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At least 343 records · Page 19Linked to original sources
PubMed2005

Protein arrays and pattern recognition: new tools to assist in the identification and management of autoimmune disease.

The occurrence of antibody patterns in connective tissue diseases has been recognized for thirty years, but the generation of multiple antibody results relied on time-consuming immunodiffusion or electrophoretic techniques. Today it is possible to study the antibody repertoire using rapid multi-analyte technologies, generally referred to as protein arrays. These arrays may use planar surfaces similar to DNA arrays, or use microspheres in suspension ("liquid arrays"). Also, many high quality autoantigens are now commercially available, including recombinant antigens. The vast amount of information that can be generated by measuring multiple antibodies for multiple patients has created demand for data processing. Software programs to aid physicians in reviewing multiple inputs as an aid to disease diagnosis and classification have been available for twenty years. Initial work used the "expert systems" approach; more recently pattern recognition has been widely evaluated because of the improvements in software programs and computational speed. The use of antibody data, generated in protein arrays, may assist in establishing diagnosis, in identifying potentially significant antibody patterns in advance of clinical symptoms, and in classifying patients based on expected disease progression.

Algorithms↗
PubMed2003

Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells.

The ability to deliver efficiently a complete genomic DNA locus to human and rodent cells will likely find widespread application in functional genomic studies and novel gene therapy protocols. In contrast to a cDNA expression cassette, the use of a complete genomic DNA locus delivers a transgene intact with its native promoter, the exons, all the intervening introns, and the regulatory regions. The presence of flanking, noncoding genomic DNA sequences could prove critical for prolonged and appropriate gene expression. We have recently developed a technology for the rapid conversion of bacterial artificial chromosome (BAC) clones into high-capacity herpes simplex virus-based amplicon vectors. Here, we express the human low-density lipoprotein receptor (LDLR), mutated in familial hypercholesterolemia (FH), from a 135-kb BAC insert. The infectious LDLR genomic locus vectors were shown to express at physiologically appropriate levels in three contexts. First, the LDLR locus was expressed appropriately in the ldl(-/-)a7 Chinese hamster ovary (CHO) cell line immediately following infectious delivery; second, the locus was maintained within a replicating episomal vector and expressed at broadly physiological levels in CHO cells for 3 months following infectious delivery; and third, the locus was efficiently expressed in human fibroblasts derived from FH patients. Finally, we show that the infectious LDLR locus retains classical expression regulation by sterol levels in human cells. This long-term expression and physiological regulation of LDLR prepares the way for in vivo functional studies of infectious delivery of BAC inserts.

Animals↗
PubMed2006

Automated target preparation for microarray-based gene expression analysis.

DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables (1) the reduction of experimentally caused result variability, (2) the separation of (important) biological variability from (undesired) experimental variation, and (3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.

Animals↗
PubMed2001

A high-throughput alphavirus-based expression cloning system for mammalian cells.

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.

Animals↗
PubMed2000

The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species.

DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible.

Claviceps↗
PubMed2006

Use of DNA microarray technology and gene expression profiles to investigate the pathogenesis, cell biology, antifungal susceptibility and diagnosis of Candida albicans.

The use of DNA microarrays is becoming the method of choice for assaying gene expression, particularly as costs and complexity are being reduced as the technology becomes more widespread and better standardized. A DNA array is nothing but a collection of probes fixed on a solid support. The probes can be PCR products of ORFs or short intragenic oligonucleotides deposited or synthesized in situ by photolithographic methods. To date, sequencing projects for fungal genomes have yielded 10 complete genomes and 21 whole shotgun sequences, including Candida albicans strain SC5314. Sequencing of the C. albicans genome has led to the construction of whole-genome DNA microarrays for in vitro transcription profiling by several universities and companies. The use of microarray or DNA chip techniques for Candida research has started recently but the number of studies using this technology is increasing rapidly, in order to address important remaining questions about pathogenesis, cell biology, antifungal susceptibility, and diagnosis.

Antifungal Agents↗
PubMed2005

Long-acting insulin analogues (insulin glargine or determir) and continuous subcutaneous insulin infusion in the treatment of type 1 diabetes mellitus in the paediatric population.

Despite many improvements in the treatment of type 1 diabetes mellitus (DM1), the non-physiological time-action profiles of conventional insulins remain a significant obstacle. In recent years, recombinant DNA technology has been used to design insulin molecules that overcome the limitations of regular and NPH insulin. The rapid insulin analogs used as prandial and the long-acting insulin analogs used as basal simulate physiological insulin profiles more closely than the older conventional insulins. The efficacy of insulin analogs now available for multiple daily injection (MDI) and continuous subcutaneous insulin infusion (CSII) therapy in DM1 has been established in pediatric patients. Insulin pumps have improved since they were first introduced. CSII therapy may provide an effective alternative for selected pediatric patients with DM1. In most studies at pediatric age, CSII therapy resulted in a improvement in HbA1c, a decreased rate of hypoglycemia without an abnormal increase in BMI, and without adversely affecting psychosocial outcomes in children and adolescents with DM1.

