platform and p-MBA-labeled immuno-Ag@Fe2O3 nanostructures with microfluidic device offers less sample and analytes demand, precise operation, increase reproducibly of spectral responses, and enables miniaturization and portability of the presented approach. In this work, we have also investigated the effect of varying expression of the EpCAM established by the Western Blot method supported by immunochemistry on the efficiency of CTCs' detection with the developed SERS method. We used four target cancer cell lines with relatively high (human metastatic prostate adenocarcinoma cells (LNCaP)), medium (human metastatic prostate adenocarcinoma cells (LNCaP)), weak (human metastatic prostate adenocarcinoma cells (LNCaP)), and no EpCAM expressions (cervical cancer cells (HeLa)) to estimate the limits of detection based on constructed calibration curves. Finally, blood samples from lung cancer patients were used to validate the efficiency of the developed method in clinical trials.

Detection of Circulating Tumor Cells Using Membrane-Based SERS Platform: A New Diagnostic Approach for 'Liquid Biopsy'.

The detection and monitoring of circulating tumor cells (CTCs) in blood is an important strategy for early cancer evidence, analysis, monitoring of therapeutic response, and optimization of cancer therapy treatments. In this work, tailor-made membranes (MBSP) for surface-enhanced Raman spectroscopy (SERS)-based analysis, which permitted the separation and enrichment of CTCs from blood samples, were developed. A thin layer of SERS-active metals deposited on polymer mat enhanced the Raman signals of CTCs and provided further insight into CTCs molecular and biochemical composition. The SERS spectra of all studied cells-prostate cancer (PC3), cervical carcinoma (HeLa), and leucocytes as an example of healthy (normal) cell-revealed significant differences in both the band positions and/or their relative intensities. The multivariate statistical technique based on principal component analysis (PCA) was applied to identify the most significant differences (marker bands) in SERS data among the analyzed cells and to perform quantitative analysis of SERS data. Based on a developed PCA algorithm, the studied cell types were classified with an accuracy of 95% in 2D PCA to 98% in 3D PCA. These results clearly indicate the diagnostic efficiency for the discrimination between cancer and normal cells. In our approach, we exploited the one-step technology that exceeds most of the multi-stage CTCs analysis methods used and enables simultaneous filtration, enrichment, and identification of the tumor cells from blood specimens.

Dark-chilling and subsequent photo-activation modulate expression and induce reversible association of chloroplast lipoxygenase with thylakoid membrane in runner bean (Phaseolus coccineus L.).

Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids. This reaction is the first step in biosynthesis of oxylipins, which play important and diverse roles in stress response. In this study, we identified four LOX genes (PcLOXA, B, C, D) in chilling-sensitive runner bean (Phaseolus coccineus L.) plant and analyzed their expression patterns during long term dark-chilling (4 °C) stress and during day/night (21ºC/4 °C) temperature fluctuations. Three of the four identified LOX genes, namely PcLOXA, PcLOXB and PcLOXD, were induced by wounding stress, while only the PcLOXA was induced by dark-chilling of both detached (wounded) leaves and whole plants. We identified PcLOXA as a chloroplast-targeted LOX protein and investigated its expression during chilling stress in terms of abundance, localization inside chloroplasts and interactions with the thylakoid membranes. The analysis by immunogold electron microscopy has shown that more than 60% of detectable PcLOXA protein was associated with thylakoids, and dark-chilling of leaves resulted in increased amounts of this protein detected within grana margins of thylakoids. This effect was reversible under subsequent photo-activation of chilled leaves. PcLOXA binding to thylakoids is not mediated by the posttranslational modification but rather is based on direct interactions of the protein with membrane lipids; the binding strength increases under dark-chilling conditions.

Molecular foundations of chilling-tolerance of modern maize.

