Glutathione and Xanthine Metabolic Changes in Tamoxifen Resistant Breast Cancer Cell Lines are Mediated by Down-Regulation of GSS and XDH and Correlated to Poor Prognosis.

Background: Tamoxifen is commonly used in the treatment of hormonal-positive breast cancer. However, 30%-40% of tumors treated with tamoxifen develop resistance; therefore, an important step to overcome this resistance is to understand the underlying molecular and metabolic mechanisms. In the present work, we used metabolic profiling to determine potential biomarkers of tamoxifen resistance, and gene expression levels of enzymes important to these metabolites and then correlated the expression to the survival of patients receiving tamoxifen. Methods: Tamoxifen-resistant cell lines previously developed and characterized in our laboratory were metabolically profiled with nuclear magnetic resonance spectroscopy (NMR) using cryogenic probe, and the findings were correlated with the expression of genes that encode the key enzymes of the significant metabolites. Moreover, the effect of significantly altered genes on the overall survival of patients was assessed using the Kaplan-Meier plotter web tool. Results: We observed a significant increase in the levels of glutamine, taurine, glutathione, and xanthine, and a significant decrease in the branched-chain amino acids, valine, and isoleucine, as well as glutamate and cysteine in the tamoxifen-resistant cells compared to tamoxifen sensitive cells. Moreover, xanthine dehydrogenase and glutathione synthase gene expression were downregulated, whereas glucose-6-phosphate dehydrogenase was upregulated compared to control. Additionally, increased expression of xanthine dehydrogenase was associated with a better outcome for breast cancer patients. Conclusion: Overall, this study sheds light on metabolic pathways that are dysregulated in tamoxifen-resistant cell lines and the potential role of each of these pathways in the development of resistance.

NMR-based metabolomics identification of potential serum biomarkers of disease progression in patients with multiple sclerosis.

Multiple sclerosis (MS) is a chronic and progressive neurological disorder, characterized by neuroinflammation and demyelination within the central nervous system (CNS). The etiology and the pathogenesis of MS are still unknown. Till now, no satisfactory treatments, diagnostic and prognostic biomarkers are available for MS. Therefore, we aimed to investigate metabolic alterations in patients with MS compared to controls and across MS subtypes. Metabolic profiles of serum samples from patients with MS (n = 90) and healthy control (n = 30) were determined by Nuclear Magnetic Resonance (1H-NMR) Spectroscopy using cryogenic probe. This approach was also utilized to identify significant differences between the metabolite profiles of the MS groups (primary progressive, secondary progressive, and relapsing-remitting) and the healthy controls. Concentrations of nine serum metabolites (adenosine triphosphate (ATP), tryptophan, formate, succinate, glutathione, inosine, histidine, pantothenate, and nicotinamide adenine dinucleotide (NAD)) were significantly higher in patients with MS compared to control. SPMS serum exhibited increased pantothenate and tryptophan than in PPMS. In addition, lysine, myo-inositol, and glutamate exhibited the highest discriminatory power (0.93, 95% CI 0.869-0.981; 0.92, 95% CI 0.859-0.969; 0.91, 95% CI 0.843-0.968 respectively) between healthy control and MS. Using NMR- based metabolomics, we identified a set of metabolites capable of classifying MS patients and controls. These findings confirmed untargeted metabolomics as a useful approach for the discovery of possible novel biomarkers that could aid in the diagnosis of the disease.

Cytotoxic and molecular differences of anticancer agents on 2D and 3D cell culture.

BACKGROUND: Cancer and multidrug resistance are regarded as concerns related to poor health outcomes. It was found that the monolayer of 2D cancer cell cultures lacks many important features compared to Multicellular Tumor Spheroids (MCTS) or 3D cell cultures which instead have the ability to mimic more closely the in vivo tumor microenvironment. This study aimed to produce 3D cell cultures from different cancer cell lines and to examine the cytotoxic activity of anticancer medications on both 2D and 3D systems, as well as to detect alterations in the expression of certain genes levels. METHOD: 3D cell culture was produced using 3D microtissue molds. The cytotoxic activities of colchicine, cisplatin, doxorubicin, and paclitaxel were tested on 2D and 3D cell culture systems obtained from different cell lines (A549, H1299, MCF-7, and DU-145). IC50 values were determined by MTT assay. In addition, gene expression levels of PIK3CA, AKT1, and PTEN were evaluated by qPCR. RESULTS: Similar cytotoxic activities were observed on both 3D and 2D cell cultures, however, higher concentrations of anticancer medications were needed for the 3D system. For instance, paclitaxel showed an IC50 of 6.234 µM and of 13.87 µM on 2D and 3D H1299 cell cultures, respectively. Gene expression of PIK3CA in H1299 cells also showed a higher fold change in 3D cell culture compared to 2D system upon treatment with doxorubicin. CONCLUSION: When compared to 2D cell cultures, the behavior of cells in the 3D system showed to be more resistant to anticancer treatments. Due to their shape, growth pattern, hypoxic core features, interaction between cells, biomarkers synthesis, and resistance to treatment penetration, the MCTS have the advantage of better simulating the in vivo tumor conditions. As a result, it is reasonable to conclude that 3D cell cultures may be a more promising model than the traditional 2D system, offering a better understanding of the in vivo molecular changes in response to different potential treatments and multidrug resistance development.

