Luc Michiels

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 D(3), 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.

Nanocrystalline diamond impedimetric aptasensor for the label-free detection of human IgE

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.

Effects of soft drink and table beer consumption on insulin response in normal teenagers and carbohydrate drink in youngsters

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.

Heat-Transfer Resistance at Solid–Liquid Interfaces: A Tool for the Detection of Single-Nucleotide Polymorphisms in DNA

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.

Structural and optical properties of DNA layers covalently attached to diamond surfaces

Label-free detection of DNA molecules on chemically vapor-deposited diamond surfaces is achieved with spectroscopic ellipsometry in the infrared and vacuum ultraviolet range. This nondestructive method has the potential to yield information on the average orientation of single as well as double-stranded DNA molecules, without restricting the strand length to the persistence length. The orientational analysis based on electronic excitations in combination with information from layer thicknesses provides a deeper understanding of biological layers on diamond. The pi-pi* transition dipole moments, corresponding to a transition at 4.74 eV, originate from the individual bases. They are in a plane perpendicular to the DNA backbone with an associated n-pi* transition at 4.47 eV. For 8-36 bases of single- and double-stranded DNA covalently attached to ultra-nanocrystalline diamond, the ratio between in- and out-of-plane components in the best fit simulations to the ellipsometric spectra yields an average tilt angle of the DNA backbone with respect to the surface plane ranging from 45 degrees to 52 degrees . We comment on the physical meaning of the calculated tilt angles. Additional information is gathered from atomic force microscopy, fluorescence imaging, and wetting experiments. The results reported here are of value in understanding and optimizing the performance of the electronic readout of a diamond-based label-free DNA hybridization sensor.

DNA Sensors with Diamond as a Promising Alternative Transducer Material

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.

DISCRISET: A battery of tests for fast waste classification - Application of tests on waste extracts

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.

Screening tests for hazard classification of complex waste materials : selection of methods

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 (H1-15) 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.

Validation of Microtox as a first screening tool for waste classification

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.

Parity-induced changes in global gene expression in the human mammary gland

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 κ, casein &bgr;, keratin 14, CCAAT/enhancer binding protein &bgr; and δ 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.