Adolescent↗
PubMed2005

Molecular biology in orthopaedics: the advent of molecular orthopaedics.

Molecular biology is the study, at the molecular level, of how genetic information is stored, inherited, and expressed and how it influences the structure and function of cells. Although molecular biology approaches have been used for decades in orthopaedic research, they are only now beginning to influence clinical practice. A variety of sophisticated techniques permit rapid and affordable DNA sequencing, gene expression profiling, gene cloning, gene manipulation, gene transfer, recombinant protein production, and other technologies of enormous biomedical importance. Success in genomics has spawned additional ambitious endeavors, including proteomics, pharmacogenetics, and bioinformatics. These techniques are providing new diagnostic, staging, prognostic, and therapeutic opportunities in all areas of medicine, including orthopaedics. With the use of molecular criteria, treatment of the orthopaedic patient may become more individualized, and greater emphasis will be placed on preventative strategies based on the patient's genetic makeup. Both surgical and nonsurgical decisions will increasingly accommodate molecular criteria.

Gene Expression↗
PubMed2002

Role of genomics in identifying new targets for cancer therapy.

The detailed map of the human genome can potentially transform future cancer therapy by merging genomics with pharmacology, thereby identifying which patients will benefit from specific therapeutic agents. Single-nucleotide polymorphisms (SNPs) provide a valuable tool for this pharmacogenetic approach to cancer therapy. The discovery of SNPs as disease markers may facilitate identification of populations at increased risk for certain cancers. In addition, SNP genetic screening may facilitate administration of appropriate treatment modalities or reveal specific genetic profiles that have importance in drug efficacy and toxicity. In addition to SNP analysis, DNA and tissue microarray analyses have the potential to transform the future of cancer therapy. For example, DNA microarrays may improve tumor classification systems as well as provide a molecular level dissection of global gene expression changes that occur in carcinogenesis. Tissue microarrays would allow one to verify candidate genes, identified from DNA microarrays, against archival tumor specimens with known clinical outcome. In addition, both microarray technologies may be combined to rapidly validate gene targets. We will review and discuss these state-of-the-art technologies including data suggesting that the combined use of these high throughput technologies will facilitate our understanding of the genetic complexities inherent in cancer and will revolutionize cancer therapy.

Drug Design↗
PubMed1994

Rapid and efficient resolution of parentage by amplification of short tandem repeats.

Short tandem repeat (STR) loci are highly informative polymorphic loci that are gaining popularity for identity testing. We have conducted parentage testing by using nine STR loci on 50 paternity trios that had been previously tested using VNTR loci. These nine unlinked STR loci are amplified in three multiplex reactions and, when examined for genetic informativeness, provide a combined average power of exclusion of 99.73% (Caucasian data). The informative value of the selected loci is based on extensive STR typing of four racial/ethnic populations. In 37 of the 50 cases, paternity could not be excluded by any of the loci. In the remaining 13 cases, paternity was excluded by at least two of the STR markers. The probability of paternity calculated for the alleged father of each matching trio was > 99% in 36 of the 37 inclusion cases. All data agreed with the results reported using VNTR loci and conventional Southern technology. Our studies validate the use of DNA typing with STR loci for parentage testing, thus providing an accurate, highly sensitive, and rapid assay.

Alleles↗
PubMed1998

Analysing genetic information with DNA arrays.

The large amount of DNA sequence information produced in recent years has created a need for high-throughput methods in biology and genetics. These include sequencing, comparing gene sequences and genotyping. DNA arrays promise a highly parallel means for analysis of DNA that is fast and cost-effective, and offers scope for application to complex systems and processes. Recent years have seen continued transfer of technology from the microelectronics industry. Rapid application of the technology to genotyping, antisense oligonucleotide selection and gene expression analysis has illustrated the general power of this approach.

Base Sequence↗
PubMed2005

Towards a nucleic acid-based diagnosis in clinical parasitology and mycology.

BACKGROUND: Multiple in-house polymerase chain reaction (PCR) assays for the diagnosis of parasitic and fungal diseases have been reported. Encouraging results have been published to anticipate or improve the diagnosis. However, the absence of standardized methods has led to discrepant results. As a consequence, these tests are not recognized as consensual diagnostic criteria. METHODS: The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, the quantitative results provided by these techniques allow to compare rapidly the efficiency of primers, probes, and DNA extraction. Therefore, one can expect a more consensual method to implement comparisons between laboratories. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, the meaning of detecting nucleic acids in a given clinical sample still needs to be defined. This requires well-designed studies with clinical consensual criteria and PCR techniques that are as similar as possible. CONCLUSIONS: The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for decision-making.

Animals↗
PubMed2006

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells.

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.

CD40 Ligand↗
PubMed2003

DNA microarrays for detecting endocrine-disrupting compounds.

