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Supplementary Table 1 from The E2F-Regulated Gene Chk1 Is Highly Expressed in Triple-Negative Estrogen Receptor−/Progesterone Receptor−/HER-2− Breast Carcinomas

We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D3, a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER−/PR−/HER-2− phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present. [Cancer Res 2007;67(14):6574–81]

Revathy Munuswamy,1,* Jana De Brandt,2,* Chris Burtin,2 Wim Derave,3 Joseph Aumann,4 Martijn A Spruit,5,6,* Luc Michiels1,* 1Faculty of Medicine and Life Sciences, Biomedical Research Institute BIOMED, Hasselt University, Hasselt, Belgium; 2Faculty of Rehabilitation Sciences, Rehabilitation Research Center REVAL, Biomedical Research Institute BIOMED, Hasselt University, Hasselt, Belgium; 3Department of Movement and Sports Sciences, Ghent University, Ghent, Belgium; 4Department of Pneumology, Jessa Hospital, Hasselt, Belgium; 5Department of Research and Development, CIRO, Horn, the Netherlands; 6Department of Respiratory Medicine, Maastricht University Medical Centre, Faculty of Health, Medicine and Life Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht, the Netherlands*These authors contributed equally to this workCorrespondence: Luc Michiels Email luc.michiels@uhasselt.beAbstract: Chronic low-grade systemic inflammation is frequently observed in patients with chronic obstructive pulmonary disease (COPD), e.g., elevated pentameric CRP (pCRP). However, pCRP can dissociate to form monomeric CRP (mCRP) which exhibits a clear pro-inflammatory behaviour in contrast to the more anti-inflammatory properties of pCRP. Therefore, mCRP may be an informative biomarker to demonstrate chronic low-grade systemic inflammation. This was confirmed by analysing serum samples from 38 patients with COPD and 18 non-COPD control persons (NCCP). mCRP was significantly elevated in patients with COPD vs. NCCP, indicating that mCRP might be considered as a new sensitive marker of chronic low-grade systemic inflammation.Keywords: COPD, low-grade systemic inflammation, pCRP, mCRP

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, we used multicolor single-molecule burst analysis microscopy to detect multiple biomarkers present on single EV. We classified the recorded signals and applied the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, we applied the method to assess both the purity and the inflammatory status of EV, and compared cell culture and plasma-derived EV isolated via different purification methods. We then applied this methodology to identify intercellular adhesion molecule-1 (ICAM-1) specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. Our methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.

Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial-derived extracellular vesicles (M-EVs) to the maintenance of microglia homeostasis and how M-EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M-EVs is limited. Here, we analysed the effects of M-EVs produced in vitro by either tumour necrosis factor alpha (TNF alpha) activated or non-activated microglia BV2 cells. We showed that M-EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M-EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M-EVs increased the protein expression of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B isoform II (LC3B-II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M-EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M-EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia-microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis. the European Research Council (ERC), Grant/Award Number: 640116 Irobi, J (corresponding author), Hasselt Univ, UHasselt, Biomed Res Inst, Hasselt, Belgium. Joy.irobi@uhasselt.be

Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis.

In this study, we show that synthetic sapphire (A1203), an established implant material, can also serve as a platform material for biosensors comparable to nanocrystalline diamond. Sapphire chips, beads, and powder were first modified with (3-aminopropyl) triethoxysilane (APTES), followed by succinic anhydride (SA), and finally single-stranded probe DNA was EDC coupled to the functionalized layer. The presence of the APTES-SA layer on sapphire powders was confirmed by thermogravimetric analyis and Fourier-transform infrared spectroscopy. Using planar sapphire chips as substrates and X-ray photoelectron spectroscopy (XPS) as surface-sensitive tool, the sequence of individual layers was analyzed with respect to their chemical state, enabling the quantification of areal densities of the involved molecular units. Fluorescence microscopy was used to demonstrate the hybridization of fluorescently tagged target DNA to the probe DNA, including denaturation- and re-hybridization experiments. Due to its high thermal conductivity, synthetic sapphire is especially suitable as a chip material for the heat-transfer method, which was employed to distinguish complementary- and non-complementary DNA duplexes containing single-nucleotide polymorphisms. These results indicate that it is possible to detect mutations electronically with a chemically resilient and electrically insulating chip material. (C) 2016 Elsevier B.V. All rights reserved.

Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-alpha induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) in vitro. Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-alpha as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes. This work was co-financed by the EU through the Interreg IV Flanders-the Netherlands project Interreg V Flanders-the Netherlands project Trans Tech Diagnostics (TTD).

