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PubMed2006

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry.

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

Acetylcholine↗

PubMed2006

Early identification of false positives in high-throughput screening for activators of p53-DNA interaction.

Naturally occurring mutant forms of p53 are deficient for specific DNA binding. However, their specific DNA binding can be reactivated. The search for small molecules that reactivate latent p53 is considered to be a cornerstone in cancer therapy. The authors describe a new homogeneous fluorescent assay approach for the characterization of binding affinities of human wild-type latent and activated p53 using DNA(*)spec(26), with and without the addition of the antibody PAb421, respectively, and fluorescence correlation spectroscopy (FCS)/2-dimensional fluorescence-intensity distribution analysis anisotropy as the detection methods. FCS was compared with 2D-FIDA anisotropy, and a very good correlation of the results with both readouts was observed (K(D)s for nonspecific DNA binding of 24.4+/-3.5 nM with 2D-FIDA anisotropy and of 29.5+/-5.5 nM with FCS). The presence of poly(dI-dC) led to a 10-fold increase of binding affinity (K(D) of 3.3+/-0.5 nM in the presence of PAb421). 2D-FIDA anisotropy was demonstrated to be the most accurate readout; hence, this detection technology was selected for a 25,000 compound member high-throughput screening (HTS) campaign. The hits obtained were qualified using a novel data evaluation algorithm that identifies false positives and moreover assesses the validity of true hits in the presence of the deteriorating artifact. This process step is of utmost importance for decreasing the attrition in fluorescence-based HTS.

Algorithms↗

PubMed2006

Gene expression profiles in Finnish twins with multiple sclerosis.

BACKGROUND: Since genetic alterations influencing susceptibility to multiple sclerosis (MS), the most common autoimmune demyelinating disease of the central nervous system (CNS), are as yet poorly understood, the purpose of this study was to identify genes responsible for MS by studying monozygotic (MZ) twin pairs discordant for MS. METHODS: In order to identify genes involved in MS development, the gene expression profiles in blood mononuclear cells obtained from eight MZ twin pairs discordant for MS were analyzed by cDNA microarray technology detecting the expression of 8 300 genes. The twins were collected from the Finnish Twin Cohort Study and both affected subjects and their healthy siblings underwent neurological evaluation and cerebral and spinal magnetic resonance imaging. Gene expressions were confirmed by relative quantitative reverse transcription PCR. RESULTS: It appeared that 25 genes were at least two-fold up-regulated and 15 genes down-regulated in 25% (2/8) of twins with MS when compared to their healthy siblings. Moreover, 6/25 genes were up-regulated in 40% of MS twins and one gene, interferon alpha-inducible protein (clone IFI-6-16) (G1P3), in 50% of them. The six most constantly expressed genes are (1) G1P3, (2) POU domain, class 3, transcription factor 1, (3) myxovirus resistance 2, (4) lysosomal-associated multispanning membrane protein-5, (5) hemoglobin alpha 2 and (6) hemoglobin beta. CONCLUSION: Over two-fold up-regulation of these six genes in almost half of MZ twins with MS suggests their role in MS pathogenesis. Studies using MZ MS twins obtained from genetically homogeneous population offer a unique opportunity to explore the genetic nature of MS.

Diseases in Twins↗

PubMed2026

Characteristics of fusion genes in breast cancer.

Fusion genes, arising from aberrant genomic rearrangements, represent critical oncogenic drivers with distinct oncogenic functions. Although relatively uncommon in breast cancer, accumulating evidence suggests that fusion genes contribute to tumor initiation, progression, and therapeutic resistance. This review first summarizes the molecular mechanisms underlying fusion gene formation, their frequency and subtype distribution, and advances in detection technologies in breast cancer. We then discuss how fusion genes reprogram oncogenic signaling pathways and mediate resistance to conventional and targeted therapies. Finally, we evaluate their translational potential as diagnostic biomarkers and therapeutic targets, emphasizing opportunities for precision oncology. By integrating current insights, this review underscores the multifaceted roles of fusion genes in breast cancer biology and highlights their promise for guiding the development of more effective, personalized treatment strategies.

Breast cancer↗

PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗

PubMed2005

Fluorescent leukotriene B4: potential applications.

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Cell Membrane↗

PubMed2005

Complete-angle projection diffuse optical tomography by use of early photons.

We present the first, to our knowledge, experimental images of complex-shaped phantoms embedded in diffuse media by use of optical tomography. Imaging is based on a complete-angle projection tomographic technique that utilizes transmitted early photons. Results are contrasted with measurements obtained at later gates as well as pseudocontinuous-wave data. The scanning system developed employs noncontact illumination and detection technologies that allow for high spatial sampling of transmitted photons. Combining this system with complete-angle illumination is found to be an important strategy toward improved imaging performance, resulting in a better-posed inversion problem. The appropriateness of reconstruction algorithms similar to those employed in x-ray computed tomography are showcased, and suggestions for model improvements are provided.

