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PubMedDate not supplied

Volumetric multiplexed transmission holography of the heart with echocardiographic data.

Three-dimensional imaging enhances delineation of cardiac anatomy and function. Currently, three-dimensional echocardiography involves complex rendering techniques to imply depth in a given image. Also, display of the final images on a video monitor neutralizes the volume information of the object. A hologram is a true, three-dimensional replica of the original object and does not entail complex data processing. Holography has not been used previously in cardiovascular imaging. In this experiment, 11 excised mammalian whole hearts and five isolated left ventricles were imaged in a water bath. Parallel, tomographic, echocardiographic images comprised the data source from which holograms were obtained by the technique of multiplexed holography. Holograms were mounted on a holographic film and viewed in a special viewing box. High-quality holograms were obtained from every data set. Excellent gray-scale discrimination allowed crisp visualization of cardiac structures and simulated diseases. Electronic sectioning and various projections optimized viewing of the three-dimensional anatomy in surgical orientations. Thus holograms can be produced from tomographic cardiac ultrasound data and could open a new avenue in three-dimensional echocardiography.

Animals↗
PubMed2004

Microarray screening of lymphocyte gene expression differences in a multiplex schizophrenia pedigree.

In order to help prioritize the selection of candidate genes and to study possible trait and not state related changes in gene expression, we compared lymphocytic gene expression patterns of five individual family members with schizophrenia and nine unaffected individuals from a large multiplex high density pedigree. We screened gene expression by microarray consisting of 1128 brain focused genes. Three criteria for selection of microarray gene differences between schizophrenia and unaffected family members were employed: a significant t-test, expression in a majority of subjects, and fold change magnitude. Gene expression levels were significantly different for nine genes between individuals with schizophrenia compared to unaffected controls, and two genes were validated by real-time PCR. The expression of the neuropeptide Y receptor Y1 gene (NPY1R localized at 4q31.3-q32) and the human guanine nucleotide-binding regulatory protein Go-alpha (GNAO1 localized at 16q13) was significantly decreased in individuals with schizophrenia compared to unaffected family controls by microarray and real-time PCR. The cytosolic malate dehydrogenase gene (MDH1 localized at 2p13.3) was also significantly increased by microarray analysis and showed a trend for increase by real-time PCR. The significant genes are discussed in terms of proximity to linkage regions, prior association studies of schizophrenia, and other reports of microarray screening of schizophrenia tissue. Evidence from these studies taken together with the present study suggests critical pathways in schizophrenia may be studied in peripheral tissue as part of the strategy in functional genomic convergence. This preliminary study needs to be repeated by screening a larger set of genes in additional families with schizophrenia. The present study offers support for examination of gene expression patterns using lymphocytic RNA for complex neuropsychiatric disorders from large cohorts of patients.

Adult↗
PubMed1997

Language processing and memory in ill and well siblings from multiplex families affected with schizophrenia.

The present study was designed to extend the investigation of genetic factors for schizophrenia to cognitive and linguistic signs of central nervous system dysfunction. Of 51 siblings studied from 19 schizophrenia multiplex families, 37 had a DSM-III-R diagnosis of schizophrenia or related schzophrenia spectrum disorder and 14 were well. Controls were 17 unrelated healthy individuals within the same social class and age range. Subjects were tested on measures of memory, attention, reading and expressive language ability. Schizophrenic and spectrum disorder siblings were significantly more impaired in tests of auditory discrimination and memory than their well siblings or controls and displayed significantly reduced syntactic complexity to their speech. While well siblings did not differ from controls on most measures, some aspects of language complexity were reduced. A familial effect was observed for tests of reading ability, attention, some syntactic measures, and short-term memory, although these were not the measures that distinguished patients from controls in this cohort, the scores were not correlated among the ill sibling pairs, and poorer scores did not segregate with schizophrenia within these families. Thus, while some measures of language, memory and attention are deviant in patients with schizophrenia, they may not be heritable and directly related to the genetics of the disorder. Instead, they may be a manifestation of, rather than a vulnerability to, the illness.

Adult↗
PubMed2000

Anesthetic management for patients with arthrogryposis multiplex congenita and severe micrognathia: case reports.