In this study we show an optical biosensor concept, based on elastic light scattering from sapphire micro-spheres. Transmitted and elastic scattering intensity of the microspheres (radius 500 mu m, refractive index 1.77) on an optical fiber half coupler is analyzed at 1510 nm. The 0.43 nm angular mode spacing of the resonances is comparable to the angular mode spacing value estimated using the optical size of the microsphere. The spectral linewidths of the resonances are in the order of 0.01 am, which corresponds to quality factors of approximately 10(5). A polydopamine layer is used as a functionalizing agent on sapphire microspherical resonators in view of biosensor implementation. The varying layer thickness on the microsphere is determined as a function of the resonance wavelength shift. It is shown that polymer functionalization has a minor effect on the quality factor. This is a promising step toward the development of an optical biosensor. (C) 2014 Elsevier B.V. All rights reserved.

A major conceptual breakthrough in cell signaling has been the finding of EV as new biomarker shuttles in body fluids. Now, one of the major challenges in using these nanometer-sized biological entities as diagnostic marker is the development of translational methodologies to profile them. SPR offers a promising label-free and real time platform with a high potential for biomarker detection. Therefore, we aimed to develop a uniform SPR methodology to detect specific surface markers on EV derived from patient with CHD. EVs having an approximate size range between 30 and 100 nm (similar to 48.5%) and 100-300 nm (similar to 51.5%) were successfully isolated. The biomarker profile of EV was verified using immunogold labeling, ELISA and SPR. Using SPR, we demonstrated an increased binding of EV derived from patients with CHD to anti-ICAM-1 antibodies as compared to EV from healthy donors. Our current findings open up novel opportunities for in-depth and label-free investigation of EV. (C) 2017 The Authors. Published by Elsevier Inc. This work was co-financed by the EU through the Interreg IV Flanders-the Netherlands project VaRiA (IVA-VLANED-3.65) and Interreg V Flanders-the Netherlands project Trans Tech Diagnostics (TTD) and Johannes Waltenberger is supported by Cells-in-Motion Cluster of Excellence (EXC 1003-CiM), University of Munster, 48149 Munster, Germany.

M13 filamentous bacteriophage has been used in displaying disease-specific antibodies, biomarkers, and peptides. One of the major drawbacks of using phage in diagnostic assays is the aspecific adsorption of proteins leading to a high background signal and decreasing sensitivity. To deal with this, we developed a genetically pure, exchangeable dual-display phage system in which biomarkers and streptavidin-binding protein (SBP) are displayed at opposite ends of the phage. This approach allows for sample purification, using streptavidin-coated magnetic beads resulting in a higher sensitivity of signal detection assays. Our dual-display cassette system approach also allows for easy exchange of both the anchor protein (SBP) and the displayed biomarker. The presented principle is applied for the detection of antibody reactivity against UH-RA. 21 which is a good candidate biomarker for rheumatoid arthritis (RA). The applicability of dual-display phage preparation using a helper plasmid system is demonstrated, and its increased sensitivity in phage ELISA assays using patient serum samples is shown. Hasselt University [08GO2BOF]

Natalie Vanden Bon,1 Bart van Grinsven,2 Mohammed Sharif Murib,2 Weng Siang Yeap,2 Ken Haenen,2,3 Ward De Ceuninck,2,3 Patrick Wagner,2,3 Marcel Ameloot,1 Veronique Vermeeren,1 Luc Michiels11Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 2Institute for Materials Research, Hasselt University, Diepenbeek, Belgium; 3IMOMEC, Diepenbeek, BelgiumAbstract: Conventional neonatal diagnosis of phenylketonuria is based on the presence of abnormal levels of phenylalanine in the blood. However, for carrier detection and prenatal diagnosis, direct detection of disease-correlated mutations is needed. To speed up and simplify mutation screening in genes, new technologies are developed. In this study, a heat-transfer method is evaluated as a mutation-detection technology in entire exons of the phenylalanine hydroxylase (PAH) gene. This method is based on the change in heat-transfer resistance (Rth) upon thermal denaturation of dsDNA (double-stranded DNA) on nanocrystalline diamond. First, ssDNA (single-stranded DNA) fragments that span the size range of the PAH exons were successfully immobilized on nanocrystalline diamond. Next, it was studied whether an Rth change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear Rth shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, Rth was shown to detect prevalent single-nucleotide polymorphisms, c.473G>A (R158Q), c.932T>C (p.L311P), and c.1222C>T (R408W), correlated with phenylketonuria, displaying an effect related to the different melting temperatures of homoduplexes and heteroduplexes.Keywords: mutation detection, heat-transfer resistance, melting temperature, nanocrystalline diamond, persistence length

Probe DNA, consisting of a 36-mer fragment was covalently immobilized on nanocrystalline chemical vapour deposition (CVD) diamond electrodes and hybridized with a 29-mer target DNA. In this paper, we report on the label-free real-time electronic monitoring of DNA denaturation upon exposure to NaOH at different flow rates and molarities, using electrochemical impedance spectroscopy as readout technology. The impedance response was separated into a denaturation time constant and a medium exchange time constant by means of a double exponential fit. It was observed that the denaturation time is dependent on the flow rate as well as on the molarity of the NaOH. Surprisingly, it was observed that at low molarities (0.05 M) the DNA does not fully denature at low flow rates. Only after flushing the flow cell a second time with 0.05 M NaOH, complete denaturation was achieved. Confocal images were obtained and plotted in 3D graphs to confirm the results. This paper provides a systematic overview of measured denaturation times for different flow rates and at different molarities of NaOH. Optimization of these parameters can be a valuable asset in the field of mutation analysis.