Algorithms↗

PubMed2006

Model system for phenotypic characterization of sequence variations in the LDL receptor gene.

BACKGROUND: Sequence variations in the LDL receptor (LDLR) gene cause defects of LDLR protein production and function through different molecular mechanisms. Here we describe a cell model system for the phenotypic characterization of sequence variations in the LDLR gene. Well-known sequence variations belonging to LDLR classes 2 to 5 (p.G565V, p.I161D, p.Y828C, and p.V429M) were studied in CHO and HepG2 cells. METHODS: Expression of LDLR protein on the cell surface was detected by use of fluorescence-conjugated antibodies against the LDLR and the LDLR activity was measured by incubating the cells with fluorescently labeled and radiolabeled LDL. The intracellular locations of the LDLR mutants and wild-type were also investigated. RESULTS: The class 2A p.G565V sequence variant exhibited an intracellular distribution of LDLR with no active receptors on the cell surface. Both the class 3 p.I161D and class 4 p.Y828C sequence variants gave surface staining but had a reduced ability to bind or internalize LDL, respectively. By determining the intracellular locations of the receptors we were able to visualize the accumulation of the class 5 p.V429M sequence variant in endosomes by means of a specific marker, as well as confirming that the class 4 p.Y828C variant was not localized in clathrin-coated pits. Flow cytometry allowed us quantitatively to determine the amount and activity of receptors. To confirm the results of binding and cell association of fluorescently labeled LDL analyzed by flow cytometry, assays using 125I-labeled LDL were performed. In addition to a useful and valid alternative to radiolabeled LDL, the unique properties of fluorescently labeled LDL allowed a variety of detection technologies to be used. CONCLUSIONS: This new approach enables phenotypic characterization of sequence variations in the LDLR gene. The assays developed may be valuable for confirming the pathogenicity of novel missense sequence variations found throughout the LDLR gene.

Animals↗

PubMed2006

Metabolite profiling in Arabidopsis.

Metabolite profiling is the multiparallel relative quantification of a mixture of compounds or compound classes using chromatography and universal detection technologies (GC-MS, LC-MS). In this respect it is an extension of classical single-target methods from which it can be distinguished by its broader view on profiling major biochemical events. This broader scope of analysis outweighs the disadvantages by making compromises in method development and the reduced accuracy for specific metabolites. This chapter exemplifies the strategies in metabolite profiling of polar compounds by gas chromatography-mass spectrometry (GC-MS). It gives experimental details on the basic steps: harvest, homogenization, extraction, fractionation, concentration, derivatization, data acquisition, raw data processing and result data tranformation.

Arabidopsis↗

PubMed2006

Analysis of polymerase chain reaction products by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) analysis is an ion-pair reversed-phase high performance liquid chromatography for performing analytical separations of DNA based on temperature: analysis is performed at a temperature sufficient to partially denature DNA heteroduplexes. The technology detects single-base changes as efficiently as short deletions and insertions. The chance that a mutation cannot be detected is 0.5%.

Automation↗

PubMed2006

Application of two-step quantitative reverse-transcription PCR to bacterial diagnostics.

Use of high copy number bacterial RNA offers several advantages in a diagnostics context compared with current deoxyribonucleic acid-based assays. The opportunity to only detect viable cells by targeting labile RNA transcripts may create an opportunity for "real-time" monitoring of pathogen load in response to a treatment regimen, while the natural amplification provided by the relative abundance of the RNA target compared with its corresponding gene opens a door to potential nonamplified direct detection technologies. In this chapter, a method is described to accurately quantify specific RNA transcripts and thus determine their potential utility as "high-copy" targets. The quantification method described also has application in gene-expression analysis.

Bacteria↗

PubMed2006

Single-nucleotide polymorphism mapping.

Single-nucleotide polymorphism (SNP) mapping is the easiest and most reliable way to map genes in Caenorhabditis elegans. SNPs are extremely dense and usually have no associated phenotype, making them ideal markers for mapping. SNP mapping has three steps. First, recombinant mutant animals are generated over a polymorphic strain (usually CB4856) using standard genetic techniques. Second, the genotype of these animals at SNP loci is determined using one of a variety of SNP detection technologies. Third, linkage between the mutant and one or more SNPs is used to position the mutant on the chromosome relative to the SNPs. This chapter presents a detailed procedure for generating recombinant animals, for assaying SNPs using restriction enzymes, and for analyzing mapping data.