It has recently been discovered that a number of synthetic chemicals and naturally occurring compounds released into the environment can influence endocrine activity. These endocrine-disrupting compounds (EDCs) are highly varied in structure, provenance, and mode of action (MOA). Many EDCs are anthropogenic, products of the chemical industry. Others are natural compounds. Although natural hormones exist at low levels in the environment, industrial sites such as pulp and paper mills and municipal sewage treatment plants can gather large amounts of natural EDCs and release them into the environment as part of their daily operations, thus increasing background environmental concentrations. The potential consequences of endocrine disruption are serious; however, comparatively little is known about the phenomenon. There is considerable debate over the true concentrations, sources, identity, and effects of potential EDCs. Investigation of the problem is hampered by the diversity of potential EDCs, which may have synergistic as well as individual effects, and the complexity of the endocrine system itself. Effective and standardized tests to accurately detect the presence of such chemicals in the environment are not available. While many tests have been proposed, they are unsatisfactory because they have only one limited endpoint (e.g., they can detect only one of many potential hormonal responses) and do not provide any mechanistic information. In addition, results from rapid screening tests are difficult to correlate with whole organism response, while larger whole organism bioassays are very costly and time consuming to perform. There is tremendous potential in the application of DNA microarray technology to screen for EDCs. DNA microarrays provide a "snapshot" of transcriptional activity in tissue samples showing which genes were actively expressed within the cells at one point in time. By combining this technology with human cell lines grown in vitro, it should be possible to conduct relatively rapid and straightforward assays to identify EDCs by observing the changes in gene expression patterns in response to exposure.

Animals↗
PubMed1990

Clinical application of novel sample processing technology for the identification of salmonellae by using DNA probes.

Two hundred and fifty clinical fecal specimens collected over a 7-month period were analyzed for the presence of salmonellae by a rapid DNA hybridization procedure. Hybridizations were performed by using a novel specimen processing protocol called wicking and a previously unreported 1,600-base-pair probe cloned from Salmonella enteritidis DNA. The probe was shown to be reactive with all 70 Salmonella serotypes tested and not reactive with 101 stock strains of other enteric bacteria. Southern analysis of 30 Salmonella isolates representing 22 serotypes suggested that the probe sequence was highly conserved, appearing as a 1,600-base-pair band in a BglII digest of isolate DNA in 29 of 30 isolates and as a 2,300-base-pair fragment in 1 of the isolates. The probe correctly identified all salmonellae (nine isolates) among 47 H2S-producing colonies tested from among 250 clinical specimens cultured on xylose-lysine-desoxycholate medium. Salmonellae grown on xylose-lysine-desoxycholate medium gave consistently higher hybridization values than did those grown on either MacConkey or Hektoen enteric agar. In addition, of eight gram-negative broth enrichments in which salmonellae were identified by conventional means, seven were probe positive. The use of this nucleic acid probe and hybridization technique provides a simple and rapid identification of Salmonella species.

Bacteriological Techniques↗
PubMed1991

Cystic fibrosis, genetics, and DNA technology.

Advances in molecular genetics are leading to changes in practice that have a direct impact on the obstetrician and gynecologist. New techniques of prenatal screening, diagnosis, and potentially therapy are rapidly evolving. Recent developments in cystic fibrosis, preimplantation genetics, fragile X syndrome, neurofibromatosis, muscular dystrophy, and Marfan syndrome are discussed.

Cystic Fibrosis↗
PubMed1990

Isolation and characterization of DNA probes for human chromosome 21.

A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al., 1988; Van Keuren et al., 1989). Methods based on using region-specific chromosome 21 DNA probes and fluorescence in situ hybridization show promise for the rapid diagnosis of trisomy 21 (Lichter et al., 1988). The continued development of chromosome 21 DNA probes and advances in the technology of molecular cytogenetics will facilitate the study of the genetic organization of chromosome 21 and its role in the pathogenesis of Down syndrome.

Chromosomes, Human, Pair 21↗
PubMed1994

Gene therapy for cystic fibrosis in humans by liposome-mediated DNA transfer: the production of resources and the regulatory process.

The number of clinical trials using gene transfer technology, either active or under discussion, is increasing rapidly. However, little information is available describing the regulatory procedures or safety specifications that must be considered before initiation of such trials in Europe. We describe the procedure used by our group to produce resources for the first stage of a phase I trial of liposome-mediated gene therapy for cystic fibrosis. The current lack of written and co-ordinated guidance from the numerous interested regulatory agencies within the UK and Europe makes determination of the appropriate safety specifications and procedures for these novel trials difficult, as does the fact that some new agencies (such as the Genetic Therapy Advisory Committee in the UK) and some which are unfamiliar with clinical trials (such as the Department of the Environment) are involved as well as the Medicines Control Agency. In addition, we estimate that the realistic cost of these trials, which in many cases will have to be covered from research budgets provided by government agencies or medical charities, could lead to delays in the clinical application of this important new therapeutic strategy.

Base Sequence↗