In this article, we report on the heat-transfer resistance at interfaces as a novel, denaturation-based method to detect single-nucleotide polymorphisms in DNA. We observed that a molecular brush of double-stranded DNA grafted onto synthetic diamond surfaces does not notably affect the heat-transfer resistance at the solid-to-liquid interface. In contrast to this, molecular brushes of single-stranded DNA cause, surprisingly, a substantially higher heat-transfer resistance and behave like a thermally insulating layer. This effect can be utilized to identify ds-DNA melting temperatures via the switching from low- to high heat-transfer resistance. The melting temperatures identified with this method for different DNA duplexes (29 base pairs without and with built-in mutations) correlate nicely with data calculated by modeling. The method is fast, label-free (without the need for fluorescent or radioactive markers), allows for repetitive measurements, and can also be extended toward array formats. Reference measurements by confocal fluorescence microscopy and impedance spectroscopy confirm that the switching of heat-transfer resistance upon denaturation is indeed related to the thermal on-chip denaturation of DNA. ispartof: ACS Nano vol:6 issue:3 pages:2712-2721 ispartof: location:United States status: published

The high prevalence of cardiovascular diseases (CVD) demands a reliable and sensitive risk assessment technique. In order to develop a fast and label-free immunosensor for C-reactive protein (CRP), a risk factor for this condition, anti-CRP antibodies were physically adsorbed to the hydrogen (H)-terminated surface of nanocrystalline diamond (NCD). An Enzyme-Linked ImmunoSorbent Assay (ELISA) reference technique showed that this was a suitable substrate for antibody-antigen recognition reactions. Electrochemical Impedance Spectroscopy (EIS) was used to electronically detect CRP recognition. The specificity of the immunosensor was demonstrated by incubation with CRP and plasminogen as reference molecule. A different impedance behavior was observed in real-time after CRP addition as compared to plasminogen addition: the impedance increased only during CRP incubation. Fitting the data showed that this corresponded with a decrease in capacitance of the molecular layer due to its increased thickness by specific CRP recognition. Sensitivity experiments in real-time showed a clear discrimination between 1 μM, 100 nM, and 10 nM of CRP after 10 min at 100 Hz. Since, 10 nM of CRP was still clearly distinguishable from buffer solution, our CRP-directed immunosensor prototype reaches a sensitivity that is within the physiologically relevant concentration range of this biomarker in healthy controls and CVD patients. Moreover, this prototype displayed real-time discriminating power between spiked and unspiked serum, and thus also shows its applicability in this biological matrix. © 2011 Elsevier B.V. All rights reserved. ispartof: Sensors and Actuators B, Chemical vol:157 issue:1 pages:130-138 status: published

The study of protein adsorption on solid surfaces is interesting for theoretical and practical bio-analytical sensing applications. In this work we combine electrochemical impedance spectroscopy, enzyme linked immunosorbent assay, and fluorescence microscopy with thin boron doped nanocrystalline diamond films to address and study the adsorption behavior of globular proteins (antibodies) on hydrophobic and hydrophilic diamond surfaces. A powerful combination of time resolved impedance spectroscopy and data modeling with equivalent circuits allow a detailed insight in the protein behavior at an interface. It is found that hydrogenated diamond is greatly favorable for impedimetric read-out but causes slight conformational loss of the protein structure and therefore also its biological activity. The oxidized surface allows faster adsorption and a high biological activity but results in smaller impedimetric response. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. ispartof: Physica Status Solidi A, Applications and Materials Research vol:208 issue:9 pages:2093-2098 status: published

Bio-electronics is a scientific field coupling the achievements in biology with electronics to obtain higher sensitivity, specificity and speed. Biosensors have played a pivotal role, and many have become established in the clinical and scientific world. They need to be sensitive, specific, fast and cheap. Electrochemical biosensors are most frequently cited in literature, often in the context of DNA sensing and mutation analysis. However, many popular electrochemical transduction materials, such as silicon, are susceptible to hydrolysis, leading to loss of bioreceptor molecules from the surface. Hence, increased attention has been shifted towards diamond, which surpasses silicon on many levels. ispartof: Sensors vol:9 issue:7 pages:5600-5636 ispartof: location:Switzerland status: published

In the framework of developing a fast and label-free immunosensor for C-reactive protein (CRP) detection, H-terminated nanocrystalline diamond (NCD) was functionalised with anti-CRP antibodies that were physically adsorbed to the surface. Impedance spectroscopy was used to electronically detect real-time CRP recognition. Different impedance behaviours were observed after CRP addition as compared to after FITC-labelled ssDNA addition at low (100 Hz) as well as at high frequencies (1 MHz). Physical interpretations of the observed impedance changes were obtained by fitting the data to an equivalent electrical circuit. Concentrations of 1 μM CRP were recognised with a reaction time of 30 minutes. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

This article reviews the Current state-of-the art of diamond-based DNA sensors. Some general concepts involved in biosensors are introduced and applied to DNA sensors. The properties of chemically vapor deposited (CVD) diamond relevant for this application are summed up, with special attention for the stability and bio-compatibility of the material. Several routes to functionalize the diamond Surface are considered. The physical properties of the obtained DNA layers are discussed in terms of surface density and molecular conformation. Possible read-out strategies are evaluated, including optical and electronic sensing. With diamond-based DNA sensors, real-time and label-free sensing is achieved.