Animals↗

PubMed2003

Clinical applications of body composition measurements using DXA.

Dual-energy X-ray absorptiometry (DXA) scanning was primarily developed for the diagnosis of osteoporosis and was initially applied to studies of the clinically important sites of the lumbar spine, femoral neck, and forearm. The rapid adoption of DXA has led to the development of different, competing generations of equipment. Improvements have been achieved through advances in X-ray generation and detection technology, modification of data acquisition protocols, and implementation of more sophisticated image analysis algorithms. As a result, DXA has been extended to allow the study of the total skeleton and its regional parts, as well as soft-tissue composition measurement. The three major components of the body: fat mass, lean mass, and bone mineral mass, can now be easily measured using a single whole body DXA scan with high precision and low scanning time. The comprehensive view of body composition provided by DXA makes it an attractive technique for a variety of clinical applications such as the prevention of cardiovascular and metabolic diseases, clinical management of different chronic diseases, and monitoring of the impact of treatment regimens on body tissues. In this article we review the contribution DXA has made to the understanding of body composition in clinical studies in adults.

Absorptiometry, Photon↗

PubMed2000

Performance evaluation of nine hormone assays on the Immulite 2000 immunoassay system.

We evaluated the analytical performance of the Immulite 2000 immunoassay analyzer (Diagnostic Products Corporation, Los Angeles, USA) based on a new detection technology, electrochemical luminescence. The evaluated analytes were thyrotropin, triiodothyronine, free thyroxine, follitropin, lutropin, prolactin, cortisol, estradiol and progesterone. We tested the assay precision, linearity, recovery, and correlation with comparison methods for these analytes. For most assays, within-run and between-day imprecisions were less than 8% and 10%, respectively. The linearity and recovery were acceptable for all assays. The correlation between the Immulite 2000 assays and comparison methods showed satisfactory results.

Hormones↗

PubMed2003

Techniques patents for SNP genotyping.

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in the human genome, accounting for more than 90% of all differences between individuals. Many complex phenotypes in humans have a significant genetic component and most of the variability is therefore likely to stem from differences in patterns of SNPs. Association studies involving the large-scale analysis of SNPs can help to identify genes affecting many human phenotype variations, including complex diseases and drug responses. SNPs therefore play a major role in all stages of the drug development process, from target identification through to clinical trials. SNPs are also the basis of pharmacogenomics, the tailoring of medicines to suit an individual's genome. Given the potential impact of SNPs on healthcare, the biotechnology industry has focussed urgently on the development of high-throughput methods for SNP genotyping. All genotyping methods are a mix and match of different allele discrimination and signal detection technologies and as such may represent the intellectual property of several individuals or organizations. In this review, we explore the patent issues surrounding SNP genotyping and how this is influencing large scale, commercially valuable projects involving SNPs.

Genome, Human↗

PubMed2004

Gentronix.

Gentronix is an innovative biotechnology company with the aim of providing cell-based assays and systems to ease or remove some of the current bottlenecks in preclinical drug discovery and development. Gentronix's first product, GreenScreen trade mark GC, is a combined genotoxicity and cytotoxicity test designed for high-throughput screening of new pharmaceutical lead compounds, giving drug development scientists a cost-effective preview of the battery of mandatory regulatory genotoxicity tests. Future products based on the company's extensive intellectual property rights portfolio will combine the company's expertise in cellular biosensor systems with novel optical detection technology to deliver a range of products for other applications within drug discovery, as well as industrial and environmental monitoring of toxic chemicals.

Biosensing Techniques↗

PubMed2003

Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis.

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.

Apolipoproteins E↗

PubMed2004

Development of a microarray assay that measures hybridization stoichiometry in moles.

Microarray data is most useful when it can be compared with other genetic detection technologies. In this report, we designed a microarray assay format that transforms raw data into a defined scientific unit (i.e., moles) by measuring the amount of array feature present and the cDNA sequence hybridized. This study profiles a mouse reference universal RNA sample on a microarray consisting of PCR products. In measuring array features, a labeled DNA sequence was designed that hybridizes to a conserved sequence that is present in every array feature. To measure the amount of cDNA sample hybridized, the RNA sample was processed to ensure consistent dye to DNA ratio for every labeled target cDNA molecule, using labeled branched dendrimers rather than by incorporation. A dye printing assay was then performed in order to correlate molecules of cyanine dye to signal intensity. We demonstrate that by using this microarray assay design, raw data can be transformed into defined scientific units, which will facilitate interpretation of other experiments, such as data deposited at the Gene Expression Omnibus and ArrayExpress.

Animals↗