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Results for “multiplex imaging”

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At least 397 records · Page 22Linked to original sources

PubMed1994

[Cyclophosphamide-responsive subclinical Sjögren's syndrome in a patient with initial peripheral and central nervous system involvement].

A 36-year old woman was admitted because of painful dysesthesia of her extremities, suggesting the presence of mononeuritis multiplex. Laboratory data was almost within normal limits, with the exception of lupus anticoagulant positivity and increase of IgM level. We considered the possibility of connective tissue diseases and examined the patient accordingly. Keratoconjunctivitis sicca without dry eye symptoms, identified by rose-bengal and fluorescence testing, was the only recognizable abnormality. Oral sicca symptoms were not revealed although lip biopsy showed infiltration by a moderate number of plasma cells and lymphocytes. Under the diagnosis of subclinical Sjögren's syndrome, the following examination was carried out. Sural nerve biopsy specimens revealed wallerian degeneration and perivascular mononuclear cell infiltration of the vasa nervorum. We therefore concluded that the peripheral neuropathy was caused by subclinical Sjögren's syndrome. Magnetic resonance imaging (MRI) of the brain demonstrated multiple small lesions with increased spin echo images (T2 weighted) in the white matter. So, this patient was suffered from not only peripheral but also central nervous system complications. The mechanism of nervous system involvement was considered to be mononuclear cell-dependent ischemic damage caused by infiltration of the vasa nervorum. Both steroid pulse therapy and oral corticosteroid administration were ineffective in treatment of the peripheral neuropathy. Alternative use of cyclophosphamide (75 mg per day) was dramatically effective in relieving peripheral nervous system disorders. This was evident in the remarkable improvement of painful dysesthesia, grip strength and motor nerve conduction velocities. This case could be considered valuable for understanding the pathophysiology of Sjögren's syndrome and associated nervous system complications.

Adult↗

PubMed2005

Odor maps in the olfactory cortex.

In the olfactory system, environmental chemicals are deconstructed into neural signals and then reconstructed to form odor perceptions. Much has been learned about odor coding in the olfactory epithelium and bulb, but little is known about how odors are subsequently encoded in the cortex to yield diverse perceptions. Here, we report that the representation of odors by fixed glomeruli in the olfactory bulb is transformed in the cortex into highly distributed and multiplexed odor maps. In the mouse olfactory cortex, individual odorants are represented by subsets of sparsely distributed neurons. Different odorants elicit distinct, but partially overlapping, patterns that are strikingly similar among individuals. With increases in odorant concentration, the representations expand spatially and include additional cortical neurons. Structurally related odorants have highly related representations, suggesting an underlying logic to the mapping of odor identities in the cortex.

Animals↗

PubMed1996

Idiopathic hypereosinophilic syndrome revealed by central nervous system dysfunction.

The idiopathic hypereosinophilic syndrome (HES) is a rare disease, characterised by persistent eosinophilia (> 1500/mm3), without underlying cause, provoking multiple organ system injury. Morbidity and mortality are mostly associated with the HES cardiopathy. Neurological signs are also frequent. Neurological dysfunction can be central (encephalopathy, organic psycho-syndrome) and peripheral (polyneuropathy, mononeuropathia multiplex, autonomic neuropathy, polymyositis). The encephalopathy is not always caused by distant thrombo-embolic events originating from the HES cardiopathy. We describe a patient with idiopathic HES central nervous system dysfunction, in the absence of cardiopathy. Furthermore we briefly discuss pathophysiological aspects, treatment modalities and the prognosis of the HES, in relation to our patient.

Anti-Inflammatory Agents↗

PubMed2001

Motion direction, speed and orientation in binocular matching.

The spatial differences between the images seen by the two eyes, called binocular disparities, can be used to recover the volumetric (three-dimensional) aspects of a scene. The computation of disparity depends upon the correct identification of corresponding features in the two images. Understanding what image features are used by the brain to solve this matching problem is one of the main issues in stereoscopic vision. Many cortical neurons in visual areas V1 (ref. 2), MT (refs 3, 4) and MST (refs 5, 6) that are tuned to binocular disparity are also tuned to orientation, motion direction and speed. Although psychophysical work has shown that motion direction can facilitate binocular matching, the psychophysical literature on the role of orientation is mixed, and it has been argued that speed differences are ineffective in aiding correspondence. Here we use a different psychophysical paradigm to show that the visual system uses similarities in orientation, motion direction and speed to achieve binocular correspondence. These results indicate that cells that multiplex orientation, motion direction, speed and binocular disparity may help to solve the binocular matching problem.