BACKGROUND: Recent progress in selective breeding of maize (Zea mays L.) towards adaptation to temperate climate has allowed the production of inbred lines withstanding cold springs with temperatures below 8 °C or even close to 0 °C, indicating that despite its tropical origins maize is not inherently cold-sensitive. RESULTS: Here we studied the acclimatory response of three maize inbred lines of contrasting cold-sensitivity selected basing on multi-year routine field data. The field observations were confirmed in the growth chamber. Under controlled conditions the damage to the photosynthetic apparatus due to severe cold treatment was the least in the cold-tolerant line provided that it had been subjected to prior moderate chilling, i.e., acclimation. The cold-sensitive lines performed equally poorly with or without acclimation. To uncover the molecular basis of the attained cold-acclimatability we performed comparative transcriptome profiling of the response of the lines to the cold during acclimation phase by means of microarrays with a statistical and bioinformatic data analysis. CONCLUSIONS: The analyses indicated three mechanisms likely responsible for the cold-tolerance: acclimation-dependent modification of the photosynthetic apparatus, cell wall properties, and developmental processes. Those conclusions supported the observed acclimation of photosynthesis to severe cold at moderate chilling and were further confirmed by experimentally showing specific modification of cell wall properties and repression of selected miRNA species, general regulators of development, in the cold-tolerant line subjected to cold stress.

Synergy of BID with doxorubicin in the killing of cancer cells.

Overexpression of the BH3-interacting domain death agonist (BID) protein sensitizes certain cancer cell lines to apoptosis induced by anticancer agents, particularly by those acting through death receptors (e.g. TRAIL). Previously, we showed that recombinant BID fused with TAT cell penetrating peptide (TAT-BID) allowed for controlled delivery of BID to different cancer cell lines and moderately sensitized some of them to TRAIL or slightly to camptothecin. In the present study, we showed that TAT-BID delivered to HeLa cells strongly sensitized them to doxorubicin, as identified by cell viability and apoptosis assays. Another cell line sensitized to doxorubicin was PC3, whereas A549 and LNCaP cells were sensitized moderately or not at all, respectively. Sensitization was more pronounced at 1 µM doxorubicin administered for 48 h than for lower doses and shorter treatments. TAT-BID and doxorubicin may thus be considered as a potential therapeutic combination for cervical carcinoma and advanced prostate cancer treatment.

Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis.

BACKGROUND: Low cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study. METHODS: Uptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay. RESULTS: TAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID. CONCLUSIONS: TAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.

Genome-wide transcriptomic analysis of response to low temperature reveals candidate genes determining divergent cold-sensitivity of maize inbred lines.

Maize, despite being thermophyllic due to its tropical origin, demonstrates high intraspecific diversity in cold-tolerance. To search for molecular mechanisms of this diversity, transcriptomic response to cold was studied in two inbred lines of contrasting cold-tolerance. Microarray analysis was followed by extensive statistical elaboration of data, literature data mining, and gene ontology-based classification. The lines used had been bred earlier specifically for determination of QTLs for cold-performance of photosynthesis. This allowed direct comparison of present transcriptomic data with the earlier QTL mapping results. Cold-treated (14 h at 8/6 °C) maize seedlings of cold-tolerant ETH-DH7 and cold-sensitive ETH-DL3 lines at V3 stage showed strong, consistent response of the third leaf transcriptome: several thousand probes showed similar, statistically significant change in both lines, while only tens responded differently in the two lines. The most striking difference between the responses of the two lines to cold was the induction of expression of ca. twenty genes encoding membrane/cell wall proteins exclusively in the cold-tolerant ETH-DH7 line. The common response comprised mainly repression of numerous genes related to photosynthesis and induction of genes related to basic biological activity: transcription, regulation of gene expression, protein phosphorylation, cell wall organization. Among the genes showing differential response, several were close to the QTL regions identified in earlier studies with the same inbred lines and associated with biometrical, physiological or biochemical parameters. These transcripts, including two apparently non-protein-coding ones, are particularly attractive candidates for future studies on mechanisms determining divergent cold-tolerance of inbred maize lines.

Time schedule-dependent effect of the CK2 inhibitor TBB on PC-3 human prostate cancer cell viability.

Inhibitors of CK2 kinase inhibit cell proliferation and induce apoptosis in numerous cancer cell lines. Due to these properties, they are considered potentially useful in anticancer therapy. In this study, we show that the exact effect of the specific CK2 inhibitor TBB on PC-3 human prostate cancer cell viability depends on the time schedule of administration: it was not observed when the treatment was directly followed by the viability assay but it appeared when the treatment and the assay were separated by a 24-h incubation without the inhibitor. Such a pattern was maintained when the TBB treatment was combined with either camptothecin or TRAIL. The time schedule-dependence of cell viability was not reflected by a similar dependence of induction of apoptosis. Despite this, the schedule in which a treatment with the CK2 inhibitor precedes that with an anticancer drug seems to be a good choice for a potential therapy against androgen-refractory prostate cancer.

