SEARCH · PubMed Health

Results for “single platform”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources

PubMed2006

A combined HIV-1 protein bead array for serology assay and T-cell subset immunophenotyping with a hybrid flow cytometer: a step in the direction of a comprehensive multitasking instrument platform for infectious disease diagnosis and monitoring.

BACKGROUND: A new generation of bench-top flow cytometers with digital signal processing to perform suspension array technology (SAT) based bead array assays as well as leukocyte immunophenotyping is now available. These hybrid instruments provide an opportunity for the development of a more cost effective multitasking platform to support infectious disease treatment in resource limited countries. METHODS: We report the development and testing of two modules compatible with the hybrid flow cytometers. The first module is an eleven HIV-1 protein bead array (PBA) for the detection of circulating antibodies and the second is a cell based T-cell enumeration assay. RESULTS: The HIV-1 PBA was tested in parallel with two enzyme immunoassays (EIAs) for the detection of plasma antibodies from 4 HIV-1 seroconversion panels and a low antibody titer panel. The PBA as well as the two EIAs performed equally for the detection of antibody positive samples from all seroconversion panels. One antibody positive sample from the low antibody titer panel was missed by the PBA together with one of the two EIAs tested. A parallel analysis of the HIV-1 PBA with Western blot (a confirmatory test for HIV infection) using plasma from nine HIV-1(+) individuals showed that the HIV-1 PBA detected more of the gp41 and gp120 antibody positive samples. Preliminary CD4 T-cell immunophenotyping results from 14 HIV(+) and 10 HIV(-) whole blood specimens with the hybrid flow cytometer platform compared well to conventional flow cytometry data. CONCLUSION: The successful combination of bead and cell based assays on a single hybrid instrument demonstrated the potential utility of a multitasking platform. The results presented are providing groundwork for future development of more cost effective modular architecture for a flexible flow cytometry based platform.

AIDS Serodiagnosis↗

PubMed2002

Base pair mismatch recognition using plasmon resonant particle labels.

We demonstrate the use of silver plasmon resonant particles (PRPs), as reporter labels, in a microarray-based DNA hybridization assay in which we screen for a known polymorphic site in the breast cancer gene BRCA1. PRPs (40-100 nm in diameter) image as diffraction-limited points of colored light in a standard microscope equipped with dark-field illumination, and can be individually identified and discriminated against background scatter. Rather than overall intensity, the number of PRPs counted in a CCD image by a software algorithm serves as the signal in these assays. In a typical PRP hybridization assay, we achieve a detection sensitivity that is approximately 60 x greater than that achieved by using fluorescent labels. We conclude that single particle counting is robust, generally applicable to a wide variety of assay platforms, and can be integrated into low-cost and quantitative detection systems for single nucleotide polymorphism analysis.

Base Pair Mismatch↗

PubMed2003

Balance in single-limb stance in healthy subjects--reliability of testing procedure and the effect of short-duration sub-maximal cycling.

BACKGROUND: To assess balance in single-limb stance, center of pressure movements can be registered by stabilometry with force platforms. This can be used for evaluation of injuries to the lower extremities. It is important to ensure that the assessment tools we use in the clinical setting and in research have minimal measurement error. Previous studies have shown that the ability to maintain standing balance is decreased by fatiguing exercise. There is, however, a need for further studies regarding possible effects of general exercise on balance in single-limb stance. The aims of this study were: 1) to assess the test-retest reliability of balance variables measured in single-limb stance on a force platform, and 2) to study the effect of exercise on balance in single-limb stance, in healthy subjects. METHODS: Forty-two individuals were examined for test-retest reliability, and 24 individuals were tested before (pre-exercise) and after (post-exercise) short-duration, sub-maximal cycling. Amplitude and average speed of center of pressure movements were registered in the frontal and sagittal planes. Mean difference between test and retest with 95% confidence interval, the intraclass correlation coefficient, and the Bland and Altman graphs with limits of agreement, were used as statistical methods for assessing test-retest reliability. The paired t-test was used for comparisons between pre- and post-exercise measurements. RESULTS: No difference was found between test and retest. The intraclass correlation coefficients ranged from 0.79 to 0.95 in all stabilometric variables except one. The limits of agreement revealed that small changes in an individual's performance cannot be detected. Higher values were found after cycling in three of the eight stabilometric variables. CONCLUSIONS: The absence of systematic variation and the high ICC values, indicate that the test is reliable for distinguishing among groups of subjects. However, relatively large differences in an individual's balance performance would be required to confidently state that a change is real. The higher values found after cycling, indicate compensatory mechanisms intended to maintain balance, or a decreased ability to maintain balance. It is recommended that average speed and DEV 10; the variables showing the best reliability and effects of exercise, be used in future studies.