The covalent attachment method for DNA on nanocrystalline diamond (NCD), involving the introduction of COOH functionalities on the surface by photoattachment of 10-undecenoic acid (10-UDA), followed by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling to NH 2-labeled ssDNA, is evaluated in terms of stability, density, and functionality of the resulting biological interface. This is of crucial importance in DNA biosensor development. The covalent nature of DNA attachment will infer the necessary stability and favorable orientation to the ssDNA probe molecules. Using confocal fluorescence microscopy, the influence of buffer type for the removal of excess 10-UDA and ssDNA, the probe ssDNA length, the probe ssDNA concentration, and the presence of the COOH-linker on the density and functionality of the ssDNA probe layer were investigated. It was determined that the most homogeneously dense and functional DNA layer was obtained when 300 pmol of short ssDNA was applied to COOH-modified NCD samples, while H-terminated NCD was resistant for DNA attachment. Exploiting this surface functionality dependence of the DNA attachment efficiency, a shadow mask was applied during the photochemical introduction of the COOH-functionalities, leaving certain regions on the NCD H-terminated. The subsequent DNA attachment resulted in a fluorescence pattern corresponding to the negative of the shadow mask. Finally, NCD surfaces covered with mixtures of the 10-UDA linker molecule and a similar molecule lacking the COOH functionality, functioning as a lateral spacer, were examined for their suitability in preventing nonspecific adsorption to the surface and in decreasing steric hindrance. However, purely COOH-modified NCD samples, patterned with H-terminated regions and treated with a controlled amount of probe DNA, proved the most efficient in fulfilling these tasks.

Animal and 'in vitro' experiences learned that human chorionic gonadotropin (hCG) is capable to protect from breast cancer. Receptors for hCG/luteinizing hormone (LH) are present on human female and male breast cancer cells. hCG decreases proliferation and invasion of breast cancer MCF-7 cells by inhibiting NF-kappa B, AP-1 activation and other genes. Doxorubicin toxicity is enhanced by conjugation with beta-hCG in MCF-7 cells. All these pieces of evidence suggest that hCG is active in human breast cancer. Direct proof however is missing. We performed a pilot study phase I trial for testing the inhibitory effects or recombinant hCG (rhCG) on primary breast cancer. Twenty-five postmenopausal women with newly diagnosed breast cancers of more than 1.5 cm were biopsied before randomization to receive either 500 microg rhCG (n=20) or placebo. After 2 weeks, surgery was done and tissues were analysed with regard to morphological, immunohistochemical and biochemical changes in tissues and plasma. rhCG reduces significantly the proliferative index and the expression of both the oestrogen receptor and progesterone receptor. rhCG does not modify the hormonal level of estradiol, progesterone, inhibin and follicle stimulating hormone (FSH) but increases significantly the level of LH. In a second pilot study, we tested the clinical efficacy through an open-label single centre study in 13 postmenopausal women with metastatic breast cancer. A 500 microg rhCG once every 2 days shows activity in postmenopausal metastatic breast cancer. The time to progression is relatively short. Response to previous hormonal treatment is indicative for rhCG activity. Given the data in primary and metastatic breast cancer rhCG further large scale investigation is highly warranted. rhCG can be an realistic option in (chemo-) prevention trials.

Kaushik Rajaram,1 Patricia Losada-Pérez,2,3 Veronique Vermeeren,1 Baharak Hosseinkhani,1 Patrick Wagner,2,4 Veerle Somers,1 Luc Michiels1 1Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, 2Institute for Materials Research (IMO), Hasselt University, 3IMEC vzw, Division IMOMEC, Diepenbeek,4Soft Matter and Biophysics Section, Department of Physics and Astronomy, KU Leuven, Leuven, Belgium Abstract: Over the last three decades, phage display technology has been used for the display of target-specific biomarkers, peptides, antibodies, etc. Phage display-based assays are mostly limited to the phage ELISA, which is notorious for its high background signal and laborious methodology. These problems have been recently overcome by designing a dual-display phage with two different end functionalities, namely, streptavidin (STV)-binding protein at one end and a rheumatoid arthritis-specific autoantigenic target at the other end. Using this dual-display phage, a much higher sensitivity in screening specificities of autoantibodies in complex serum sample has been detected compared to single-display phage system on phage ELISA. Herein, we aimed to develop a novel, rapid, and sensitive dual-display phage to detect autoantibodies presence in serum samples using quartz crystal microbalance with dissipation monitoring as a sensing platform. The vertical functionalization of the phage over the STV-modified surfaces resulted in clear frequency and dissipation shifts revealing a well-defined viscoelastic signature. Screening for autoantibodies using antihuman IgG-modified surfaces and the dual-display phage with STV magnetic bead complexes allowed to isolate the target entities from complex mixtures and to achieve a large response as compared to negative control samples. This novel dual-display strategy can be a potential alternative to the time consuming phage ELISA protocols for the qualitative analysis of serum autoantibodies and can be taken as a departure point to ultimately achieve a point of care diagnostic system. Keywords: dual-display phage, surface plasmon resonance, quartz crystal microbalance, dissipation monitoring, streptavidin-binding protein, rheumatoid arthritis&nbsp