Bortezomib advanced mechanisms of action in multiple myeloma, solid and liquid tumors along with its novel therapeutic applications.

Bortezomib (BTZ) is a first-in-class reversible and selective proteasome inhibitor. It inhibits the ubiquitin proteasome pathway that leads to the degradation of many intracellular proteins. Initially, BTZ was FDA approved for the treatment of refractory or relapsed multiple myeloma (MM) in 2003. Later, its usage was approved for patients with previously untreated MM. In 2006, BTZ was approved for the treatment of relapsed or refractory Mantle Cell Lymphoma (MCL) and, in 2014, for previously untreated MCL. BTZ has been extensively studied either alone or in combination with other drugs for the treatment of different liquid tumors especially in MM. However, limited data evaluated the efficacy and safety of using BTZ in patients with solid tumors. In this review, we will discuss the advanced and novel mechanisms of action of BTZ documented in MM, solid tumors and liquid tumors. Moreover, we will shed the light on the newly discovered pharmacological effects of BTZ in other prevalent diseases.

Surface Plasmon Resonance Sensitivity Enhancement Based on Protonated Polyaniline Films Doped by Aluminum Nitrate.

Complex composite films based on polyaniline (PANI) doped hydrochloric acid (HCl) incorporated with aluminum nitrate (Al(NO3)3) on Au-layer were designed and synthesized as a surface plasmon resonance (SPR) sensing device. The physicochemical properties of (PANI-HCl)/Al(NO3)3 complex composite films were studied for various Al(NO3)3 concentrations (0, 2, 4, 8, 16, and 32 wt.%). The refractive index of the (PANI-HCl)/Al(NO3)3 complex composite films increased continuously as Al(NO3)3 concentrations increased. The electrical conductivity values increased from 5.10 µS/cm to 10.00 µS/cm as Al(NO3)3 concentration increased to 32 wt.%. The sensitivity of the SPR sensing device was investigated using a theoretical approach and experimental measurements. The theoretical system of SPR measurement confirmed that increasing Al(NO3)3 in (PANI-HCl)/Al(NO3)3 complex composite films enhanced the sensitivity from about 114.5 [Deg/RIU] for Au-layer to 159.0 [Deg/RIU] for Au-((PANI-HCl)/Al(NO3)3 (32 wt.%)). In addition, the signal-to-noise ratio for Au-layer was 3.95, which increased after coating by (PANI-HCl)/Al(NO3)3 (32 wt.%) complex composite layer to 8.82. Finally, we conclude that coating Au-layer by (PANI-HCl)/Al(NO3)3 complex composite films enhances the sensitivity of the SPR sensing device.

Glycolytic flux control by drugging phosphoglycolate phosphatase.

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.

Novelty detection for metabolic dynamics established on breast cancer tissue using 2D NMR TOCSY spectra.

Most metabolic profiling approaches focus only on identifying pre-known metabolites on NMR TOCSY spectrum using configured parameters. However, there is a lack of tasks dealing with automating the detection of new metabolites that might appear during the dynamic evolution of biological cells. Novelty detection is a category of machine learning that is used to identify data that emerge during the test phase and were not considered during the training phase. We propose a novelty detection system for detecting novel metabolites in the 2D NMR TOCSY spectrum of a breast cancer-tissue sample. We build one- and multi-class recognition systems using different classifiers such as, Kernel Null Foley-Sammon Transform, Kernel Density Estimation, and Support Vector Data Description. The training models were constructed based on different sizes of training data and are used in the novelty detection procedure. Multiple evaluation measures were applied to test the performance of the novelty detection methods. Depending on the training data size, all classifiers were able to achieve 0% false positive rates and total misclassification error in addition to 100% true positive rates. The median total time for the novelty detection process varies between 1.5 and 20 seconds, depending on the classifier and the amount of training data. The results of our novel metabolic profiling method demonstrate its suitability, robustness and speed in automated metabolic research.