Arthrogryposis multiplex congenita (AMC) is a spectrum syndrome of multiple persistent limb contractures often accompanied by associated anomalies, including cleft palate, genitourinary defects, gastroschisis, and cardiac defects. Pediatric patients with AMC frequently present for multiple surgeries requiring general endotracheal anesthesia. We describe our anesthetic experience with the laryngeal mask airway and endotracheal tube in two neonates with AMC and severe micrognathia. We discuss AMC and outline the problems encountered in difficult airway management.

Anesthesia↗
PubMed2002

Luminescent quantum dots for multiplexed biological detection and imaging.

Recent advances in nanomaterials have produced a new class of fluorescent labels by conjugating semiconductor quantum dots with biorecognition molecules. These nanometer-sized conjugates are water-soluble and biocompatible, and provide important advantages over organic dyes and lanthanide probes. In particular, the emission wavelength of quantum-dot nanocrystals can be continuously tuned by changing the particle size, and a single light source can be used for simultaneous excitation of all different-sized dots. High-quality dots are also highly stable against photobleaching and have narrow, symmetric emission spectra. These novel optical properties render quantum dots ideal fluorophores for ultrasensitive, multicolor, and multiplexing applications in molecular biotechnology and bioengineering.

Biological Assay↗
PubMed1997

Optimizing multiplex and LA-PCR with betaine.

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the internet. This month's column discusses the use of additives for optimizing the amount and quality of product obtained through multiplex and 'long and accurate' polymerase chain reaction (LA-PCR). For details on how to partake in the newsgroup, see the accompanying box.

Artifacts↗
PubMed1999

Multiplex detection of single-nucleotide variations using molecular beacons.

We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis.

Adenosine↗
PubMed1997

Arthrogryposis multiplex congenita and bilateral mid-brain infarction following maternal overdose of co-proxamol.

We report a case of arthrogryposis multiplex congenita secondary to fetal hypokinesia in a 41-week gestation infant following antenatal central nervous system injury. The mother's pregnancy was complicated by an episode of attempted self harm, with an overdose of co-proxamol at 22 weeks of gestational age, and by the use of cocaine in combination with excess alcohol intake. Magnetic resonance imaging showed bilateral mid-brain cysts and marked atrophy of the basal ganglia and thalami.

Acetaminophen↗
PubMed2003

Multiplex phase cycling.

We discuss a new class of phase cycling procedures, in which a set of individual phase-shifted transients are stored separately in the computer and processed afterwards to yield the separated NMR signals from two or more coherence transfer pathways. In the case of two-dimensional double-quantum spectroscopy, this multiplex acquisition procedure allows the acquisition of pure-absorption spectra in only 62.5% of the time needed by previous methods.

Journal Article↗
PubMed1998

Cytologic-pathologic correlations: fine needle aspiration of three cases of steatocystoma multiplex.

Steatocystoma multiplex is a rare condition characterized by multiple subcutaneous nodules classically located on the anterior chest. Diagnosis usually is made clinically or by biopsy. The lesions may be numerous, and surgical treatment may not be practical or the results may be unsatisfactory. Three patients with multiple subcutaneous nodules underwent fine needle aspiration, which produced smears with acellular debris. A diagnosis was possible in the first patient because of close collaboration with a dermatologist. The lesions in the other patients were recognized clinically and were confirmed cytologically by fine needle aspiration. A syringe holder allows the use of thin needles (22-gauge) to aspirate very small lesions (4 to 5 mm). Fine needle aspiration may provide a useful alternative to surgical methods in the treatment of these lesions because it is inexpensive, well-tolerated by the patients, and has no associated morbidity.

Adult↗
PubMedDate not supplied

Genetic network identification by high density, multiplexed reversed transcriptional (HD-MRT) analysis in steroidogenic axis model cell lines.