A biochemical method to immobilize DNA on synthetic diamond for biosensor applications is developed. Nanocrystalline diamond is grown using microwave plasma-enhanced chemical vapour deposition. On the hydrogen-terminated surface 10-undecenoic acid is tethered photochemically under 254 nm illumination, followed by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide crosslinker-mediated attachment of amino modified DNA. The attachment is functionally confirmed by comparison of supernatant fluorescence and gel electrophoresis. The linking procedure allowed for 35 denaturation and rehybridisation steps. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The protective effect of an early first full-term pregnancy in relation to breast cancer risk is well established, but the molecular and cell-specific changes in the human mammary gland involved remain unclear. To identify the molecular changes associated with pregnancy-induced differentiation, we analysed the global gene expression profiles of normal mammary tissues from both a parous and a nulliparous woman, using serial analysis of gene expression. This approach allowed us to identify sets of genes, known and unknown, that are differentially expressed in parous versus age-matched nulliparous mammary gland tissues. The normal mammary gland of a multiparous woman is characterized by several known differentiation markers such as casein kappa, casein beta, keratin 14, CCAAT/enhancer binding protein beta and delta and adipsin. Candidate genes involved in cytoarchitectural remodelling and growth inhibition with a potential role in pregnancy-induced protection against breast cancer were also observed. Several genes that are highly expressed in the nulliparous mammary gland and that are lost after pregnancy, encode for growth promoting, cytoskeletal and extracellular matrix proteins. One of these genes, the small breast epithelial mucin, is almost completely downregulated upon a first full-term pregnancy but is known to be expressed in more than 90% of invasive ductal carcinomas.

The interplay between cancer cells and the normal surrounding tissue is believed to influence the biological behavior of the tumor. However, the presence of multiple cell types within the prelevated tumor specimen may attenuate changes that occur specifically in the malignant cells within their microenvironment. To study gene expression of the malignant cells in situ, we used a new microdissection method to separate ductal carcinoma in situ (DCIS) cells from the surrounding stroma, immunological infiltrates, and endothelial cells. We applied an adapted microSAGE protocol, without total mRNA amplification, to study their gene expression profile. Three thousand two hundred one different transcripts were identified in a total of 29 534 observed tags. Of these unique tags, 88.3% matched known GenBank sequences and 11.7% represented unknown transcripts. As compared to a total DCIS SAGE library, microdissection combined with SAGE revealed additional genes expressed only in normal surrounding, probably stromal, cells and not or significantly less in DCIS tumor cells. This study demonstrates that microdissection can be combined with SAGE as a tool to study transcriptomes. This approach provides important new information on differential gene expression both in tumor cells and normal surrounding tissue. Several of the observed differences indeed disappear when the total tumor mass is analyzed.

Limburgs Univ Centrum, Mat Res Inst, B-3590 Diepenbeek, Belgium. Linburgs Univ Centrum, Biomed Res Inst, B-3590 Diepenbeek, Belgium.Wenmackers, S, Limburgs Univ Centrum, Mat Res Inst, Wetenschapspk 1, B-3590 Diepenbeek, Belgium.

Aptamers, short synthetic ssDNA or RNA molecules with a specific three-dimensional structure, are promising recognition elements in biosensor technology. In vitro generation of aptamers with high sensitivity and specificity toward a broad range of analytes has been achieved using the systematic evolution of ligands by exponential enrichment (SELEX) process. This iterative pathway of aptamer generation consists of sequential positive and counterselection steps. The present research aimed to select two sets of ssDNA aptamers which both are able to bind to different functional groups on the cyclopentanoperhydrophenanthrene ring of 17β-estradiol (E2). By repetitively switching between positive selection steps using E2 as target molecule and counterselection steps with nortestosterone as countermolecule, aptamers were successfully selected against the hydroxylated aromatic A ring of E2. Additionally, an aptamer which binds the upper segments of the B, C and D ring of the cyclopentanoperhydrophenanthrene ring of E2 was generated after repetitively swapping between positive selection steps with E2 as target molecule and counterselection steps with dexamethasone as countermolecule. Epitope specificity of the aptamers was demonstrated by evaluating their binding responses toward a number of steroid hormones structurally related to E2. The selected aptamers with affinities for different functional groups of E2 can potentially be applied to develop a cross-reactive aptasensor. This aptasensor introduces a promising tool for the future of in-field real-time monitoring of a wide range of steroid hormones.