Adult↗

PubMed2004

Transfected cell microarrays for the expression of membrane-displayed single-chain antibodies.

The expression of recombinant antibody fragments on the surface of mammalian cells has recently emerged as a therapeutic strategy, particularly in the treatment of a number of cancers. Screening technologies that allow for the facile characterization of fragments expressed on the cell surface would hasten the identification and isolation of reagents to be used as therapeutics. In this report, we describe a cellular microarray-based platform for the comparative functional analysis of single-chain antibodies (scFvs) expressed on the plasma membrane of mammalian cells. Using the anti-fluorescein monoclonal antibody 4-4-20 as a model system, the native binding site and three mutants were expressed as scFvs on the membrane of HEK 293T/17 cells in a microarray format. Collectively, the equilibrium dissociation constants of the soluble forms of the wild-type scFv and the three mutants spanned nearly 3 orders of magnitude. Expression of the scFvs on the surface of mammalian cells was achieved by the deposition of plasmid DNAs in micrometer-sized spots onto the surface of a glass microscope slide. The addition of cells to the printed array resulted in the expression of the scFvs in clusters of cells in spatially discrete locations. Ligand binding assays performed with a fluorescein-bovine serum albumin conjugate demonstrated the ability of the transfected cell microarray to differentiate the relative binding affinities of the expressed scFvs. Further, the apparent affinities of the membrane-displayed scFvs were within 10-fold of those reported for the soluble forms of the scFvs. The assays described herein demonstrate the potential for cellular microarrays to be used for the high-throughput screening of potential therapeutic reagents. More generally, our work details the utility of transfected cell microarrays in mediating the functional characterization of expressed membrane receptor proteins.

Antibodies↗

PubMed1988

A study of section wrinkling on single-hole coated grids using TEM and SEM.

To prevent section wrinkles usually encountered with the use of coated single-hole grids, a simple method was developed. Formvar film resting on a platform with holes (3.5 mm diameter) was heated with a slide warmer (60-65 degrees C). The bottom of a glass petri dish was inverted over the platform to keep the ambient air at the desired temperature. Sections were picked up from the boat of the diamond knife with a single-hole grid and deposited at the orifice of the platform and allowed to dry. The grids were then carefully pushed through the orifice of the platform with the blunt head of a nail (3 mm diameter).

Animals↗

PubMed2005

Platform influence on DNA microarray data in postmortem brain research.

In addition to the substantial biological diversity among humans, our limited ability to reliably measure expression changes of small magnitude significantly reduces our capacity to obtain convergent sets of transcriptome data in postmortem brain. In particular, differences in the structure and sensitivity/reproducibility of microarray platforms, and in the variety of tools used to analyze microarray data, strongly influence experimental outcome. In order to better understand the sensitivity, dynamic range, and reproducibility of three common DNA microarray platforms, we compared two human postmortem samples on cDNA microarrays with dual-fluorescence, oligonucleotide GeneChips (Affymetrix), and single-color gel matrix deposited CodeLink oligonucleotide arrays. All three microarray platforms reported a good dynamic range and high correlation in replicate experiments, but they failed to consistently identify the same genes as differentially expressed between the same samples. Given their reproducibility and proven accuracy, different microarray platforms appear to be measuring different things by nature of their design and function. This needs to be taken into account when comparing data across studies.

