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PubMed2005

Universal labeling chemistry for nucleic acid detection on DNA chips.

An efficient strategy for nucleic acid labeling and analysis on deoxyribonucleic acid (DNA) chips has been developed. This approach, which combines the fragmentation and the labeling steps, is based on the reactivity of the phosphates of DNA and ribonucleic acid (RNA) fragments and is using reporter molecules bearing a bromomethyl- or aryldiazomethane-reactive group. In this chapter, we describe the preparation of the reactive label and protocols for efficient labeling of any nucleic acid sequence, DNA or RNA, prior to their hybridization, detection, and analysis on DNA chips.

Nucleic Acid Hybridization↗
PubMed2004

Q-PCR in combination with ChIP assays to detect changes in chromatin acetylation.

Quantitative polymerase chain reaction (Q-PCR) allows for the accurate and reproducible determination of the amount of target DNA in a sample through the measurement of PCR product accumulation in "real time." This method determines starting target DNA quantity over a large assay dynamic range and requires no post-PCR sample manipulation. When used in combination with the method of chromatin immunoprecipitation (ChIP), the amount of protein binding to a specific region of DNA can be accurately and rapidly determined. A method for quantifying the presence of acetylated histones H3 and H4 on different regions of a target locus using Q-PCR after ChIP is described.

Acetylation↗
PubMed2006

Contraction study of a single cardiac muscle cell in a microfluidic chip.

This chapter introduces a microfluidic method to study the contraction of a single cardiac muscle cell (cardiomyocyte). This method integrates single-cell selection, cell retention, dye loading, chemical stimulation, and fluorescence measurement for intracellular calcium on one microfluidic chip. Before single-cell experiments, the bonded chip was modified in order to make the channel deep enough to accommodate a large, single cardiomyocyte. After the modification, a single heart muscle cell could be selected and retained at a cell retention structure. Fluo-4 AM was loaded in the cell for the measurement of intracellular calcium ion concentration in the cell. Subsequently, caffeine was introduced into the chamber to induce the contraction of the cardiomyocyte. During contraction, fluorescence measurement was used to monitor the intracellular calcium level,and an optical imaging system was used to monitor the shape to confirm the contraction. The resting [Ca2+]i of cardiomyocyte was determined and was consistent with the value of approx 100 nM in the literature.

Aniline Compounds↗
PubMed2006

Histone modifications and transcription factor binding on chromatin ChIP-PCR assays.

Chromatin, the eukaryotic template of genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on the N-termini of histones, such as acetylation, methylation, phosphorylation, and ubiquitination. Distinct histone modifications generate synergistic or antagonistic interaction affinities for novhistone proteins, which in turn dictate dynamic transitions between transcriptionally active or silent states of chromatin. Besides transcription, numerous biological processes, including DNA replication, DNA repair, and recombination, are regulated by chromatin-associated factors. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between the structural or regulatory proteins and DNA and its role in regulating diverse cellular processes in vivo by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material and detection of the associated DNA. Here we illustrate the overall experimental procedure by taking ChIP analysis of the human telomerase reverse transcriptase promoter as an example.

Animals↗
PubMed2000

Steam pretreatment of Douglas-fir wood chips. Can conditions for optimum hemicellulose recovery still provide adequate access for efficient enzymatic hydrolysis?

Douglas-fir sapwood and heartwood were impregnated with SO2 and steam exploded at three severity levels, and the cellulose-rich, water-insoluble component was enzymatically hydrolyzed. The high-severity conditions resulted in near complete solubilization and some degradation of hemicelluloses and a significant improvement in the efficiency of enzymatic digestibility of the cellulose component. At lower severity, some of the hemicellulose remained unhydrolyzed, and the cellulose present in the pretreated solids was not readily hydrolyzed. The medium-severity pretreatment conditions proved to be a good compromise because they improved the enzymatic hydrolyzability of the solids and resulted in the recovery of the majority of hemicellulose in a monomeric form within the water-soluble stream. Sapwood-derived wood chips exhibited a higher susceptibility to both pretreatment and hydrolysis and, on steam explosion, formed smaller particles as compared to heartwood-derived wood chips.

Cellulase↗
PubMed2005

Preparation of a visual protein chip for detection of IgG against Treponema pallidum.

