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At least 199 records · Page 11Linked to original sources

PubMed2004

Cellular dielectric spectroscopy: a powerful new approach to label-free cellular analysis.

The past decade has seen a number of significant changes in identifying higher quality lead compounds earlier in the drug discovery process. Cell-based assay technologies yielding high-content information have emerged to achieve this goal. Although most of these systems are based on fluorescence detection, this article describes the development and application of an innovative cellular assay technology based on radio frequency spectrometry and bioimpedance measurements. Using this technique, the authors have discovered a link between cellular bioimpedance changes and receptor-mediated signal transduction events. By performing dielectric spectroscopy of cells across as pectrum of frequencies (1 KHz to 110 MHz), a series of receptor-specific, frequency-dependent impedance patterns is collected. These raw data patterns are used to determine the identity of the cellular receptor-signaling pathway being tested and to quantify stimulation endpoints and kinetics. The authors describe the application of this technology to the analysis of ligand-induced cellular responses mediated by the 3 major classes of G-protein-coupled receptors (GPCRs) and protein tyrosine kinase receptors. This single assay platform can be used with ease to monitor G(s), G(i), and G(q) GPCRs without the need for chimeric or promiscuous G-proteins, fluorophors, or tagged proteins. In contrast to other methods of monitoring cellular signal transduction, this approach provides high information content in a simplified, noninvasive, and biologically relevant fashion.

Animals↗

PubMed2006

Photonic bus with loop signal routing and multichannel wavelength selection on a single silicon-on-insulator platform.

We have demonstrated a silicon-on-insulator-based photonic bus with integrated splitters, a demultiplexer, attenuators, and a multiplexer for loop signal routing and multichannel optical wavelength selection. The integrated photonic bus has demonstrated a 33+ dB optical cross talk, a 32+ dB extinction ratio, and a rise time of 7.6 micros with two-stage Mach-Zehnder attenuators.

Journal Article↗

PubMed2004

Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies.

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.

Cell Adhesion↗

PubMed2006

Role and challenges of proteomics in pharma and biotech: technical, scientific and commercial perspective.

Contemporary proteomics, currently in its exponential growth phase, is a bewildering array of tools. Proteomic methods are the result of a convergence of rapidly improving mass spectrometry technologies, protein chemistry and separation sciences, genomics and bioinformatics. Strides in improving proteomics technologies to map and measure proteomes and subproteomes are being made. However, no single proteomic platform appears ideally suited to address all research needs or accomplish ambitious goals satisfactorily. However, proteomics is in a unique position to contribute to protein discovery and to public health in terms of better biomarkers, diagnostics and treatment of disease. While the potential is great, many challenges and issues remain to be solved. Fundamental issues, such as biological variability, pre-analytic factors and analytical reproducibility, remain to be resolved. Neither an all-genetic approach nor an all-proteomic approach will solve biological complexity. Proteomics will be the foundation for constructing and extracting useful knowledge to pharma and biotech depicted in the following path: data --> structured data --> information --> information architecture --> knowledge --> useful knowledge.

Animals↗

PubMed2001

Flow cytometric platform for high-throughput single nucleotide polymorphism analysis.

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

Flow Cytometry↗

PubMed2004

Detection of tumor markers with ProteinChip technology.

The early diagnosis of cancer at a curable stage is crucial for the successful treatment of this disease. Most of the currently used tumor assays appear too late and rely on single biomarkers with high false-negative and/or false-positive rates. As an additional burden for the patient, the traditional assays often require biopsy material instead of less invasively taken samples like serum. With the hope for more reliable DNA- and RNA-based screening tools, the research activities of the past 20 years have focused on the genomic characteristics of cancer cells. But, up to now, the output from this strategy has been disappointingly low and the disillusionment is paired with a return to proteins as the real key players in all physiological and pathological processes. Meanwhile, comparative protein profiling is generally acknowledged as a promising way for the detection of specific and predictive protein patterns reflecting certain stages of cancer without dependency on single markers. To meet the new technological demands, the ProteinChip Biomarker System was developed for the Expression Difference Mapping analysis of several hundreds of samples per day on a single, uncomplicated platform; with software support for the construction of multi-marker predictive models. The Interaction Discovery Mapping platform is introduced as the next methodical step for investigations about protein binding partners of possible importance in diagnosis and therapy. This review summarizes the current state in cancer diagnosis, provides an introduction into the ProteinChip technology, and gives an update on publications and research collaborations in SELDI-based tumor marker discovery.

Animals↗

PubMed1993

Tutorial: using Microsoft Excel for health care CQI.

The transition from QA to CQI in health care calls for decentralized data collection and analysis in conjunction with the use of personal computers to support CQI team activities. In this article, we discuss recent advances in PC software and hardware technology that make possible revolutionary changes in health care quality data management. We also present detailed examples of how to use Microsoft Excel 4.0 as a single software platform to support commonly used CQI tools and analyses.

Computer Graphics↗

PubMed2001

Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry.

BACKGROUND: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside beta-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample. METHODS: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions. RESULTS: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39-70 nmol. mg(-1). h(-1) compared with 3.7-5.1 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0-6.8 nmol. mg(-1). h(-1) in cell lysates from healthy patients compared with 0.1-0.2 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 microg) for both ASM and GCG. CONCLUSIONS: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.

Biotinylation↗

PubMed2001

Integrating metabolism and toxicity in multi-organ systems.