Brain↗

PubMed2004

Refining the results of a whole-genome screen based on 4666 microsatellite markers for defining predisposition factors for multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a complex genetic background. In order to identify loci associated with the disease, we had performed a genome screen initially using 6000 microsatellite markers in pooled DNA samples of 198 MS patients and 198 controls. Here, we report on the detailed reanalysis of this set of data. Distinctive features of microsatellites genotyped in pooled DNA causing false-positive association or masking existing association were met by improved evaluation and refined correction factors in the statistical analyses. In order to assess potential errors introduced by DNA pooling and genotyping, we resurveyed the experiment in a subset of microsatellite markers using de novo-composed DNA pools. True MS associations of markers were verified via genotyping all individual DNA samples comprised in the pools. Microsatellites share characteristically superb information content but they do not lend themselves to automation in very large scale formats. Especially after DNA pooling many artifacts of individual marker systems require special attention and treatment. Therefore, in the near future comprehensive whole-genome screens may rather be performed by typing single nucleotide polymorphisms on chip-based platforms.

DNA Primers↗

PubMed2026

Simultaneous quantitative detection of multiple low-frequency variants by high-dynamic-range capillary electrophoresis.

Sensitive and quantitative detection of low-frequency variants across multiple loci is critical for nucleic acid-based diagnostics, yet clinical implementation requires a balance among sensitivity, multiplexing capacity, cost, and operational simplicity. We previously developed a high-dynamic-range capillary electrophoresis system capable of detecting variants at allele frequencies below 1%; however, its application was limited to single-locus analysis. Here, we expanded this platform to multiplex detection by incorporating mobility-shift strategies into the assay design. This approach enabled simultaneous analysis of 15 hotspot variants across three clinically relevant loci: KRAS codons 12 and 13 and GNAS codon 201. Validation using synthetic oligonucleotides, formalin-fixed paraffin-embedded tissue, and liquid specimens demonstrated high quantitative accuracy over clinically relevant variant allele frequency ranges, with measured values closely matching expected values (R2 > 0.97). The assay showed high concordance with targeted amplicon sequencing and digital polymerase chain reaction for all variants at variant allele frequencies ≥1%, while also detecting selected variants below this threshold. Collectively, these results establish a multiplexed high-dynamic-range capillary electrophoresis assay for simultaneous, quantitative detection of low-frequency variants, offering a scalable and cost-effective approach for disease-focused gene panels in clinical laboratory settings.

HiDy↗

PubMed2006

Oncolytic HSV armed with platelet factor 4, an antiangiogenic agent, shows enhanced efficacy.

Oncolytic herpes simplex viruses (HSV) have emerged as a promising platform for cancer therapy. However, efficacy as single agents has thus far been unsatisfactory. Tumor vasculature is critical in supporting tumor growth, but successful antiangiogenic approaches often require maintaining constant levels of antiangiogenic products. We hypothesized that oncolytic HSV has the potential to destroy tumor vasculature and that this effect can be enhanced by combination with antiangiogenic gene transfer. We examined the strategy of arming oncolytic HSV with an antiangiogenic transgene, platelet factor 4 (PF4). The PF4 transgene was inserted into oncolytic HSV G47Delta utilizing a bacterial artificial chromosome construction system. Whereas bG47Delta-empty showed robust cell killing and migration inhibition of proliferating endothelial cells (HUVEC and Py-4-1), the effect was further enhanced by PF4 expression. Importantly, enhanced potency did not impede viral replication. In vivo, bG47Delta-PF4 was more efficacious than its nonexpressing parent bG47Delta-empty at inhibiting tumor growth and angiogenesis in both human U87 glioma and mouse 37-3-18-4 malignant peripheral nerve sheath tumor models. Enhancing the antiangiogenic properties of oncolytic HSV through the expression of antiangiogenic factors such as PF4 is a powerful new strategy that targets both the tumor cells and tumor vasculature.

