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PubMed2005

Rapid pKa estimation using vacuum-assisted multiplexed capillary electrophoresis (VAMCE) with ultraviolet detection.

A rapid approach for estimating the pK(a) value of small organic molecules was developed using vacuum-assisted multiplexed capillary electrophoresis (VAMCE) with ultraviolet detection. The VAMCE method employed a 96-capillary array, arranged in a standard 8 x 12 microtiter plate configuration, with each row of capillaries filled with 12 individual running buffers of equal ionic strength (I = 50 mM) covering a pH range from 2.2 to 10.7. A separate compound was injected hydrodynamically into each row of capillaries allowing the estimation of pK(a) values for eight compounds in a single run. The application of a vacuum during the separation generated a bulk fluid flow and allowed the electrophoretic separation to be completed within 5 min. The complete VAMCE method, conditioning, and electrophoretic separation was optimized to allow the pK(a) estimation for between 128 to 168 compounds in an 8-h period. The VAMCE method provided a reliable approach for estimating pK(a) values both within- and between-day. The pK(a) values for a series of 96 compounds estimated by VAMCE agreed well with some of literature pK(a) values with an average absolute difference of 0.22 log units.

Electrophoresis, Capillary↗
PubMed2003

Color multiplex polymerase chain reaction for quantitative analysis of epidermal growth factor receptor genes in colorectal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane cell surface receptor, which belongs to the c-erbB family of tyrosine kinases. Chimeric anti-EGFR monoclonal antibody is an investigational therapy for advanced adenocarcinoma of the colon. Anti-EGFR is believed to be most effective against those neoplasms with elevated EGFR levels. Possible mechanisms for over expression of EGFR include gene amplification, transcriptional upregulation, or decreased degredation of the EGFR protein. METHODS: We analyzed a series of 36 specimens of colonic adenocarcinoma with known levels of EGFR protein expression for amplification of the gene sequence of EGFR. Carcinomas were considered positive for EGFR expression when greater than 10% of the neoplastic cells stained at a level of 2+ or 3+. Foci of adenocarcinoma were microdissected from paraffin sections and quantitative real-time PCR (polymerase chain reaction) performed using a thermal cycler with real time fluorescence capability (Light cycler(TM), Roche Diagnostics, Indianapolis, IN). A relative quantitation assay comparing the EGFR gene to the control albumin gene was performed by 2-color multiplexing. RESULTS: Usable data on gene amplification status were obtained in 31 of the 36 samples. The average EGFR/albumin gene copy number ratio for the 31 samples of colon adenocarcinoma in which PCR results were obtained was 1.13 +/- 0.55 with a range of 0.26-2.29. The average EGFR/albumin gene copy number ratio obtained for 16 normal DNA leukocyte samples used to establish the efficiency curves was 1.03 +/- 0.31 with a range of 0.49-1.51. CONCLUSIONS: EGFR protein is overexpressed in a significant percentage of colonic adenocarcinomas. As with Her-2/neu overexpression, overexpression of EGFR serves as a basis for specific antibody therapy in a subset of carcinomas. Unlike Her-2/neu, where most overexpression is secondary to gene amplification, overexpression of EGFR appears to be unrelated to gene amplification.

Adenocarcinoma↗
PubMed1986

Quadrature-multiplexed rf excitation for information doubling in NMR imaging.

A quadrature multiplexed pulse sequence to double the data information in a given acquisition time is proposed for NMR tomography. This technique takes advantage of the Hermitian property of the Fourier transform operation in NMR imaging. The proposed technique uses two rf pulses with 90 degrees phase difference simultaneously applied for the excitation of the spins in two different slices. The method results in truly simultaneous data acquisition coming from two different anatomical slices which can be separated into the real and imaginary parts of the image domain. This technique, therefore, also allows us either to reduce the experiment time by half or double the number of slices in the same examination time, depending on the applied imaging scheme. As a preliminary study, simultaneous dual-slice imaging of a human head using a conventional single-slice imaging scheme was performed.

Fourier Analysis↗
PubMed2003

Mononeuropathy multiplex in association with livedoid vasculitis.

Livedoid vasculitis is a chronic dermatological disorder associated with petechiae and recurrent, unusually shaped ulcers that heal to form hyperpigmentated areas and atrophie blanche. This condition is more correctly termed a vasculopathy, rather than a vascultis, and is often associated with an underlying hypercoagulable disorder. We report a patient with livedoid vasculitis and mononeuropathy multiplex. We propose that peripheral nervous system involvement arises from multifocal areas of ischemia due to fibrin and thrombin deposition within both the wall and lumen of vasa nervorum.

Adult↗
PubMed1990

Mononeuropathy multiplex associated with acute hepatitis B infection.

Immunologic syndromes are associated with hepatitis B viral (HBV) infection. However, mononeuropathy multiplex (MM), a syndrome in which immune factors may play a role, is rare in acute HBV infection. Few cases of MM associated with HBV infection have been reported. We report a case of acute HBV infection in which MM involving the median, ulnar and lateral femoral cutaneous nerves complicated the course of the acute illness. Skin biopsy demonstrated vasculitic changes which may accompany the immunologic factors in HBV infection.