Transcriptional network analysis in steroidogenic axis cell lines requires an understanding of cellular network composition and complexity. Previous studies have shown that absence of transcriptional network components in a cell line compromises that cell line's functional capacity for transcriptional regulation. Our goal was to analyze qualitatively steroidogenic axis-derived cell lines' expression of a putative transcriptional network involved in human and mouse development. To pursue this analysis we used Northern blots and a high density-multiplexed reverse transcription-polymerase chain reaction (HD-MRT-PCR) approach. Our results revealed that, while some members of this putative network were universally expressed, only a minority of the non-constitutive targeted transcripts were present in any single line. Based on our data and previously published results for contextual expression of these transcription factors, a model was constructed possessing the topology suggestive of a scale-free network: certain network members were highly connected nodes and would represent critical sites of vulnerability. The importance of these highly connected nodes for network function is supported by the severe phenotypes exhibited by human patients and animal models when these genes are mutated. We conclude that knowledge of network composition in specific cell lines is essential for their use as models to investigate functional interactions within selected subnetworks.

Alternative Splicing↗
PubMed2002

Typing of Y chromosome single nucleotide polymorphisms in a Japanese population by a multiplexed single nucleotide primer extension reaction.

We have developed a new method for typing single nucleotide polymorphisms (SNPs) on the human Y chromosome based on a multiplexed single nucleotide primer extension. This method has the advantage that several SNPs are typed rapidly and simultaneously. We examined 15 different SNP loci on Y chromosome, M9, M105, M122, M125, M128, M130, SRY465, IMS-JST006241, IMS-JST006841, IMS-JST002611, IMS-JST003305, IMS-JST008425, IMS-JST021354, IMS-JST021355 and IMS-JST055457, in 159 Japanese males. From the typing results of these 15 loci, we found 13 haplotypes. Gene diversity for each locus ranged from 0.025 to 0.486 and the haplotype diversity was estimated to be 0.838. This method could be readily applied for personal identification and paternity testing.

Journal Article↗
PubMed2002

High-throughput cell analysis using multiplexed array technologies.

The desire for more biologically relevant data from primary screening has resulted in a dramatic increase of cell-based assays in HTS labs. Consequently, new cell-array technologies are being developed to increase the quality and quantity of data emerging from such screens. These technologies take the form of both positional and non-positional formats, each with their own advantages. Notably, screens using these technologies generate databases of high-quality data that can be analyzed in ways currently not possible. The power of cell-based assays combined with new array and analytical technologies will enable the condensation of serial drug discovery processes, thereby decreasing the time and cost of taking a hit compound into clinical trials. Here, we compare array strategies being developed towards the goal of integrating multiplexed cell-based assays into HTS.

Animals↗
PubMed2002

Miniaturised multiplexed immunoassays.

Miniaturised immunoassays are of general interest for applications that require the simultaneous determination of different parameters from a minute sample of material. Apart from planar microarray-based systems, bead-based flow cytometric approaches are well suited for the multiplexed detection of target molecules, especially when the number of parameters that have to be determined in parallel is limited.

Animals↗
PubMed1999

Multiplex semi-quantitative reverse transcriptase-polymerase chain reaction of low abundance neuronal mRNAs.

The sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) has provided molecular biology research with an exquisitely sensitive and fast technique for studying gene expression. This method is particularly useful to study transcripts in the nervous system, which are on average present at low levels and the amount of tissue or cells to be analyzed is often limited. Here, we describe a RT-PCR assay which allows the simultaneous detection and semi-quantitation of several transcripts (multiplex). Multiple PCR primer pairs are used to detect different target transcripts in a single reaction, together with a pair of primers able to amplify the hypoxantine-phosphoribosyl-transferase (HPRT), a gene constitutively expressed at low levels throughout the nervous system. HPRT levels remain constant also during neurogenesis and it is thus apt to be used in developmental neurobiology. This internal standard is the mRNA of reference to evaluate sample variation in RT and PCR reactions and to monitor the degradation and recovery of RNAs. Normalization with respect to HPRT cDNA allows to estimate the relative abundance of each target mRNA.

Animals↗
PubMed2002

Multiplex real-time NASBA for monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA.