Angiogenesis Inhibitors↗

PubMed2003

Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system.

Antagonists to the human metabotropic glutamate receptor subtype 5a(mGluR(5a)) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson's disease. To discover novel antagonists to the mGluR(5a), a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.

Calcium↗

PubMed2001

Analysis of cellular adhesion by microarray expression profiling.

Microarrays of oligonucleotides or cDNAs can be used to establish the expression profiles of numerous genes in a single experiment. We have established a microarray platform to identify genes in a number of different pathological conditions, particularly those with an inflammation component. This platform utilised the output of an eosinophil sequencing project in which 1069 sequences were identified that were not represented in the public domain. An eosinophil model cell line, AML14.3D10, was used to investigate cell adhesion. The transcription profile of adhered and non-adhered AML 14.3D10 cells was shown to be both technically and biologically reproducible. A number of genes were found differentially expressed in the adhered vs. non-adhered populations. In the adhered population, the expression of these genes was restricted compared to brain, lung, kidney and especially bone marrow. However, the differentially regulated genes were not among those genes most restricted to eosinophils. We discuss the implications of transcription profiling on gene annotation and its potential utility for the identification of targets for drug intervention.

Base Sequence↗

PubMed2025

Attomolar Detection of HIV-1 with Label-Free RCA-rCRISPR on Smartphone.

Human Immunodeficiency Virus-1 (HIV) remains a major global public health challenge, having led to over 42.3 million deaths since its discovery in the early 1980s. Despite progress in prevention and treatment, around 60% of people with HIV (PWH) remain undiagnosed in resource-limited regions, disproportionately affecting vulnerable populations and underserved communities across the world. This illustrates the critical need for accessible, accurate, and equipment-free diagnostic tools to enhance detection and thus provide opportunities to curb its spread. Here, we developed a low-cost, robust, and label-free rolling circle amplification (RCA)-rCRISPR diagnostic platform for detecting HIV viral load with minimal instrumentation. Our strategy, combining the integration of RNA-detecting RCA reaction with plasmid reporter-based ratiometric CRISPR (rCRISPR), enables sensitive detection of unprocessed RNA targets without the need for intensive sample pre-treatment. This label-free RCA-rCRISPR diagnostic platform detected HIV RNA down to single-digit aM sensitivity (~3000 copies/mL) from PWH-derived HIV samples ex vivo. Unlike typical RCA, which requires sample fragmentations to break long RNA target sequences, our design harnesses the triple functions of the phi29 DNA polymerase (namely exonuclease activity, polymerization, and strand displacement), enabling the detection of the entire HIV genome without pre-fragmentation. For point-of-care (POC) applications, we constructed an all-in-one smartphone-based minigel electrophoresis device to facilitate equipment-free HIV viral load testing, making it accessible to resource-limited communities. Additionally, the assay has demonstrated the ability for point mutation detection (BRAF mutation in canine urothelial carcinoma), showcasing the robustness of our strategy for broad disease diagnostic applications.

HIV↗

PubMed2005

"Lineage addiction" in human cancer: lessons from integrated genomics.