A rapid, visual protein chip method for the detection of IgG against Treponema pallidum was developed using Staphylococcus protein A-modified gold nanoparticles. After recombinant T. pallidum antigen Tpp47 was arrayed on aldehyde-coated slides, qualitative and semiquantitative assay results were obtained from the hybridization signal and the subsequent gray stain values. The Tpp47-specific serum antibody was bound to immobilized Tpp47 antigen on the functionalized glass slides. The Staphylococcus protein A-modified gold nanoparticle probe exclusively recognized anti-Tpp47 IgG. The "sandwich" format hybridization signal was then amplified with silver staining, a high sensitivity visual detection technique. It took 3.5 h to prepare the detection protein chip, but only 20 min for real sample detection. The lowest titer of detectable antibody for this method was 1:128, which correlates to approx 1 ng/mL. Furthermore, the results can only be seen unaided, which drastically reduces the cost of detection, but can also be kept for a long time. The data also confirmed the proposed method's specificity, sensitivity, and convenience. As a result, the method could be applied as an alternative diagnostic tool in the clinical diagnosis of T. pallidum.

Antibody Specificity↗
PubMed2000

Identification and characterization of genes responsive to apoptosis: application of DNA chip technology and mRNA differential display.

Apoptosis (programmed cell death) is a genetically programmed active cell death process for maintaining homeostasis under physiological conditions and for responding to various stimuli. Many human diseases have been associated with either increased apoptosis (such as AIDS and neurodegenerative disorders) or decreased apoptosis (such as cancer and autoimmune disorders). In an attempt to understand apoptosis signaling pathway and genes associated with apoptosis, we established two cell model systems on which apoptosis is induced either by DNA damaging agent, etoposide or by redox agent, 1,10-phenanthroline (OP). DNA chip profiling or mRNA differential display (DD) was utilized to identify genes responsive to apoptosis induced by these two agents. In etoposide model with chip hybridization, we defined signaling pathways that mediate apoptosis in p53 dependent manner (through activation of p53 target genes such as Waf-1/p21, PCNA, GPX, S100A2 and PTGF-beta) as well as in p53-independent manner (through activation of ODC and TGF-beta receptor, among others). In OP model with DD screening, we cloned and characterized two genes: glutathione synthetase, encoding an enzyme involved in glutathione synthesis and Sensitive to Apoptosis Gene (SAG), a novel evolutionarily conserved gene encoding a zinc RING finger protein. Both genes appear to protect cells from apoptosis induced by redox agents. Further characterization of SAG revealed that it is a growth essential gene in yeast and belongs to a newly identified gene family that promotes protein ubiquitination and degradation. Through this activity, SAG regulates cell cycle progression and many other key biological processes. Thus, SAG could be a valid drug target for anti-cancer and anti-inflammation therapies.

Amino Acid Sequence↗
PubMed1996

[Implantable patient chip].

Currently, enormous innovative developments are taking place in the area of micro-electronics. In particular, advances in the sector of chip card controller ICs make it appear possible to quickly implement theoretical applications which, only just a few months ago would have been considered utopian and would have ended up gathering dust in the developer's drawer. The present paper describes a technical possibility, so far not implementable in practice, for always having personal emergency data available. Only a year or two ago any idea of implanting electronic circuitry employing memory modules would have been immediately dropped again. At the present state of development in the area of microelectronics, however, structures measuring less than 5 microns in length have become a matter of routine, and a personal memory chip in biotolerable packaging and measuring no more than about (5 x 5 x 1) mm3 can now be implanted subcutaneously. At the present time, further application based on the use of the same technological facilities, such as, for example, the cyclic recording and storage of the heart rate, are under discussion.

Biosensing Techniques↗
PubMed2002

Electro-magnetic base technology for estremely sensitive immunosensors and DNA-chips.

We report on the development of an innovative electro-magnetic base technology for estremely sensitive sensors allowing the electrical detection of biological analytes like antigenes or DNA as well as a simple multiple detection of binding forces occurring at specific bonds between proteins. The technology is based on the strong impact of specifically captured magnetic microbeads on an electrical current generated in a fluid by a small sensor chip with an array of activated microelectrodes. The new technological principle with the on-chip detection of analytes will be suitable for large scale applications due to its mass production compatible technologies and allow an alternative way to monitor relevant substances without the consumption of critical additional solutions and reagents.