Metabolic Profiling of Thymic Epithelial Tumors Hints to a Strong Warburg Effect, Glutaminolysis and Precarious Redox Homeostasis as Potential Therapeutic Targets.

Thymomas and thymic carcinomas (TC) are malignant thymic epithelial tumors (TETs) with poor outcome, if non-resectable. Metabolic signatures of TETs have not yet been studied and may offer new therapeutic options. Metabolic profiles of snap-frozen thymomas (WHO types A, AB, B1, B2, B3, n = 12) and TCs (n = 3) were determined by high resolution magic angle spinning 1H nuclear magnetic resonance (HRMAS 1H-NMR) spectroscopy. Metabolite-based prediction of active KEGG metabolic pathways was achieved with MetPA. In relation to metabolite-based metabolic pathways, gene expression signatures of TETs (n = 115) were investigated in the public "The Cancer Genome Atlas" (TCGA) dataset using gene set enrichment analysis. Overall, thirty-seven metabolites were quantified in TETs, including acetylcholine that was not previously detected in other non-endocrine cancers. Metabolite-based cluster analysis distinguished clinically indolent (A, AB, B1) and aggressive TETs (B2, B3, TCs). Using MetPA, six KEGG metabolic pathways were predicted to be activated, including proline/arginine, glycolysis and glutathione pathways. The activated pathways as predicted by metabolite-profiling were generally enriched transcriptionally in the independent TCGA dataset. Shared high lactic acid and glutamine levels, together with associated gene expression signatures suggested a strong "Warburg effect", glutaminolysis and redox homeostasis as potential vulnerabilities that need validation in a large, independent cohort of aggressive TETs. If confirmed, targeting metabolic pathways may eventually prove as adjunct therapeutic options in TETs, since the metabolic features identified here are known to confer resistance to cisplatin-based chemotherapy, kinase inhibitors and immune checkpoint blockers, i.e., currently used therapies for non-resectable TETs.

Automated metabolic assignment: Semi-supervised learning in metabolic analysis employing two dimensional Nuclear Magnetic Resonance (NMR).

Metabolomics is an expanding field of medical diagnostics since many diseases cause metabolic reprogramming alteration. Additionally, the metabolic point of view offers an insight into the molecular mechanisms of diseases. Due to the complexity of metabolic assignment dependent on the 1D NMR spectral analysis, 2D NMR techniques are preferred because of spectral resolution issues. Thus, in this work, we introduce an automated metabolite identification and assignment from 1H-1H TOCSY (total correlation spectroscopy) using real breast cancer tissue. The new approach is based on customized and extended semi-supervised classifiers: KNFST, SVM, third (PC3) and fourth (PC4) degree polynomial. In our approach, metabolic assignment is based only on the vertical and horizontal frequencies of the metabolites in the 1H-1H TOCSY. KNFST and SVM show high performance (high accuracy and low mislabeling rate) in relatively low size of initially labeled training data. PC3 and PC4 classifiers showed lower accuracy and high mislabeling rates, and both classifiers fail to provide an acceptable accuracy at extremely low size (≤9% of the entire dataset) of initial training data. Additionally, semi-supervised classifiers were implemented to obtain a fully automatic procedure for signal assignment and deconvolution of TOCSY, which is a big step forward in NMR metabolic profiling. A set of 27 metabolites were deduced from the TOCSY, and their assignments agreed with the metabolites deduced from a 1D NMR spectrum of the same sample analyzed by conventional human-based methodology.

In Vitro Spatio-Temporal NMR Metabolomics of Living 3D Cell Models.

Three-dimensional cell cultures are of growing importance in biochemical research as they represent tissue features more accurately than standard two-dimensional systems, but to investigate these challenging new models an adaptation of established analytical techniques is required. Spatially resolved data for living organoids are needed to gain insight into transport processes and biochemical characteristics of domains with different nutrient supply and waste product removal. Within this work, we present an NMR-based approach to obtain dynamically radial metabolite profiles for cell spheroids, one of the most frequently used 3D models. Our approach combines an easy to reproduce custom-made measurement design, maintaining physiological conditions without inhibition of the NMR experiment, with spatially selective NMR pulse sequences. To overcome the inherently low sensitivity of NMR spectroscopy we excited slices instead of smaller cube-like voxels in combination with an efficient interleaved measurement approach and employed a commercially available cryogenic NMR probe. Finally, radial metabolite profiles could be obtained via double Abel inversion of the measured one-dimensional intensity profiles. Applying this method to Ty82 cancer cell spheroids demonstrates the achieved spatial resolution, for instance confirming exceedingly high lactic acid and strongly decreased glucose concentrations in the oxygen-depleted core of the spheroid. Furthermore, our approach can be employed to investigate fast and slow metabolic changes in single spheroids simultaneously, which is shown as an example of a spheroid degrading over several days after stopping the nutrient supply.