Genome-era advances in the field of oncology endorse the notion that many tumors may prove vulnerable to targeted therapeutic avenues once their salient molecular alterations are elucidated. Accomplishing this requires both detailed genomic characterization and the ability to identify in situ the critical dependencies operant within individual tumors. To this end, DNA microarray platforms such as high-density single-nucleotide polymorphism (SNP) arrays enable large-scale cancer genome characterization, including copy number and loss-of-heterozygosity analyses at high resolution. Clustering analyses of SNP array data from a large collection of tumor samples and cell lines suggest that certain copy number alterations correlate strongly with the tissue of origin. Such lineage-restricted alterations may harbor novel cancer genes directing genesis or progression of tumors from distinct tissue types. We have explored this notion through combined analysis of genome-scale data sets from the NCI60 cancer cell line collection. Here, several melanoma cell lines clustered on the basis of increased dosage at a region of chromosome 3p containing the master melanocyte regulator MITF. Combined analysis of gene expression data and additional functional studies established MITF as an amplified oncogene in melanoma. MITF may therefore represent a nodal point within a critical lineage survival pathway operant in a subset of melanomas. These findings suggest that, like oncogene addiction, "lineage addiction" may represent a fundamental tumor survival mechanism with important therapeutic implications.

Chromosomes, Human, Pair 3↗

PubMed1997

Interaction of leg stiffness and surfaces stiffness during human hopping.

When mammals run, the overall musculoskeletal system behaves as a single linear "leg spring". We used force platform and kinematic measurements to determine whether leg spring stiffness (k(leg)) is adjusted to accommodate changes in surface stiffness (ksurf) when humans hoop in place, a good experimental model for examining adjustments to k(leg) in bouncing gaits. We found that k(leg) was greatly increased to accommodate surfaces of lower stiffnesses. The series combination of k(leg) and ksurf [total stiffness (ktot)] was independent of ksurf at a given hopping frequency. For example, when humans hopped at a frequency of 2 Hz, they tripled their k(leg) on the least stiff surface (ksurf = 26.1 kN/m; k(leg) = 53.3 kN/m) compared with the most stiff surface (ksurf = 35,000 kN/m; k(leg) = 17.8 kN/m). Values for ktot were not significantly different on the least stiff surface (16.7 kN/m) and the most stiff surface (17.8 kN/m). Because of the k(leg) adjustment, many aspects of the hopping mechanics (e.g., ground-contact time and center of mass vertical displacement) remained remarkably similar despite a > 1,000-fold change in ksurf. This study provides insight into how k(leg) adjustments can allow similar locomotion mechanics on the variety of terrains encountered by runners in the natural world.

Aged↗

PubMed2004

High-resolution global profiling of genomic alterations with long oligonucleotide microarray.

Cancer represents the phenotypic end point of multiple genetic lesions that endow cells with a full range of biological properties required for tumorigenesis. Among the hallmark features of the cancer genome are recurrent regional gains and losses that, upon detailed characterization, have provided highly productive discovery paths for new oncogenes and tumor suppressor genes. In this study, we describe the use of an oligonucleotide-based microarray platform and development of requisite assay conditions and bioinformatic mining tools that permits high-resolution genome-wide array-comparative genome hybridization profiling of human and mouse tumors. Using a commercially available 60-mer oligonucleotide microarray, we demonstrate that this platform provides sufficient sensitivity to detect single-copy difference in gene dosage of full complexity genomic DNA while offering high resolution. The commercial availability of the microarrays and associated reagents, along with the technical protocols and analytical tools described in this report, should provide investigators with the immediate capacity to perform DNA analysis of normal and diseased genomes in a global and detailed manner.

Animals↗

PubMed2006

Dominant-negative fibroblast growth factor receptor expression enhances antitumoral potency of oncolytic herpes simplex virus in neural tumors.