Rhythmic diel pattern of gene expression in juvenile maize leaf.

BACKGROUND: Numerous biochemical and physiological parameters of living organisms follow a circadian rhythm. Although such rhythmic behavior is particularly pronounced in plants, which are strictly dependent on the daily photoperiod, data on the molecular aspects of the diurnal cycle in plants is scarce and mostly concerns the model species Arabidopsis thaliana. Here we studied the leaf transcriptome in seedlings of maize, an important C4 crop only distantly related to A. thaliana, throughout a cycle of 10 h darkness and 14 h light to look for rhythmic patterns of gene expression. RESULTS: Using DNA microarrays comprising ca. 43,000 maize-specific probes we found that ca. 12% of all genes showed clear-cut diel rhythms of expression. Cluster analysis identified 35 groups containing from four to ca. 1,000 genes, each comprising genes of similar expression patterns. Perhaps unexpectedly, the most pronounced and most common (concerning the highest number of genes) expression maxima were observed towards and during the dark phase. Using Gene Ontology classification several meaningful functional associations were found among genes showing similar diel expression patterns, including massive induction of expression of genes related to gene expression, translation, protein modification and folding at dusk and night. Additionally, we found a clear-cut tendency among genes belonging to individual clusters to share defined transcription factor-binding sequences. CONCLUSIONS: Co-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ between taxa.

Yeast transcription factor Oaf1 forms homodimer and induces some oleate-responsive genes in absence of Pip2.

Genes encoding peroxisomal proteins in the yeast Saccharomyces cereviasiae are induced in the presence of oleate in growth medium. This induction is known to be mediated by the binding of a heterodimer of transcription factors Oaf1 and Pip2 to an upstream activating sequence called ORE (oleate response element). By analyzing expression of nine ORE-containing genes we show that the presence of an ORE sequence is not sufficient to confer oleate inducibility, as three such genes were in fact expressed constitutively. Moreover, some of the oleate-inducible genes undergo activation even in the absence of Pip2. Using coimmunoprecipitation we show that, when Pip2 is missing, Oaf1 may form homodimers which apparently substitute for the Oaf1-Pip2 heterodimer.

Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae.

Murine DNA methyltransferases Dnmt1 and Dnmt3a were expressed in the yeast Saccharomyces cerevisiae. Adjustment to yeast preferences of the nucleotide sequences upstream and downstream of the translation initiation sites of both cDNAs was needed to obtain significant levels of the methyltransferases. Both proteins were correctly localized to the nucleus and their presence had no measurable influence on the functioning of yeast cells. Both Dnmt1 and Dnmt3a expressed in yeast cells were enzymatically active in vitro, and in vivo in the genomic DNA of the transgenic S. cerevisiae ca. 0.06% and 0.4%, respectively, of cytosines became methylated. This level of DNA methylation is about 100- to 10-fold less than that observed in mammalian cells. The constructed system may be used to investigate the in vivo specificity of individual mammalian DNA methyltransferases and to search for additional factors needed to allow more efficient in vivo methylation of chromatin-contained DNA and to study their mechanism of action.

Genomic structure of two ras family genes in the slime mold Physarum polycephalum.

Genomic structure of two Physarum polycephalum ras family genes, Ppras2 and Pprap1, has been determined, including the upstream region of the latter. The genes are interrupted by three and four introns, respectively. The first intron of Ppras2 has the same location within the coding sequence as the first intron in another ras homolog from this organism, Ppras1 [Trzcinska-Danielewicz, J., Kozlowski, P., and Toczko, K. (1996). "Cloning and genomic sequence of the Physarum polycephalum Ppras1 gene, a homologue of the ras protooncogene", Gene 169, pp. 143-144]. All introns, ranging from 53 to ca. 460 base pairs, have the canonical 5' and 3' ends, are greatly enriched in pyrimidines in the coding strand and have frequent pyrimidines-only tracts. These latter features seem to be responsible for the difficulties in cloning and sequencing of parts of these genes. Short sequences shared with P. polycephalum transposon-like repeats are common in the introns, indicating a possible role of transposition in intron evolution. In all three ras family genes phase zero introns are located mostly between sequences coding for regular protein secondary structure elements.