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Maize is a subtropical, cold-sensitive species. However, some varieties of this species have the potential to withstand long-term low temperatures, even at the seedling stage. The molecular basis of this phenomenon has not been determined. In a chromatin-level study, we compared the cold-stress reaction of seedlings of two maize inbred lines showing contrasting levels of cold sensitivity. The cold-tolerant line was selected based on field data and previous physiological and transcriptomic level studies. The first condition of gene expression—chromatin accessibility—was assessed by formaldehyde-aided isolation of regulatory elements method and DNA sequencing. Potentially expressed genes and cis-regulatory sequences open for interaction with transcription factors have been defined. The results of this study suggest that during cold stress, the tolerant maize line shifted resources from growth to defense. This shift was shown by potential hormone-level events—degradation of growth-promoting gibberellins and synthesis of jasmonic and abscisic acids. This finding is congruent with the xeromorphic morphology of seedlings of the cold-tolerant line and their ability to regrow when stress ceases. It is a common reaction of cold-tolerant maize lines. Moreover, in the cold-tolerant line, several genes from the low-temperature signaling pathways were potentially expressed. Additionally, numerous stress-response AP2/EREBP-binding cis-motifs were accessible in the cold-tolerant line. Differently in the cold-sensitive B73 line, MADS-binding cis-motifs were the most abundant. Development of the photosynthetic apparatus is crucial for the survival of maize seedlings at low temperature. Our results suggest efficient photosynthesis in seedlings of the cold-tolerant line, as was described earlier in physiological-level analyses.

Surface-enhanced Raman scattering (SERS) has been intensively used recently as a highly sensitive, non-destructive, chemical specific, and label-free technique for a variety of studies. Here, we present a novel SERS substrate for: (i) the standard ultra-trace analysis, (ii) detection of whole microorganisms, and (iii) spectroelectrochemical measurements. The integration of electrochemistry and SERS spectroscopy is a powerful approach for in situ investigation of the structural changes of adsorbed molecules, their redox properties, and for studying the intermediates of the reactions. We have developed a conductive SERS platform based on photovoltaic materials (PV) covered with a thin layer of silver, especially useful in electrochemical SERS analysis. These substrates named Ag/PV presented in this study combine crucial spectroscopic features such as high sensitivity, reproducibility, specificity, and chemical/physical stability. The designed substrates permit the label-free identification and differentiation of cancer cells (renal carcinoma) and pathogens (Escherichia coli and Bacillus subtilis). In addition, the developed SERS platform was adopted as the working electrode in an electrochemical SERS approach for p-aminothiophenol (p-ATP) studies. The capability to monitor in real-time the electrochemical changes spectro-electro-chemically has great potential for broadening the application of SERS.

Isolation and detection of circulating tumor cells (CTCs) from human blood plays an important role in non- invasive screening of cancer evolution and in predictive therapeutic treatment. Here, we present the novel tool utilizing: (i) the microfluidic device with (ii) incorporated photovoltaic (PV) based SERS-active platform, and (iii) shell-isolated nanoparticles (SHINs) for simultaneous separation and label-free analysis of circulating tumour cells CTCs in the blood specimens with high specificity and sensitivity. The proposed microfluidic chip enables the efficient size – based inertial separation of circulating cancer cells from the whole blood samples. The SERS-active platform incorporated into the microfluidic device permits the label-free detection and identification of isolated cells through the insight into their molecular and biochemical structure. Additionally, the silver nanoparticles coated with an ultrathin shell of silica (Ag@SiO2) was used to improve the detection accuracy and sensitivity of analysed tumor cells via taking advantages of shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS). The empirical analysis of SHINERS spectra revealed that there are some differences among studied (HeLa), renal cell carcinoma (Caki-1), and blood cells. Unique SHINERS features and differences in bands intensities between healthy and cancer cells might be associated with the variations in the quantity and quality of molecules such as lipid, protein, and DNA or their structure during the metastasis cancer formation. To demonstrate the statistical efficiency of the developed method and improve the differentiation for circulating tumors cells detection the principal component analysis (PCA) has been performed for all SHINERS data. PCA method has been applied to recognize the most significant differences in SHINERS data among the three analyzed cells: Caki-1, HeLa, and blood cells. The proposed approach challenges the current multi-steps CTCs detection methods in the terms of simplicity, sensitivity, invasiveness, destructivity, time and cost of analysis, and also prevents the defragmentation/damage of tumor cells and thus leads to improving the accuracy of analysis. The results of this research work show the potential of developed SERS based tool for the separation of tumor cells from whole blood samples in a simple and minimally invasive manner, their detection and molecular characterization using one single technology.

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&mdash, prostate cancer (PC3), cervical carcinoma (HeLa), and leucocytes as an example of healthy (normal) cell&mdash, 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.

The circulating tumor cells (CTCs) isolation and characterization has a great potential for non-invasive biopsy. In the present research, the surface&ndash, 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&rsquo, 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.

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.