BACKGROUND: The monitoring for HCMV mRNA expression in whole blood provides an accurate and informative diagnostic approach. METHODS AND MATERIALS: A multiplex real-time NASBA with three molecular beacons (MR-NASBA) was developed for the simultaneous detection and quantification of HCMV-encoded immediate early-1 (IE1) and late pp67 mRNA. The assay was evaluated using RNA from in vitro HCMV-infected cells and sequential whole blood samples (100 microl) of HCMV infected lung transplant recipients. RESULTS: The MR-NASBA showed equal performance compared with standard NASBA assays (sensitivity of 1-3 x 10(3) RNA molecules in 100 microl blood and a linear range of 10(3)-10(6) RNA molecules). The standard IE1 Q-RNA provides a reliable internal system control. No interference was observed between the individual beacon signals. The simultaneous 'one-tube' quantification of IE1-RNA levels combined with qualitative detection of pp67-RNA is feasible without loss of assay performance in clinical whole blood specimens. CONCLUSION AND COMMENTS: MR-NASBA may be suitable for monitoring HCMV-activity in transplant recipients to aid in fine-tuning of antiviral intervention in high risk populations employing a traffic light diagnostic approach: no HCMV RNA signal (green light: safe) reflects absent or fully latent infection requiring no antiviral intervention; an 'IE1-RNA only' signal (yellow light: alert) indicates an emerging or subclinical active infection, opting for preemptive treatment in high risk populations; simultaneous 'IE1-RNA plus pp67-RNA' detection (red light:danger) indicates disseminating productive infection requiring immediate antiviral treatment.

Biomarkers↗
PubMed2005

Sensitive detection of deletions of one or more exons in the neurofibromatosis type 2 (NF2) gene by multiplexed gene dosage polymerase chain reaction.

Mutation detection in the neurofibromatosis type 2 (NF2) gene is challenging because when combining mutation detection methods such as single-strand conformational polymorphism and heteroduplex analysis, denaturing gradient gel electrophoresis, and direct sequencing of aberrant polymerase chain reaction (PCR) fragments only 30 to 60% of the constitutional mutations are detected. Because large deletions and complete chromosome rearrangements are also described methods such as microarray-comparative genomic hybridization and fluorescence in situ hybridization are also used. The one type of mutation often missed corresponds to deletions encompassing one or few exons. To detect this type we have developed a swift and reliable method. We perform a gene dosage analysis with two fluorescent multiplex PCR assays that amplify 15 of the 17 NF2 exons. The labeled PCR products are quantified and gene dose is calculated with respect to controls. We tested the reliability of this method with DNA from eight NF2 patients with known heterozygous NF2 deletions, eight controls and four unknown NF2 patients. In all of the patients with known heterozygous deletions we found in several exons a reduction of gene dosage to 50 to 69%. In one NF2 patient with previously unknown mutation and a severe phenotype we found the gene dosage of two exons reduced by 50% indicating a deletion of these two exons on one allele. This finding was validated by reverse transcriptase-PCR on fibroblast and schwannoma cell cultures of this patient and cDNA sequencing. Our gene dosage assay will detect deletions of one or more exons as well as gross deletions of the whole coding region of the gene. It can complement the existing screening methods because it is faster and easier.

DNA Mutational Analysis↗
PubMed2003

Detection of FLT3 internal tandem duplication and D835 mutations by a multiplex polymerase chain reaction and capillary electrophoresis assay.

FLT3 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells and plays an important role in stem cell survival and differentiation. Two different types of functionally important FLT3 mutations have been identified. Internal tandem duplication mutations arise from duplications of the juxtamembrane portion of the gene and result in constitutive activation of the FLT3 protein. This alteration has been identified in approximately 20% to 30% of patients with acute myelogenous leukemia and appears to be associated with a worse prognosis. The second type of FLT3 mutation, missense mutations at aspartic acid residue 835, occurs in approximately 7.0% of acute myelogenous leukemia cases. These mutations also appear to be activating and to portend a worse prognosis. Identification of FLT3 mutations is important because it provides prognostic information and may play a pivotal role in determining appropriate treatment options. We have developed an assay to identify both internal tandem duplication and D835 FLT3 mutations in a single multiplex polymerase chain reaction. After amplification, the polymerase chain reaction products are analyzed by capillary electrophoresis for length mutations and resistance to EcoRV digestion. Here we describe the performance characteristics of the assay, assay validation, and our clinical experience using this assay to analyze 147 clinical specimens.

Alleles↗