Biosensing Techniques↗
PubMed1991

Choice by rats for enriched versus standard home cages: Plastic pipes, wood platforms, wood chips, and paper towels as enrichment items.

The purpose of the present study was to determine whether simple additions to the home cages of rats made those cages preferable to standard housing arrangements. Results indicated that most rats preferred cages with wood platforms, wood chips, and paper towels to otherwise identical cages without these items. Wood chips were not, however, practical with the cages used in the present study. Plastic pipes caused no problems but were not preferred by most animals. Both wood platforms and paper towels created no problems and appeared to be useful as enrichment items. The latter were preferred to the former in a direct comparison.

Journal Article↗
PubMed1988

Investigation of possible health effects of community exposure to fermenting wood chips.

We conducted a case-control study of emergency room (ER) patients to evaluate whether asthma is caused by living near a wood-chip fueled power plant that released wood-chip fermentation products. Only eight (29 per cent) of 28 asthma patients seen in the ER during an 11-week period lived within 1.5 miles of the plant compared with 18 (34 per cent) of 54 control patients matched for severity of diagnosis and seen during the same period (Mantel-Haenszel odds ratio controlling for age = 0.96).

Adult↗
PubMed2003

Self-assembled DNA-conjugated polymer for novel DNA chip.

We developed DNA-conjugated polymer for DNA chip fabrication. A 30 mer probe DNA and disulfide bridges were covalently attached to the polymer side chain. The DNA-conjugated polymer can be specifically adsorbed on a gold substrate surface by a self-assembly technique. The interaction between fully matched DNA and DNA-conjugated polymer was investigated by surface plasmon resonance (SPR) technique. The DNA-conjugated polymer-modified gold surface highly recognized fully matched DNA, rather than unmatched DNA. Therefore, DNA-conjugated polymer can be used for novel DNA chip fabrication.

Base Sequence↗
PubMed2004

DNA-conjugated polymers for self-assembled DNA chip fabrication.

We developed two DNA-conjugated polymers, one based on polyallylamine and the other on polyacrylic acid, for use in DNA chips. A 30-mer single-stranded DNA probe and thioctic acid were covalently attached to polyallylamine as sidechains. The same single-stranded DNA and 3-(pyridyldithio)propionyl hydrazide were covalently attached to polyacrylic acid as sidechains. Both DNA-conjugated polymers could be specifically immobilized onto a gold sensor substrate by a self-assembly technique. The interactions between fully matched DNA and each DNA-conjugated polymer were investigated by surface plasmon resonance. A gold surface modified with either DNA-conjugated polymer recognized fully matched DNA much better than unmatched DNA. The hybridization selectivity and efficiency of DNA-conjugated polyallylamine was optimized by adjusting the pH so as to reduce the effects of cationic polymer sidechains. The hybridization selectivity and efficiency of DNA-conjugated polymers were higher than those of a conventional immobilized thiol-based DNA. The coating of DNA-conjugated polymers reduced nonspecific adsorption of DNA by the gold substrate. DNA-conjugated polyacrylic acid was more selective toward fully matched DNA than was DNA-conjugated polyallylamine. Therefore, DNA-conjugated polymers show promise for application in novel DNA chips.

Base Sequence↗
PubMed2006

Microfluidic polymer chip integrated with an ISFET detector for cationic surfactant assay in dental rinses.

A cationic surfactant ion-selective field-effect transistor (cationic surfactant-ISFET) has been developed based on the tetraphenylborate derivative known as sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate. The cationic surfactant-ISFET shows an almost Nernstian response to tetradecyldimethylbenzylammonium chloride (Zephiramine) over a concentration range between 1.0 x 10(-6) M and 1.0 x 10(-3) M, with a slope of 58.5 +/- 1.7 mV/decade. The cationic surfactant-ISFET can be used over a range of pH values, between pH 3 and 9. The cationic surfactant-ISFET shows excellent selectivity for Zephiramine over small inorganic cations, but shows similar selectivity for other cationic surfactants, such as hexadecyltrimethylammonium and stearyltrimethylammonium ions. A microfluidic polymer chip was integrated with the cationic surfactant-ISFET, and this was fabricated using polystyrene plates and stainless wires as a template for the channel. Cationic surfactant-ISFETs used in a batch system and microchips integrated with cationic surfactant-ISFETs showed very similar performance in terms of low detection limits, slope sensitivity and the stability of the potential response. The microfluidic polymer chip was then applied to the determination of cationic surfactants in dental rinses.