HR-MAS NMR Based Quantitative Metabolomics in Breast Cancer.

High resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly used for profiling of breast cancer tissue, delivering quantitative information for approximately 40 metabolites. One unique advantage of the method is that it can be used to analyse intact tissue, thereby requiring only minimal sample preparation. Importantly, since the method is non-destructive, it allows further investigations of the same specimen using for instance transcriptomics. Here, we discuss technical aspects critical for a successful analysis - including sample handling, measurement conditions, pulse sequences for one- and two dimensional analysis, and quantification methods - and summarize available studies, with a focus on significant associations of metabolite levels with clinically relevant parameters.

Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation.

Tamoxifen (TAM) is commonly used for cell type specific Cre recombinase-induced gene inactivation and in cell fate tracing studies. Inducing a gene knockout by TAM and using non-TAM exposed mice as controls lead to a situation where differences are interpreted as consequences of the gene knockout but in reality result from TAM-induced changes in hepatic metabolism. The degree to which TAM may compromise the interpretation of animal experiments with inducible gene expression still has to be elucidated. Here, we report that TAM strongly attenuates CCl4-induced hepatotoxicity in male C57Bl/6N mice, even after a 10 days TAM exposure-free period. TAM decreased (p < 0.0001) the necrosis index and the level of aspartate- and alanine transaminases in CCl4-treated compared to vehicle-exposed mice. TAM pretreatment also led to the downregulation of CYP2E1 (p = 0.0045) in mouse liver tissue, and lowered its activity in CYP2E1 expressing HepG2 cell line. Furthermore, TAM increased the level of the antioxidant ascorbate, catalase, SOD2, and methionine, as well as phase II metabolizing enzymes GSTM1 and UGT1A1 in CCl4-treated livers. Finally, we found that TAM increased the presence of resident macrophages and recruitment of immune cells in necrotic areas of the livers as indicated by F4/80 and CD45 staining. In conclusion, we reveal that TAM increases liver resistance to CCl4-induced toxicity. This finding is of high relevance for studies using the tamoxifen-inducible expression system particularly if this system is used in combination with hepatotoxic compounds such as CCl4.

Impact of intratumoral heterogeneity of breast cancer tissue on quantitative metabolomics using high-resolution magic angle spinning 1 H NMR spectroscopy.

High-resolution magic angle spinning (HR MAS) nuclear magnetic resonance (NMR) spectroscopy is increasingly being used to study metabolite levels in human breast cancer tissue, assessing, for instance, correlations with prognostic factors, survival outcome or therapeutic response. However, the impact of intratumoral heterogeneity on metabolite levels in breast tumor tissue has not been studied comprehensively. More specifically, when biopsy material is analyzed, it remains questionable whether one biopsy is representative of the entire tumor. Therefore, multi-core sampling (n = 6) of tumor tissue from three patients with breast cancer, followed by lipid (0.9- and 1.3-ppm signals) and metabolite quantification using HR MAS 1 H NMR, was performed, resulting in the quantification of 32 metabolites. The mean relative standard deviation across all metabolites for the six tumor cores sampled from each of the three tumors ranged from 0.48 to 0.74. This was considerably higher when compared with a morphologically more homogeneous tissue type, here represented by murine liver (0.16-0.20). Despite the seemingly high variability observed within the tumor tissue, a random forest classifier trained on the original sample set (training set) was, with one exception, able to correctly predict the tumor identity of an independent series of cores (test set) that were additionally sampled from the same three tumors and analyzed blindly. Moreover, significant differences between the tumors were identified using one-way analysis of variance (ANOVA), indicating that the intertumoral differences for many metabolites were larger than the intratumoral differences for these three tumors. That intertumoral differences, on average, were larger than intratumoral differences was further supported by the analysis of duplicate tissue cores from 15 additional breast tumors. In summary, despite the observed intratumoral variability, the results of the present study suggest that the analysis of one, or a few, replicates per tumor may be acceptable, and supports the feasibility of performing reliable analyses of patient tissue.