A microcavity-based deoxyribonucleic acid (DNA) optical biosensor is demonstrated for the first time using synthetic sapphire for the optical cavity. Transmitted and elastic scattering intensity at 1510 nm are analyzed from a sapphire microsphere (radius 500 µm, refractive index 1.77) on an optical fiber half coupler. The 0.43 nm angular mode spacing of the resonances correlates well with the optical size of the sapphire sphere. Probe DNA consisting of a 36-mer fragment was covalently immobilized on a sapphire microsphere and hybridized with a 29-mer target DNA. Whispering gallery modes (WGMs) were monitored before the sapphire was functionalized with DNA and after it was functionalized with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The shift in WGMs from the surface modification with DNA was measured and correlated well with the estimated thickness of the add-on DNA layer. It is shown that ssDNA is more uniformly oriented on the sapphire surface than dsDNA. In addition, it is shown that functionalization of the sapphire spherical surface with DNA does not affect the quality factor (Q . ≈ 04) of the sapphire microspheres. The use of sapphire is especially interesting because this material is chemically resilient, biocompatible, and widely used for medical implants. ispartof: Journal of Biomedical Optics vol:19 issue:9 ispartof: location:United States status: published

The PvuII polymorphism of the estrogen receptor (ESR) gene and its relation to bone mineral density (BMD), fracture history, and muscle strength was studied in 313 postmenopausal (76 +/- 5 years) women of Caucasian origin, of whom 142 had suffered from a fragility fracture after the age of 50 years (14 with fracture of the hip, 38 of the spine, 45 of the wrist, and 85 of other bones). The ESR genotype distribution was similar in women with and without a history of fragility fracture (PP 21%, Pp 43%, pp 36% compared with PP 18%, Pp 47%, pp 35%). We did not find a correlation between the ESR genotypes and BMD at the lumbar spine, the femoral neck, or the proximal forearm. No association was found with grip or quadriceps strength. We further evaluated the relationship between the vitamin D receptor (VDR) and ESR haplotypes and BMD in a random subgroup of 270 elderly women. No differences were found in women with the BBpp versus the bbPP haplotype in the femoral neck (mean difference +/- SD, in Bbpp compared with bbPP groups: -0.05 +/- 0.15 g/cm2), the spine (0.01 +/- 0.13 g/cm2), or the forearm (0.04 +/- 0.08 g/cm2). The significant association of quadriceps strength with VDR genotypes (25% lower in BB compared with bb genotype, p0.05) was not influenced by ESR haplotypes. We conclude that in elderly Caucasian women the PvuII ESR polymorphism is not associated with osteoporosis, fracture history, nor muscle strength and does not influence the association of bone density and muscle strength with polymorphism of the VDR.

There is ample evidence that breast cancer susceptibility is induced during the developmental stages of the human breast where, in a manner related to sex-steroid hormones, insulin plays an important role. In turn, nutrition might be implicated. Regular soft drinks and table beer, both carbohydrate-containing drinks, are candidates affecting insulin concentrations. Eleven teenagers, between the ages of 13 and 17 years, consumed a soft drink and a table beer in a crossover study. The blood levels of insulin and glucose were related to anthropomorphometric and endocrine factors. In contrast to table beer, consumption of regular soft drinks induced a fast and dramatic increase in both glucose and insulin concentration within a maximum 1 hour after consumption. The insulin response was linearly correlated to the body mass index (BMI). Children with a small increase in BMI are highly sensitive to regular soft drinks with regard to glucose and insulin response. The finding suggests a vicious circle of high caloric drinks, increase in BMI and insulin response. It is one of the nutritional pathways which might affect susceptibility for breast cancer in youngsters. Table beer, a drink with fermented sugars, does not share these effects on carbohydrate metabolism.

SummaryPhenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%–23% (P10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.

We have studied backward-wave phonon echoes at 9.3 GHz in several chalcogenide glasses. A difference in the input-power dependence of the echopower between the different chalcogenide glasses has been observed and is discussed with respect to the mean coordination of the samples.