Depth Perception↗

PubMed2026

Cumulative microscopy reveals cellular states in fibroblasts from patients with genetic disorders.

Analysis of cellular states and signaling trajectories can provide insights into causes of disease. We developed cumulative microscopy, a method to perform cyclical imaging without elution or quenching steps. Cumulative microscopy computationally extracts individual signals from accumulating fluorescence during sequential imaging. We use cumulative microscopy to quantitatively assess cell cycle and stress markers in individual primary fibroblasts from patients with rare genetic proliferative disorders with increased cancer risk. Neural network-based analysis of cumulative microscopy data suggests that cells from patients with Cartilage-hair hypoplasia (CHH), but not Mulibrey Nanism (MUL), show replication stress. We analyze cell states and cell trajectories and find that a subset of cells from patients with CHH show spontaneous replication stress, followed by cell cycle exit in both G1 and G2 phases. We note that replication stress potentially could underlie both proliferative defects and increased cancer risk in CHH patients and conclude that cumulative microscopy is an efficient, quantitative, and generalizable approach to multiplex microscopy.

Humans↗

PubMed1999

A highly accurate, low cost test for BRCA1 mutations.

The hereditary breast and ovarian cancer syndrome is associated with a high frequency of BRCA1 mutations. However, the widespread use of BRCA1 testing has been limited to date by three principal concerns: the fear of loss of health and life insurance, the uncertain clinical value of a positive test result, and the current lack of an inexpensive and sensitive screening test for BRCA1 mutations. We have developed an inexpensive system for gene mutational scanning, based on a combination of extensive multiplex PCR amplification and two dimensional electrophoresis. The efficiency of this system, as a screening test for BRCA1 mutations, was evaluated in a panel of 60 samples from high risk women, 14 of which contained a previously identified mutation in BRCA1. All 14 mutations were identified, as well as an additional five that had previously escaped detection. In addition to the 19 mutations, a total of 15 different polymorphic variants were scored, most of which were recurring. All were confirmed by nucleotide sequencing. The cost of screening per sample was calculated to be approximately US$70 for the manual technique used in this study, and may be reduced to approximately US$10 with the introduction of commercially available PCR robotics and fluorescent imaging. Implementation of this method of mutation screening in the research and clinical setting should permit rapid accrual of quantitative data on genotype-phenotype associations for the evaluation of diagnostic testing.

Breast Neoplasms↗

PubMed1988

Multiplex DNA sequencing.

The increasing demand for DNA sequences can be met by replacement of each DNA sample in a device with a mixture of N samples so that the normal throughput is increased by a factor of N. Such a method is described. In order to separate the sequence information at the end of the processing, the DNA molecules of interest are ligated to a set of oligonucleotide "tags" at the beginning. The tagged DNA molecules are pooled, amplified, and chemically fragmented in 96-well plates. The resulting reaction products are fractionated by size on sequencing gels and transferred to nylon membranes. These membranes are then probed as many times as there are types of tags in the original pools, producing, in each cycle of probing, autoradiographs similar to those from standard DNA sequencing methods. Thus, each reaction and gel yields a quantity of data equivalent to that obtained from conventional reactions and gels multiplied by the number of probes used. To date, even after 50 successive probings, the original signal strength and the image quality are retained, an indication that the upper limit for the number of reprobings may be considerably higher.

Automation↗

PubMed2001

Array-based ELISAs for high-throughput analysis of human cytokines.

In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.

Anti-Inflammatory Agents↗

PubMed1998

An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH).

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromosomes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.

Chromosome Aberrations↗

PubMed2026

Multiplexed RT-LAMP Assays in Handheld Devices for In-Situ Detection of Chikungunya, Dengue, Mayaro, and Zika Viruses.

Mosquito-borne viruses pose a significant global health challenge, particularly in resource-limited settings where multiple viruses often cause illnesses with similar symptoms that require different treatment. We introduce the first 7-plex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in a hand-held device capable of detecting the presence of Chikungunya virus (CHIKV), dengue virus serotypes (DENV 1-4), Mayaro virus (MAYV), and Zika virus (ZIKV) in a single test. If the result is positive from the single-plex device for the 7-plex assay, 3-plex and 4-plex devices are then used to identify the exact virus within a specimen. In-situ detection is achieved by integrating valve-enabled, paper-based sample preparation with fluorescence detection using a blue LED flashlight as a light source and a yellow plastic film as a filter, allowing visual discrimination between positive and negative samples by the naked eye or by recording images using a smart phone. The detection limits ranged from 2 genome equivalents (GE)/reaction (for ZIKV) to 92 GE/reaction (for DENV-3) across 7 types of viruses when 1 μL of viral RNA was used. We observed 90% overall agreement between the point-of-care (POC) device and lab-based reverse transcription polymerase chain reactions (RT-PCR) when blinded clinical specimens were tested. This assay and device have a potential to address critical surveillance gaps in endemic regions, enabling timely detection of multiple mosquito-borne viruses to guide appropriate clinical management and public health countermeasures in settings where laboratory resources are scarce.