Glycerol-3-phosphate Acyltransferase 1 Promotes Tumor Cell Migration and Poor Survival in Ovarian Carcinoma.

Glycerophosphodiesterase EDI3 (GPCPD1; GDE5; GDPD6) has been suggested to promote cell migration, adhesion, and spreading, but its mechanisms of action remain uncertain. In this study, we targeted the glycerol-3-phosphate acyltransferase GPAM along with choline kinase-α (CHKA), the enzymes that catabolize the products of EDI3 to determine which downstream pathway is relevant for migration. Our results clearly showed that GPAM influenced cell migration via the signaling lipid lysophosphatidic acid (LPA), linking it with GPAM to cell migration. Analysis of GPAM expression in different cancer types revealed a significant association between high GPAM expression and reduced overall survival in ovarian cancer. Silencing GPAM in ovarian cancer cells decreased cell migration and reduced the growth of tumor xenografts. In contrast to these observations, manipulating CHKA did not influence cell migration in the same set of cell lines. Overall, our findings show how GPAM influences intracellular LPA levels to promote cell migration and tumor growth. Cancer Res; 77(17); 4589-601. ©2017 AACR.

Metabolic profiling of ob/ob mouse fatty liver using HR-MAS 1H-NMR combined with gene expression analysis reveals alterations in betaine metabolism and the transsulfuration pathway.

Metabolic perturbations resulting from excessive hepatic fat accumulation are poorly understood. Thus, in this study, leptin-deficient ob/ob mice, a mouse model of fatty liver disease, were used to investigate metabolic alterations in more detail. Metabolites were quantified in intact liver tissues of ob/ob (n = 8) and control (n = 8) mice using high-resolution magic angle spinning (HR-MAS) 1H-NMR. In addition, after demonstrating that HR-MAS 1H-NMR does not affect RNA integrity, transcriptional changes were measured by quantitative real-time PCR on RNA extracted from the same specimens after HR-MAS 1H-NMR measurements. Importantly, the gene expression changes obtained agreed with those observed by Affymetrix microarray analysis performed on RNA isolated directly from fresh-frozen tissue. In total, 40 metabolites could be assigned in the spectra and subsequently quantified. Quantification of lactate was also possible after applying a lactate-editing pulse sequence that suppresses the lipid signal, which superimposes the lactate methyl resonance at 1.3 ppm. Significant differences were detected for creatinine, glutamate, glycine, glycolate, trimethylamine-N-oxide, dimethylglycine, ADP, AMP, betaine, phenylalanine, and uridine. Furthermore, alterations in one-carbon metabolism, supported by both metabolic and transcriptional changes, were observed. These included reduced demethylation of betaine to dimethylglycine and the reduced expression of genes coding for transsulfuration pathway enzymes, which appears to preserve methionine levels, but may limit glutathione synthesis. Overall, the combined approach is advantageous as it identifies changes not only at the single gene or metabolite level but also deregulated pathways, thus providing critical insight into changes accompanying fatty liver disease. Graphical abstract A Evaluation of RNA integrity before and after HR-MAS 1H-NMR of intact mouse liver tissue. B Metabolite concentrations and gene expression levels assessed in ob/ob (steatotic) and ob/+ (control) mice using HR-MAS 1H-NMR and qRT-PCR, respectively.

Application of surface plasmon resonance imaging technique for the detection of single spherical biological submicrometer particles.

Recent proof-of-principle studies demonstrated the suitability of the surface plasmon resonance imaging (SPRi) technique for the detection of individual submicrometer and nanoparticles in solutions. In the current study, we used the SPRi technique for visualization of the binding of round-shaped viruses (inactivated influenza A virus) and virus-like particles (human immunodeficiency virus (HIV)-based virus-like particles) to the functionalized sensor surface. We show the applicability of the SPRi technique for the detection of individual virus-like particles in buffers without serum as well as in buffers containing different concentrations of serum. Furthermore, we prove the specificity of visualized binding events using two different pseudotypes of HIV virus-like particles. We also demonstrate the applicability of the SPRi technique for the determination of relative particle concentrations in solutions. Moreover, we suggest a technical approach, which allows enhancing the magnitude of binding signals. Our studies indicate that the SPRi technique represents an efficient research tool for quantification and characterization of biological submicrometer objects such as viruses or virus-like particles, for example.

Looking into Living Cell Systems: Planar Waveguide Microfluidic NMR Detector for in Vitro Metabolomics of Tumor Spheroids.