The Waste Framework Directive (WFD; 2008/98/EG) describes how waste materials are to be classified as hazardous or not. For complex waste materials chemical analyses are often not conclusive and the WFD provides the possibility to assess the hazardous properties by testing on the waste materials directly. As a methodology WFD refers to the protocols described in the CLP regulation (regulation on Classification, Labeling and Packaging of chemicals) but the toxicity tests on mammals are not acceptable for waste materials. The DISCRISET project was initiated to investigate the suitability of alternative toxicity tests that are already in use in pharmaceutical applications, for the toxicological hazard assessment of complex waste materials. Results indicated that Microtox was a good candidate as a first screening test in a tiered approached hazard assessment. This is now further validated in the present study. The toxic responses measured in Microtox were compared to biological responses in other bioassays for both organic and inorganic fractions of the wastes. Both fractions contribute to the toxic load of waste samples. Results show that the Microtox test is indeed a good and practical screening tool for the organic fraction. A screening threshold (ST) of 5 geq/l as the EC50 value in Microtox is proposed as this ST allows to recognize highly toxic samples in the screening test. The data presented here show that the Microtox toxicity response at this ST is not only predictive for acute toxicity in other organisms but also for sub lethal toxic effects of the organic fraction. This limit value has to be further validated. For the inorganic fraction no specific biotest can be recommended as a screening test, but the use of direct toxicity assessment is also preferable for this fraction as metal speciation is an important issue to define the toxic load of elutriate fractions. A battery of 3 tests (Microtox, Daphnia and Algae) for direct toxicity assessment of this fraction is recommended in literature, but including tests for mechanistic toxicity might be useful.

A system of five sets of multiplex polymerase chain reaction amplifications followed by denaturing gel electrophoresis analysis allows rapid analysis of all 13 exons of the phenylalanine hydroxylase gene.

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms. This research is funded by the FWO project G.0581.12N. The authors gratefully thank S. Muyldermans and N. Devoogdt (Vrije Universiteit Brussel, Belgium) for the vector pHEN6(c):pelB-NbVCAM1-His6, and J.-P. Noben and E. Rooyackers for the MS measurements. We further acknowledge the Hercules Foundation for the project “LC-MS@UHasselt: Linear Trap Quadrupole-Orbitrap mass spectrometer”, and the Interreg IV-A project “BioMiMedics” which is financed by the EU and the province of Limburg (Belgium). We further thank the Interuniversity Attraction Poles program (P7/05) initiated by the Belgian Science Policy Office (BELSPO).

In this study we describe the development of an alternative methodology for hazard characterization of waste materials. Such an alternative methodology for hazard assessment of complex waste materials is urgently needed, because the lack of a validated instrument leads to arbitrary hazard classification of such complex waste materials. False classification can lead to human and environmental health risks and also has important financial consequences for the waste owner. The Hazardous Waste Directive (HWD) describes the methodology for hazard classification of waste materials. For mirror entries the HWD classification is based upon the hazardous properties (H115) of the waste which can be assessed from the hazardous properties of individual identified waste compounds or if not all compounds are identified from test results of hazard assessment tests performed on the waste material itself. For the latter the HWD recommends toxicity tests that were initially designed for risk assessment of chemicals in consumer products (pharmaceuticals, cosmetics, biocides, food, etc.). These tests (often using mammals) are not designed nor suitable for the hazard characterization of waste materials. With the present study we want to contribute to the development of an alternative and transparent test strategy for hazard assessment of complex wastes that is in line with the HWD principles for waste classification. It is necessary to cope with this important shortcoming in hazardous waste classification and to demonstrate that alternative methods are available that can be used for hazard assessment of waste materials. Next, by describing the pros and cons of the available methods, and by identifying the needs for additional or further development of test methods, we hope to stimulate research efforts and development in this direction. In this paper we describe promising techniques and argument on the test selection for the pilot study that we have performed on different types of waste materials. Test results are presented in a second paper. As the application of many of the proposed test methods is new in the field of waste management, the principles of the tests are described. The selected tests tackle important hazardous properties but refinement of the test battery is needed to fulfil the a priori conditions.

The Hazardous Waste Directive (HWD, Council Directive 91/689/EC, 1991) provides a framework for classification of hazardous waste, based on 15 Hazard (H)-criteria. For complex wastes the HWD foresees the application of toxicity tests on the waste material itself to assess its toxic properties. However, these proposed test methods often involve mammalian testing, which is not acceptable from an ethical point of view, nor is it feasible economically. The DISCRISET project was initiated to investigate the use of alternative chemical and biological fast screening tests for waste hazard classification. In the first part of the project, different methods were reviewed and a testing strategy was proposed to minimize time and cost of analysis by a tiered approach. This includes as a first tier chemical analysis followed by a general acute toxicity screen as a second tier and as a third tier mechanistic toxicity tests to assess chronic toxicity (genotoxicity, hormone disturbance, teratogenic effects, immunologic activity). In this phase of the project, selected methods were applied to 16 different waste samples from various sources and industries. The first tier chemical tests are recommended for the full characterization of the leachate fraction (inorganics) but not for the organic fraction of samples. Here the chemical characterization is only useful if toxic content is known or suspected. As second tier the fast bacterial test Microtox is validated as a general toxicity screen for the organic fraction (worst case organic extract). Samples that are not classified in tier 1 or 2 are then further investigated in the third tier by the mechanistic toxicity tests and tested for their potential chronic toxicity: immune activity (TNF-α upregulation) is indicative for corrosive, irritating or sensitising effects (H4/H8/H15), reproductive effects (H10) are indicated by hormone disturbance and early life stage abnormalities in fish larvae when exposed to the extracts and mutagenicity and carcinogenicity (H7, H11) are indicated by SOS response induction and increased mutation frequency in the Ames test when exposed to the extracts. Results indicate that the combination of chemical tests and bioassays allows important hazardous properties to be addressed and the tiered approach ensures that the tests are performed quickly and economically. The suggested strategy provides a solid and ethical alternative to the methods described in the HWD and is a vast improvement on the current, arbitrary classification.