In maize seedlings, severe cold results in dysregulation of circadian pattern of gene expression causing profound modulation of transcription of genes related to photosynthesis and other key biological processes. Plants live highly cyclic life and their response to environmental stresses must allow for underlying biological rhythms. To study the interplay of a stress and a rhythmic cue we investigated transcriptomic response of maize seedlings to low temperature in the context of diurnal gene expression. Severe cold stress had pronounced effect on the circadian rhythm of a substantial proportion of genes. Their response was strikingly dual, comprising either flattening (partial or complete) of the diel amplitude or delay of expression maximum/minimum by several hours. Genes encoding central oscillator components behaved in the same dual manner, unlike their Arabidopsis counterparts reported earlier to cease cycling altogether upon cold treatment. Also numerous genes lacking circadian rhythm responded to the cold by undergoing up- or down-regulation. Notably, the transcriptome changes preceded major physiological manifestations of cold stress. In silico analysis of metabolic processes likely affected by observed gene expression changes indicated major down-regulation of photosynthesis, profound and multifarious modulation of plant hormone levels, and of chromatin structure, transcription, and translation. A role of trehalose and stachyose in cold stress signaling was also suggested. Meta-analysis of published transcriptomic data allowed discrimination between general stress response of maize and that unique to severe cold. Several cis- and trans-factors likely involved in the latter were predicted, albeit none of them seemed to have a major role. These results underscore a key role of modulation of diel gene expression in maize response to severe cold and the unique character of the cold-response of the maize circadian clock.

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. 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. 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.

Populations of three sympatric species (Daphnia cucullata, Daphnia galeata, and Daphnia hyalina (Daphnia longispina complex)) inhabiting three lakes in northeastern Poland (Roś, Mikołajskie, Wigry) were checked for interspecific hybrids. Hybrid Daphnia coexisting with parental individuals were detected in each of the investigated lakes. Several classes of hybrids (F1, F2, backcrosses), including D. cucullata×galeata×hyalina individuals, have been detected. Hybrids constituted a substantial (13%–38%) part of the total abundance of individuals of the D. longispina complex. Deficiency of heterozygotes (hybrids) detected may indicate the existence of barriers for gene flow between the sympatric Daphnia taxa.

Maize yield is limited by its cold-sensitivity, the molecular mechanisms of which are poorly understood. We investigated transcriptome changes in maize leaves subjected to 14 °C for 4 (alarm phase) or 28 (acclimation phase) hours using Maize Oligonucleotide Array Project chips containing ca. 57,000 probes. To facilitate global analyses, we assigned Gene Ontology annotations to 10,171 probes using rice and Arabidopsis proteomes as basis. Additionally, gene-by-gene analysis of differentially expressed genes was done basing on annotations provided by microarray producer to indicate those most promising for further studies. Microarray results were validated by qRT-PCR. Chilling affected 440 (4 h) or 155 (28 h) transcripts. After 4 h, 59 transcripts showed aberrant response to photoperiod, suggesting diurnal clock disruption as a mechanism by which chilling affects maize physiology. GO-based analysis indicated modification of cell wall properties as another early response. Few photosynthesis-related genes were repressed by chilling, despite the popular notion that photosynthetic apparatus is a major target of cold. Conversely, induction of chloroplast-related genes not directly engaged in photosynthesis was evident at later stages of the response. Numerous transcripts strongly affected by chilling were of unknown function; they may encode novel activities crucial in maize response to chilling and deserve detailed studies.

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.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-771) contains supplementary material, which is available to authorized users.

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., Plant Molecular Biology, 85 (3), ISSN:0167-4412, ISSN:1573-5028

The expression patterns of two true ras genes, Ppras1 and Ppras2and one rap gene, Pprap1were examined in four Physarum polycephalum developmental stages: uninucleate amoebae, plasmodia (multinucleate syncytia), spherules (a vegetative, dormant stage) and fruiting bodies. Ppras1 and Pprap1 are expressed in all stages examined with the maximum levels of their transcripts in amoebae and fruiting bodies, respectively, and the minimum levels in plasmodia, whereas the Ppras2 transcript is only detectable in amoebae and fruiting bodies. The results obtained indicate that P. polycephalum is an organism possessing a developmentally regulated ras gene family and presents a convenient system to study the role of ras/rap genes in control of growth and differentiation of lower eukaryotic organisms.Copyright 1997 Academic Press Limited Copyright 1997Academic Press Limited

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.

The slime mold Physarum polycephalum is a morphologically simple organism with a large and complex genome. The exon-intron organization of its genes exhibits features typical for protists and fungi as well as those characteristic for the evolutionarily more advanced species. This indicates that both the taxonomic position as well as the size of the genome shape the exon-intron organization of an organism. The average gene has 3.7 introns which are on average 138 bp, with a rather narrow size distribution. Introns are enriched in AT base pairs by 13% relative to exons. The consensus sequences at exon-intron boundaries resemble those found for other species, with minor differences between short and long introns. A unique feature of P.polycephalum introns is the strong preference for pyrimidines in the coding strand throughout their length, without a particular enrichment at the 3'-ends.

We have cloned the genomic copy of the Ppras1 gene, a homologue of the ras proto-oncogene, from the true slime mold Physarum polycephalum. Ppras1 contains five small introns, four of which have a high content of pyrimidines. The (dC)-homopolymers present in introns 4 and 5 may be responsible for the observed recA-independent deletion in Ppras1 upon amplification of the Ppras1-bearing plasmid by choramphenicol. Although Ppras1 exhibits amino acid and nucleotide homologies with the DdrasG gene, a homologue of ras from another slime mold, Distyostelium discoideum, locations and sequences of their introns are quite different. This discordance suggests that introns of the ras genes in these species were acquired independently.