Nucleic Acid Amplification Techniques↗

PubMed1993

A novel candidate autoantigen in a multiplex family with multiple sclerosis: prevalence of T-lymphocytes specific for an MBP epitope unique to myelination.

Although the major isoform of myelin basic protein (MBP) in the healthy adult CNS is the 18.5-kDa protein, other isoforms containing exon 2 encoded protein (21.5 kDa and 20.2 kDa) exist and are expressed primarily during myelin formation. Since remyelination is a prominent feature in MS lesions, we examined the frequencies of T cell lines (TCLs) specific for epitopes within exon 2 encoded MBP (X2MBP), and also within 18.5-kDa MBP, in members of a multiplex family with MS. TCLs specific for X2MBP were as prevalent as TCLs specific for immunodominant epitopes within 18.5-kDa MBP. In addition, while frequencies of TCLs specific for 18.5-kDa MBP were no different between the affected and unaffected, the frequency of X2MBP-specific TCLs correlated with disease.

Aged↗

PubMed2003

Peptidomics: A new approach to affinity protein microarrays.

Protein microarrays for diagnostic and proteomic analyses are being developed using a number of different techniques for each of the steps required including immobilisation methods, assay and detection systems. This is extremely different to the development of DNA microarrays which is now a well established technology that has demonstrated the capabilities of transcriptomics to deliver validated differential transcripts. As mRNA and protein levels do not always correlate, protein microarrays would seem to be an obvious successor to DNA arrays. Unlike nucleic acids, however, protein targets are typically nonhomogeneous in physicochemical properties and affinity capture agents are often poorly characterised making the experiments difficult to perfect and reproduce. Moreover, running multiple affinity assays in parallel (multiplexing) is compromised by the heterogeneity of antibody affinities to their protein targets. In the peptidomic approach presented here the assayed mixture of proteins is enzymatically digested prior to affinity capture to form a mixture of short peptides that are more similar in their physicochemical properties than intact proteins. These peptides can be predicted by in silico digestion of individual proteins, e.g. from protein databases allowing design of nonhomologous reagents for the screening of affinity agent libraries. The use of mass spectrometry (e.g. matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for a direct confirmation of the identity of the species captured, provides a further advantage compared to the more usual method of detection in which fluorescently labelled captured species are scanned to give a spatially resolved image of the array.

Peptides↗

PubMed2005

A new genetic test for the rapid identification of shiga-toxines producing (STEC), enteropathogenic (EPEC) E. coli isolates from children.

Routine bacteriological techniques do not allow detection of the most frequent enteric pathogens in young children: enteropathogenic Escherichia coli (EPEC) and shigatoxinogenic E. coli (STEC/EHEC). Since there is no correlation between serotype and pathotype, a genotypic determination is therefore necessary for the identification of these pathogenic strains. We evaluated the Genotype EHEC test (Hain Life Science, Germany), a new rapid system based on DNA multiplex amplification and further hybridization for the detection of shigatoxin stx1, stx2 genes, intimin eae gene and invasin ipaH gene harbored by Shigella and enteroinvasive E. coli (EIEC). E. coli strains of various serogroups isolated from children with acute gastroenteritis, hemorrhagic colitis or hemolytic-uremic syndrome were tested. Their genotypes were first determined by standard in-house PCR. The strains collection included 11 STEC/EHEC (serogroups O157, O111, O26, O91, O-untypable) and nine EPEC (serogroups O26, O157, O55, O126, O127, O-untypable). The same strains were tested with Genotype EHEC. For all the strains, the hybridization banding pattern obtained by Genotype EHEC correlated with their expected genotypic characteristics. No specific equipment is required, except a thermocycler. Absence of electrophoresis system, of ethidium bromide staining and imaging system is a clear-cut advantage of Genotype EHEC. In addition, the short testing time (less than 2 h) optimizes treatment orientation. The Genotype EHEC test allows an easy and reliable identification of EHEC, STEC, EPEC and also EIEC. As such, it is a useful tool for the rapid diagnosis of diarrheal diseases.