PURPOSE: Oncolytic herpes simplex viruses (HSV) appear to be a promising platform for cancer therapy. However, efficacy as single agents has thus far been unsatisfactory. Fibroblast growth factor (FGF) signaling is important for the growth and migration of endothelial and tumor cells. Here, we examine the strategy of arming oncolytic HSV with a dominant-negative FGF receptor (dnFGFR) that targets the FGF signaling pathway. EXPERIMENTAL DESIGN: A mouse Nf1:p53 malignant peripheral nerve sheath tumor (MPNST) cell line expressing dnFGFR was generated by transfection. The effects of dnFGFR expression on cell growth and migration in vitro and tumor formation in vivo were determined. The dnFGFR transgene was then inserted into oncolytic HSV G47Delta using a bacterial artificial chromosome construction system. Antitumoral and antiangiogenic activities of bG47Delta-dnFGFR were examined. RESULTS: MPNST 61E4 cells expressing dnFGFR grew less well than parental control cells. bG47Delta-dnFGFR showed enhanced killing of both tumor (human U87 glioma and F5 malignant meningioma cells and murine MPNST 61E4 and 37-3-18-4 cells) and proliferating endothelial cells (human umbilical vascular endothelial cell and Py-4-1) in vitro compared with the control vector bG47Delta-empty without inhibiting viral replication. In vivo, bG47Delta-dnFGFR was more efficacious than its nonexpressing parent bG47Delta-empty at inhibiting tumor growth and angiogenesis in both human U87 glioma and mouse 37-3-18-4 MPNST tumors in nude mice. CONCLUSIONS: By using multiple therapeutic mechanisms, including destruction of both tumor cells and tumor endothelial cells, an oncolytic HSV encoding dnFGFR enhances antitumor efficacy. This strategy can be applied to other oncolytic viruses and for clinical translation.

Angiogenesis Inhibitors↗

PubMed2000

The effect of familiarity on distraction and single cue use after hippocampal damage.

Spatial disorientation frequently occurs in conditions such as Alzheimer's disease that damage the hippocampus, a brain structure necessary for learning and memory. Use of a single cue to mark a submerged escape platform in the Morris water test can reduce spatial disorientation in rats. If the cue used is a familiar one, disoriented rats perform the wayfinding task as well as control animals. However, in a real-world environment, cues rarely occur alone. This study examined whether rats with bilateral hippocampal lesions familiar with a cue performed the Morris water test as well as controls and faster than lesioned rats unfamiliar with the cue when a distracter was present. Bilateral electrolytic lesions were made in male rats that had received either familiarity with a cue or handling only. Familiar lesioned rats were introduced to the distracter on test days 1 (FB1) or 3 (FB3), and unfamiliar lesioned rats on day 3 (UB3). No significant differences were found for FB1 or FB3 rats and their respective controls. FB1 rats increased mean swim times on day 1 compared to preoperative day 4 (16.44 +/- 4.3 vs. 4.06 +/- 2.1 s, respectively, p < 0.03) but quickly adapted to the distracter. FB3 rats were slower than FB1 rats on day 3 (6.81 +/- 1.0 vs. 4.56 +/- 0.3 s, respectively, p < 0.05). UB3 rats were impaired on the task compared to FB1 rats on days 3-7 (p < 0.05). These findings suggest that cue familiarity is effective in the presence of a distracter and that the response of disoriented rats to a distracter is influenced by the amount of prior experience with the cue.

Analysis of Variance↗

PubMed2001

Method for non-invasively recording electrocardiograms in conscious mice.

BACKGROUND: The rapid increase in the development of mouse models is resulting in a growing demand for non-invasive physiological monitoring of large quantities of mice. Accordingly, we developed a new system for recording electrocardiograms (ECGs) in conscious mice without anesthesia or implants, and created Internet-accessible software for analyzing murine ECG signals. The system includes paw-sized conductive electrodes embedded in a platform configured to record ECGs when 3 single electrodes contact 3 paws. RESULTS: With this technique we demonstrated significantly reduced heart rate variability in neonates compared to adult mice. We also demonstrated that female mice exhibit significant ECG differences in comparison to age-matched males, both at baseline and in response to beta-adrenergic stimulation. CONCLUSIONS: The technology we developed enables non-invasive screening of large numbers of mice for ECG changes resulting from genetic, pharmacological, or pathophysiological alterations. Data we obtained non-invasively are not only consistent with what have been reported using invasive and expensive methods, but also demonstrate new findings regarding gender-dependent and age-dependent variations in ECGs in mice.

Adrenergic beta-Agonists↗