Calibration↗
PubMed2000

Long-term effects of clinical outcome with low and high dose in the Captopril in Heart Insufficient Patients Study (CHIPS).

BACKGROUND: Although angiotensin-converting enzyme inhibitors are recommended as first line therapy in patients with chronic heart failure, the target doses proven to be effective in major morbidity and mortality trials (e.g. captopril 50 mg b.i.d), are generally not used in daily practice in Belgium. AIM: The objective of this study (CHIPS, Captopril in Heart Insufficient Patients Study) was to compare the long-term effects of a low dose (25 mg b.i.d.) and a high dose (50 mg b.i.d.) of captopril in mild to moderate heart failure. After a titration period of at least 10 days, patients who tolerated 50 mg b.i.d., were randomly assigned to receive either the low dose or the high dose of captopril and followed up to 2 years. RESULTS: 298 patients were included and were followed up for a mean of 12 months. Progression in heart failure seems to be favourably influenced by therapy with high dose in comparison to low dose; a relative difference of 29% in the rates of heart failure worsening was observed between the two doses, 31.5% and 22.4% for low and high dose (p = 0.088), respectively. Treatment with high dose showed also a trend to benefit as compared to low dose in reducing the number of hospitalizations for all causes from 22.4 to 14.5% (p = 0.1) and for congestive heart failure from 14.7 to 7.2% (p = 0.06); moreover, the incidence of fatal and nonfatal cardiac events showed a trend in favour of the high dose of 22% (p = 0.142). The total number of adverse events was comparable for both doses, but dizziness and hypotension were a little more frequently reported in the high-dose group. Serum creatinine values showed no significant changes either in the low-dose or in the high-dose group. CONCLUSION: In the CHIPS-study, in comparison to a low dose, therapy with a high dose of captopril tends to improve the long-term clinical outcome of patients with mild to moderate heart failure without significantly more toxicity.

Adolescent↗
PubMed2003

Mobility-shift analysis with microfluidics chips.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Computer Simulation↗
PubMed2003

Visualisation of gene expression data - the GE-biplot, the Chip-plot and the Gene-plot.

Visualisation methods for exploring microarray data are particularly important for gaining insight into data from gene expression experiments, such as those concerned with the development of an understanding of gene function and interactions. Further, good visualisation techniques are useful for outlier detection in microarray data and for aiding biological interpretation of results, as well as for presentation of overall summaries of the data. The biplot is particularly useful for the display of microarray data as both the genes and the chips can be simultaneously plotted. In this paper we describe several ordination techniques suitable for exploring microarray data, and we call these the GE-biplot, the Chip-plot and the Gene-plot. The general method is first evaluated on synthetic data simulated in accord with current biological interpretation of microarray data. Then it is applied to two well-known data sets, namely the colon data of Alon et al. (1999) and the leukaemia data of Golub et al. (1999). The usefulness of the approach for interpreting and comparing different analyses of the same data is demonstrated.

Journal Article↗
PubMed1990

MR imaging findings in patients with bone-chip allografts.

Increasing use of MR to evaluate primary bone neoplasms has stimulated numerous articles on the initial assessment of these lesions. However, scant MR literature has been written about the postoperative MR appearance. We studied the MR appearance of lesions treated by curettage followed by packing with cancellous bone-chip allograft. We retrospectively reviewed the MR scans of 18 consecutive patients treated in this way. Pathologic diagnoses of these lesions were confirmed according to commonly accepted criteria. The allograft sites showed a distinctive pattern of speckled bright signal on T1-weighted images in eight cases. Thirteen of 18 grafts showed a whorled or speckled pattern of increased signal on the T2-weighted images. Only four allograft regions had predominantly low signal on both T1- and T2-weighted images. Two of 18 patients had recurrent tumor proved by open biopsy. MR images in these cases showed areas of homogeneous signal that replaced areas of speckled hyperintensity on both T1- and T2-weighted images. In both these recurrences the tumor had signal intensity similar to that seen on the preoperative MR study. We conclude that knowledge of the MR appearance of cancellous bone chip allografts is important to avoid misinterpreting areas of high signal on T1- or T2-weighted images as areas of recurrence of tumor and/or hemorrhage.

Bone Neoplasms↗