Acute Disease↗
PubMed1994

Paraneoplastic pseudo-obstruction, mononeuropathy multiplex, and sensory neuronopathy.

A patient with mononeuropathy multiplex, sensory neuronopathy, and diffuse intestinal pseudo-obstruction consisting of gastroparesis and impaired small and large bowel motility was found to have a small cell carcinoma of the lung. The constellation of findings were indicative of paraneoplastic neuropathy, which was confirmed with the appropriate antibody studies demonstrating antineuronal nuclear antibodies. Identification of paraneoplastic neuropathy is important, since early treatment of the primary cancer, which is virtually always a small cell carcinoma of the lung, may halt the progression of the disorder.

Aged↗
PubMed2005

Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation-dependent probe amplification (MLPA).

OBJECTIVE: To test whether multiplex ligation-dependent probe amplification (MLPA) can be used for the detection of aneuploidy of chromosomes 13, 18, 21, X, and Y in uncultured amniocytes. METHODS: We performed a prospective study based on 527 amniotic fluid samples. Chromosome copy numbers were determined by analysing the relative amount of PCR product of chromosome-specific MLPA probes. Results were available within 48 h and were compared with those of karyotyping. RESULTS: There were 517 conclusive MLPA tests. In 514 tests, results were concordant with those of karyotyping. There were two cases of 69,XXX triploidy that could not be detected by MLPA and there was one false-positive result. Here, MLPA indicated a 47,XXY fetus, whereas the karyotype was 46,XY. We correctly identified all 23 cases of autosomal trisomy and the single case of monosomy X in samples collected from 16 up to 36 weeks of gestation. In 10 cases (2%), the result was inconclusive owing to an insufficient amount of DNA. CONCLUSION: Sensitivity, specificity, and failure rate of MLPA were comparable to those of FISH and QF-PCR. Aneuploidy screening in uncultured amniocytes by MLPA is feasible in a clinical diagnostic setting, yielding an informative and rapid result in 98% of cases.

Amniocentesis↗
PubMed2001

Preimplantation genetic diagnostic protocols for alpha- and beta-thalassaemias using multiplex fluorescent PCR.

Haemoglobinopathies including alpha- and beta-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for beta-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for beta-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting beta-thalassaemia mutations (codon 41-42 and IVSI-110) and one for alpha-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of beta-globin and delta-globin was also found to be an important issue that necessitated careful design of primers for the beta-globin gene.

Adult↗
PubMedDate not supplied

Antenatal sonographic diagnosis of arthrogryposis multiplex congenita.

Arthrogryposis Multiplex Congenita (AMC) was suspected on ultrasound examination of a fetus at 30.5 weeks of gestation. The criteria for establishing this prenatal diagnosis as well as the importance of establishing the diagnosis at any gestational age are discussed. The diagnosis of AMC was confirmed at birth in this case.

Adult↗
PubMed1993

Early prenatal sonographic diagnosis of neuropathic arthrogryposis multiplex congenita with osseous heterotopia.

A prenatal diagnosis of arthrogryposis multiplex congenita (AMC) has been carried out on a 19-week-old fetus by means of echography. The ultrasonographic characteristics were unnatural position of the four limbs associated with articular anomalies together with absence of active fetal movements. A therapeutic interruption of pregnancy was performed and the diagnosis was confirmed. At autopsy, architectural disorder of the motor neurons of the anterior medullary horn revealed a neuropathic pathogenesis of the arthrogryposis. Moreover, at the lumbar level the spinal cord was progressively replaced by heterotopic bony tissue which caused a more severe deformity of the lower limbs compared with the upper. The aspects of anatomo-pathological, genetic, and differential diagnosis are discussed showing the precocity of the prenatal diagnosis and the peculiarity of the aetiology of our case.

Abortion, Therapeutic↗
PubMed2003

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.

Animals↗
PubMed2004

Photoaptamer arrays applied to multiplexed proteomic analysis.

Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.

Antibodies↗
PubMed2006

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho-specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.

Antibodies, Phospho-Specific↗
PubMed2006

Autoantigen arrays for multiplex analysis of antibody isotypes.

We describe here a microarray-based method for multiplexed, antigen-specific assessment of immunoglobulin (Ig) subclasses. We used 1152-feature arrays composed of 140 antigens or antigen fragments to detect isotype-specific mAb, to quantitatively monitor changes in isotype mAb concentration, and to profile antigen-specific antibody isotype production in a murine model of autoimmunity. This platform can be easily adapted to a variety of applications, and has the potential to elucidate mechanisms that govern development and evolution of antibody responses in in vivo and in vitro systems.

Animals↗
PubMed2003

Parallel high-throughput accurate mass measurement using a nine-channel multiplexed electrospray liquid chromatography ultraviolet time-of-flight mass spectrometry system.