Child↗

PubMed2007

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.

Animals↗

PubMed2007

Use of the CellCard System for analyzing multiple cell types in parallel.

The CellCard system enables the analysis of multiple cell types within a single microtiter well. In doing so, the CellCard system not only determines the effect of an experimental condition on a cell type of interest, but also the relative selectivity of that response across nine other cell types. In addition, this approach of cellular multiplexing is a means of miniaturization without the necessity of microfluidic devices. The standard 96-well plate generates ten 96-well plates of data (or, the equivalent of a 960-well plate). Taken together, the CellCard technology enables multiple cell types to be assayed within a single microtiter well allowing for the simultaneous determination of cellular activity and compound selectivity. This chapter will describe a method by which multiple cell types can be simultaneously assayed for biological parameters of interest.

Animals↗

PubMed2004

[A novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology].

Multiplex amplifiable probe hybridization (MAPH) has recently been developed to detect gene copy number changes in total genome in several genetic disease. Here we reported a novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology for DNA fragment copy number measurement. A set of amplifiable probes were prepared by locus-specific forward and reverse primers synthesized with the T7 and T3 promoter sites at their respective 5'-ends to the interest DNA fragments and purified by Qiagen PCR products purification kit. The set of probes were then hybridized with genome DNA immobilized on the nylon membrane. The selective probes after MAPH were collected with streptavidin coated magnetic beads and amplified by a biotin labeled universal primers. The biotinylated PCR products were then intended for hybridization to the corresponding oligonucleotide arrays consisted of 10 exons of DMD gene probes and positive,negative control probes immobilized on glass slides. After hybridization, the slides were washed by hybridization buffer and stained by streptavidin-Cy3. The hybridization fluorescence images were scanned by the ScanArray software and further quantitatively analyzed by the ImageJ software. Here we used oligonucleotide array technology to replace the agarose gel analysis for detecting the biotinylated PCR products so that extendable ability of parallel assay in our method had greatly been improved. One control female, one control male and one DMD patient were tested in this study. The results demonstrate the feasibility of high-throughput detection and relative quantification of DNA fragment copy numbers changes in total genome DNA combination of MAPH and high-density oligonucleotide arrays in single reaction.

Gene Dosage↗

PubMed2002

A comparison of techniques to optimize measurement of voltage changes in electrical impedance tomography by minimizing phase shift errors.

In electrical impedance tomography, errors due to stray capacitance may be reduced by optimization of the reference phase of the demodulator. Two possible methods, maximization of the demodulator output and minimization of reciprocity error have been assessed, applied to each electrode combination individually, or to all combinations as a whole. Using an EIT system with a single impedance measuring circuit and multiplexer to address the 16 electrodes, the methods were tested on resistor-capacitor networks, saline-filled tanks and humans during variation of the saline concentration of a constant fluid volume in the stomach. Optimization of each channel individually gave less error, particularly on humans, and maximization of the output of the demodulator was more robust. This method is, therefore, recommended to optimize systems and reduce systematic errors with similar EIT systems.

Artifacts↗

PubMed2006

Urothelial cancer biomarkers for detection and surveillance.

Cancer is a complex process, and the US cancer-specific death rate has not changed in the last 50 years. Cure of the disease usually results from early diagnosis and treatment. Urothelial carcinoma (UC) has the highest recurrence rate of any cancer and is the second most common cancer of the genitourinary tract. It usually does not present at a metastatic stage, but only 50% of patients treated with cystectomy survive > or =5 years. There is a UC surveillance protocol, which includes cystoscopy, imaging, and cytology, to detect progression and allow early treatment of life-threatening UC. Many patients may not complete the surveillance protocol, and the cost of these studies is increasing. In addition, questions about the efficacy of these modalities have been raised. Therefore, bladder urinary tumor markers have been developed to aid in the diagnosis and surveillance of UC. Because urothelial cells are bathed in the urine and are continually shed, UC presents a unique opportunity to monitor bladder neoplasia in a noninvasive fashion. Currently, there are many research bladder tumor markers, but only a few are commercially available US Food and Drug Administration (FDA)-approved products. The commercially available bladder tumor markers and potential future markers are discussed. The ideal urinary bladder tumor test is still unavailable, but the eventual "gold standard" will consist of multiplex assays that analyze nucleic acids and proteins for detection. In addition, these tests would also reveal to the clinician both prognostic information and therapeutic targets for personalized medical treatment.

Biomarkers, Tumor↗