Health care, food quality control, and environmental management often rely on the detection of abnormal molecules, in the body, in food, and in the environment, respectively. More and more, these fields are moving towards point-of-care detection, since increased analysis speed, and hence, decreased cost, are becoming important determining factors for funding. In parallel, but also to allow for this speedy and on-site analysis, the scientific world has evolved into the ‘nano’-scale. This was made possible by the dawn of bio-electronics: a scientific field coupling the achievements in molecular biology with the advances in electronics. The goal of this field is to interrogate the functional activity of bioreceptor molecules, i.e. the recognition and/or metabolization of their targets, with electronics to increase the detection speed for, and sensitivity to, certain pathogens, pollutants, and genetic mutations. To enable the electronic interrogation of these bioreceptor molecules, they need to be attached to, or embedded into, a solid support or transducer with a favourable orientation and density, ensuring the retention of their biological functionality. The resulting analytical device is called a biosensor. The transducer ‘translates’ the biological recognition event between the receptor molecule and its target into a readable signal. Many biosensors have become established in the clinical and scientific world. However, still few of them have made it to point-of-care applications. The success and applicability of biosensors as point-of-care tools is based on five requirements. They need to be sensitive, specific, fast, cheap, and portable. Electronic biosensors based on (semi-)conductive transducers are hence preferred for point-of-care applications, since they are fast in signal generation and cheap to produce. Many popular semiconductive transduction materials, such as silicon (Si) and germanium (Ge), however, are susceptible to hydrolysis, leading to a loss of bioreceptor molecules from the surface, and hence, to instability of the sensor platform. This negatively influences the sensitivity and specificity of the sensor. This explains our increased attention towards diamond, which surpasses Si and Ge on many levels. It can be made into a semiconductor, preferred for electronic applications, it is chemically and mechanically very stable, it can be functionalized with bioreceptor molecules (DNA, aptamers, antibodies, whole cells), and it is biocompatible since it is only composed of carbon (C).

In this article, we report on the electronic monitoring of DNA denaturation by NaOH using electrochemical impedance spectroscopy in combination with fluorescence imaging as a reference technique. The probe DNA consisting of a 36-mer fragment was covalently immobilized on nanocrystalline-diamond electrodes and hybridized with different types of 29-mer target DNA (complementary, single-nucleotide defects at two different positions, and a non-complementary random sequence). The mathematical separation of the impedimetric signals into the time constant for NaOH exposure and the intrinsic denaturation-time constants gives clear evidence that the denaturation times reflect the intrinsic stability of the DNA duplexes. The intrinsic time constants correlate with calculated DNA-melting temperatures. The impedimetric method requires minimal instrumentation, is label-free and fast with a typical time scale of minutes and is highly reproducible. The sensor electrodes can be used repetitively. These elements suggest that the monitoring of chemically induced denaturation at room temperature is an interesting approach to measure DNA duplex stability as an alternative to thermal denaturation at elevated temperatures, used in DNA-melting experiments and single nucleotide polymorphism (SNP) analysis. ispartof: Lab on a Chip vol:11 issue:9 pages:1656-1663 ispartof: location:England status: published

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH 2 )-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer–IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.

The FAU gene is the cellular homologue of the fox sequence in the Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV). FAU (for FBR-MuSV a ssociated ubiquitously expressed gene) encodes the ribosomal protein S30 fused to a ubiquitin-like protein. A cosmid clone containing the human FAU gene was used for fluorescence in situ hybridization to metaphase chromosomes. The obtained localization to 11q13 was confirmed by hybridization against a panel of somatic cell hybrids containing different parts of chromosome 11 on a hamster background. FAU was then further mapped, both on a panel of radiation-reduced somatic cell hybrids designed to carry different parts of the 11q13 region and by pulsed-field gel electrophoresis. This fine mapping assigned FAU close to the skeletal muscle glycogen phosphorylase gene (PYGM), in a region that contains several oncogenes as well as the putative tumor suppressor genes MEN1 and ST3.

The authors acknowledge financial support by the Research Foundation-Flanders (FWO-Vlaanderen) in the framework of the project G.0159.07 'Structural and electronic properties of biologically modified graphene-based layers', a collaboration between Hasselt University, University of Antwerp and Catholic University Leuven. Furthermore, we acknowledge financial support from the federal Belgian BELSPO project IUP VI 'Quantum effects in clusters and nanowires'. S. W. is a postdoctoral research fellow of FWO-Vlaanderen and V. V. is a postdoc of the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT). We also want to thank Thomas Cleij and Milos Nesladek for fruitful discussions and helpful advice.