A nine-channel multiplexed electrospray (MUX) liquid chromatography ultraviolet time-of-flight mass spectrometry (LC/UV/TOFMS) system has been used to simultaneously measure accurate masses of eluting components from eight parallel gradient LC columns. Accuracies better than 5 and 10 ppm were achieved for 50 and 80% of samples, respectively, from a single batch analysis of ten plates (960 samples) of a Fmoc-Asp(OtBu)-OH and reserpine mixture. Combinatorial library compounds were analyzed using this parallel high-throughput system in both positive and negative modes to rigorously verify expected products and identify side products. A mass accuracy of 10 ppm root mean square (RMS) is routinely obtained for combinatorial library samples from this high-throughput accurate mass LC/MS system followed by automated data processing. This mass accuracy is critical in revealing combinatorial synthesis problems that would be missed by unit mass measurement.

Alanine↗
PubMed1989

New theory of hippocampal function: associated rehearsal of multiplexed coded symbols.

A new theory of the role of the hippocampus in the selective storage of information in long-term memory is presented. This theory is based on the very recent discovery that neurons in the mammalian cerebral cortex transmit extremely precise copies of patterns of discharge in time when specific sensory inputs are presented, patterns that are interpreted to code for or symbolize specific items of information. The theory incorporates and provides an explanation for both the complex and unique internal structure of the hippocampal formation (the hippocampus and associated dentate gyrus) and the roles of many of the direct and indirect connections that structure makes with other brain structures. It also explains the deficits in learning that result from damage to the hippocampus and/or tracts that provide inputs to (or outputs from) this body as well as the role of the hippocampal formation in mapping the relationship of an individual to objects in its environment. The proposed enplanation is as follows. The hippocampal formation functions as a coordinated structure that, specifically, generates multiple copies of two different kinds of symbols (i.e., specific patterns of trains of nerve discharges in time). These two kinds of patterns are respectively provided by the entorhinal cortex through the perforant-alvear pathways and by the septal region, through the fornix, one of the two inputs to and the only output from the hippocampal formation. These two separate and different kinds of patterns are used to make multiplexed patterns that are ultimately transmitted to the cingulate gyrus and from there to other cortical memory storage locations. This transmission of amplified representations of different symbols occurs through the fornix. From there they are either transmitted to the mammilary bodies in the hypothalamus and from there to the anterior thalamic nuclei or, alternatively, directly to the anterior thalamic nuclei, bypassing the mammilary bodies. These thalamic nuclei in turn project the information to the cingulate gyrus of the cortex. The effect of the transmission of these mixtures of symbols is to cause the coordinated rehearsal and selective storage of relationships between separate inputs (specifically, patterns of discharge that symbolize different aspects of input) that are of probable significance to the survival of the system. The repeated presentation of such specific combinations of representations (symbols) then causes rehearsal-consolidation of these symbol associations as more permanent memories.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Multiplex PCR assay for Cylindrospermopsis raciborskii and cylindrospermopsin-producing cyanobacteria.

Water bodies are routinely monitored for the presence of potentially toxic cyanobacteria; however, the methodology for confirming toxicity is currently complex and expensive. Here we describe the application of gene-based technology to rapidly identify cylindrospermopsin-producing cyanobacteria, specifically, Cylindrospermopsis raciborskii. A multiplex polymerase chain reaction (PCR) test was developed that simultaneously identified polyketide synthase (pks) and peptide synthetase (ps) determinants associated with cylindrospermopsin production and distinguished C. raciborskii from other cylindrospermopsin-producing cyanobacteria of the species Anabaena bergii and Aphanizomenon ovalisporum, by targeting the rpoC1 gene. Twenty-one C. raciborskii, 5 A. bergii, 10 Aph. ovalisporum isolates and 3 environmental samples all yielded PCR results consistent with their toxicological status, as assessed by high-performance liquid chromatography coupled to mass spectrometry or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and C. raciborskii was always correctly identified. The PCR test is a rapid, reliable, and economical way of assessing the toxic potential of cyanobacterial blooms formed by these organisms.

Alkaloids↗
PubMed1993

Measurement of gene expression by multiplex competitive polymerase chain reaction.

We have developed a polymerase chain reaction (PCR)-based method to measure glutathione peroxidase (GSH-Px) mRNA levels. Expression was measured by multiplex competitive PCR amplification of (a) cDNA from GSH-Px and the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (b) two internal standards consisting of single-base mutants of GSH-Px and GAPDH cDNA that cause either a loss (GSH-Px) or a gain (GAPDH) of an EcoRI restriction endonuclease recognition site. RNA extracted from a human papillomavirus-immortalized human bronchial epithelial cell line (BEP2D) was reverse transcribed. Serial dilutions of cDNA were PCR amplified in the presence of GSH-Px and GAPDH primers and quantified amounts of mutated internal standards. The amplified DNA was restriction digested with EcoRI and electrophoresed on an agarose gel stained with ethidium bromide, separating native from mutated products. Densitometry was performed to quantitate the bands. Our studies demonstrate that this technique measures the relative expression of GSH-Px to GAPDH precisely and reproducibly for studies done with the same master mixture and dilution of internal standards. Ratios of relative gene expression varied less than 25% from the mean. This technique will be useful to measure changes in gene expression, particularly when the amount of study sample is limited or the level of gene expression is low.

Base Sequence↗