The complex cell metabolism and its link to oncogenic signaling pathways have received huge interest within the last few years. But the lack of advanced analytical tools for the investigation of living cell metabolism is still a challenge to be faced. Therefore, we designed and fabricated a novel miniaturized microslot NMR detector with on-board heater integrated with a microfluidic device as NMR sample holder. For the first time, a tumor spheroid of 500 µm diameter and consisting of 9000 cells has been studied noninvasively and online for 24 h. The dynamic processes of production and degradation of 23 intra- and extracellular metabolites were monitored. Remarkably high concentrations of lactate and alanine were observed, being an indicator for a shift from oxidative to glycolytic metabolism. In summary, this methodical development has proven to be a successful analytical tool for the elucidation of cellular functions and their corresponding biochemical pathways. Additionally, the planar geometry of the microslot NMR detector allows the hyphenation with versatile lab-on-a chip (LOC) technology. This opens a new window for metabolomics studies on living cells and can be implemented into new application fields in biotechnology and life sciences.

"Grafting-from" polymerization of PMMA from stainless steel surfaces by a RAFT-mediated polymerization process.

The synthesis of grafted PMMA homopolymer films is reported using a surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization from a RAFT-agent immobilized on a silanized stainless steel surface. Therefore, stainless steel surfaces were hydroxylated with piranha solution followed by silanization with 3-aminopropylsilane (APS). The pendant primary amino groups of the cross-linked polysiloxane layer were reacted with 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid N-hydroxysuccinimide ester to produce a surface with covalently immobilized RAFT agents. PMMA homopolymers of different molecular weights between 13 060 and 45 000 g/mol were then prepared by a surface-initiated RAFT polymerization. Molecular weight (MW) and polydispersity index (PDI) were determined from sacrificial polymerization in solution. The different steps of stainless steel surface modification and the ultrathin films were investigated using atomic force microscopy (AFM), static, X-ray photoelectron spectroscopy (XPS), attenuated total reflectance infrared spectroscopy (ATR-IR), and ellipsometry.

Study on the reversible changes of the surface properties of an L-cysteine self-assembled monolayer on gold as a function of pH.

A stimuli-response biological surface of L-cysteine was prepared on a polycrystalline gold surface from aqueous solution. The effect of the pH value of the rinsing solution on the surface composition was studied with X-ray photoelectron spectroscopy (XPS). Qualitative and quantitative analysis of the amino, carboxyl, and thiol functional groups of these self-assembled monolayers indicate that L-cysteine molecules exist in the neutral and zwitterionic forms and that they are sensitive to the pH of the rinsing solution. In addition, the wetting properties of the functionalized surface were studied by contact angle (CA) analysis: they were also dependent on the pH of the rinsing solution. Furthermore, it was shown that this functionalization process was reversible.

Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors.

Workers occupationally exposed to cadmium, cobalt and lead have been reported to have increased levels of DNA damage. To analyze whether in vivo relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene array study using primary normal human bronchial epithelial cells. Cells were incubated with 15 microg/l Cd(II), 25 microg/l Co(II) and 550 microg/l Pb(II) either with individual substances or in combination. Differentially expressed genes were filtered out and used to identify enriched GO categories as well as KEGG pathways and to identify transcription factors whose binding sites are enriched in a given set of promoters. Interestingly, combined exposure to Cd(II), Co(II) and Pb(II) caused a coordinated response of at least seven stress response-related transcription factors, namely Oct-1, HIC1, TGIF, CREB, ATF4, SRF and YY1. A stress response was further corroborated by up regulation of genes involved in glutathione metabolism. A second major response to heavy metal exposure was deregulation of the cell cycle as evidenced by down regulation of the transcription factors ELK-1 and the Ets transcription factor GABP, as well as deregulation of genes involved in purine and pyrimidine metabolism. A third and surprising response was up regulation of genes involved in steroid metabolism, whereby promoter analysis identified up regulation of SRY that is known to play a role in sex determination. A forth response was up regulation of xenobiotic metabolising enzymes, particularly of dihydrodiol dehydrogenases 1 and 2 (AKR1C1, AKR1C2). Incubations with individual heavy metals showed that the response of AKR1C1 and AKR1C2 was predominantly caused by lead. In conclusion, we have shown that in vivo relevant concentrations of Cd(II), Co(II) and Pb(II) cause a complex and coordinated response in normal human bronchial epithelial cells. This study gives an overview of the most responsive genes.