A wide range of experimental and predictive approaches are currently employed by the pharmaceutical industry in an attempt to avoid developing molecules with likely toxicities. Experimental systems may be adapted to handle the increasing number of molecules that require screening. The cost implication of such extensive screening is, however, immense and in silico models must therefore become the first lines of investigation to assess the metabolic and toxicological potential of new compound libraries. In silico systems have been based on the relationship between the chemical structure of novel molecules and toxicological outcomes in terms of types, but not always, locations of lesions. There should be sufficient published and in-house data available on the distribution of diverse molecules to identify the rules that govern organ, cellular and subcellular distribution of molecules (and their metabolites) as the basis of tissue and cell targeting and, more importantly, the possibility of toxicological processes. In addition, the extensive cell biology knowledge needs to be linked to current in silico systems to enhance our understanding of metabolic activation and other processes that affect likely toxicological consequences in different tissues and specific cell types. There is a need to provide a single knowledge platform that can access all current in silico products that use different datasets. The closer integration of these diverse tools will help predict other approaches to help identify the most likely toxicological potential of novel molecules, and ensure the 'least' toxic molecules are developed.

Animals↗

PubMed2002

Real time control of the integrated urban wastewater system using simultaneously simulating surrogate models.

The urban wastewater system (sewer and treatment plant) has a major impact on the river water quality of urban streams. To minimise this impact, real time control is a valuable option. Since the ultimate goal of any control strategy is to optimise the quality of the river system, it is useful to take pollutant immissions into account when determining the control strategy and/or the setpoints of the controller. However, a simultaneously simulating model of the complete system is needed in order to allow design and evaluation of such control strategies. In this work an integrated model of the urban wastewater system is created. This has been accomplished by implementing surrogate models of the three subsystems within a single software platform. The coupled submodels are subsequently used in a semi-hypothetical case study to optimise the resulting river water quality. An ammonia sensor in the river has been used to control the amount of water treated biologically in the treatment plant. It was shown that this integrated control could lower the peak ammonia concentration in the part of the river downstream of the treatment plant. Hence, a proof of principle has been given that the use of measurements in the river to perform control actions in the sewer system and the treatment plant is a promising option.

Cities↗

PubMed2001

No mission<-->no margin: it's that simple.

The authors describe their experience in developing a strategy-focused organization using the balanced scorecard methodology. They achieved this at Duke Children's Hospital by aligning the clinicians and administrators around a single integrated platform that linked improving business processes with achieving quality clinical outcomes. By organizing in this manner, they reduced cost by dollars 30 million and increased net margin by dollars 15 million while improving outcomes and staff satisfaction. This article describes a methodology to achieve strategic control of the organization, increase the knowledge of key stakeholders, and transform the organization to optimize the organization's performance.

Benchmarking↗

PubMed2006

Normalization of microarray data: single-labeled and dual-labeled arrays.

DNA microarray is a powerful tool for high-throughput analysis of biological systems. Various computational tools have been created to facilitate the analysis of the large volume of data produced in DNA microarray experiments. Normalization is a critical step for obtaining data that are reliable and usable for subsequent analysis such as identification of differentially expressed genes and clustering. A variety of normalization methods have been proposed over the past few years, but no methods are still perfect. Various assumptions are often taken in the process of normalization. Therefore, the knowledge of underlying assumption and principle of normalization would be helpful for the correct analysis of microarray data. We present a review of normalization techniques from single-labeled platforms such as the Affymetrix GeneChip array to dual-labeled platforms like spotted array focusing on their principles and assumptions.

Cluster Analysis↗

PubMed2025

ShortCake: an integrated platform for efficient and reproducible single-cell analysis.

SUMMARY: Recent advances in single-cell analysis have introduced new computational challenges. Researchers often need to use multiple analysis tools written in different programming languages while managing version conflicts between related packages within a single workflow. For the research community, minimizing the time spent on environment setup and installation issues is essential. We present ShortCake, a containerized platform that integrates a suite of single-cell analysis tools written in R and Python. ShortCake isolates competing Python tools into separate virtual environments that can be easily accessed within a Jupyter notebook. This enables users to effortlessly transition between various environments, including R, even within a single notebook. Additionally, ShortCake offers multiple "flavors," enabling users to select container images tailored to their specific needs. ShortCake provides a unified environment with fixed versions of various tools, thus streamlining workflows, reducing setup time, and improving reproducibility. AVAILABILITY AND IMPLEMENTATION: The ShortCake image is available on DockerHub (https://hub.docker.com/r/rnakato/shortcake) and Zenodo (DOIs: 10.5281/zenodo.17116765 and 10.5281/zenodo.17118158). The source code is available on GitHub (https://github.com/rnakato/ShortCake).

Single-Cell Analysis↗

PubMed2003

Genome-wide loss of heterozygosity analysis from laser capture microdissected prostate cancer using single nucleotide polymorphic allele (SNP) arrays and a novel bioinformatics platform dChipSNP.

Oligonucleotide arrays that detect single nucleotide polymorphisms were used to generate genome-wide loss of heterozygosity (LOH) maps from laser capture microdissected paraffin-embedded samples using as little as 5 ng of DNA. The allele detection rate from such samples was comparable with that obtained with standard amounts of DNA prepared from frozen tissues. A novel informatics platform, dChipSNP, was used to automate the definition of statistically valid regions of LOH, assign LOH genotypes to prostate cancer samples, and organize by hierarchical clustering prostate cancers based on the pattern of LOH. This organizational strategy revealed apparently distinct genetic subsets of prostate cancer.

Alleles↗

PubMed2003

Immediate occlusal loading of single lower molars using Brånemark System Wide-Platform TiUnite implants: an interim report of a prospective open-ended clinical multicenter study.

BACKGROUND: First and second molars in the lower jaw are the most commonly missing teeth. As in other jaw regions with tooth loss, implant therapy is an attractive concept. Owing to the wide dimension of the molar teeth, wide implants seem to be a natural choice. However, wide implants in posterior mandibular regions sometimes present a problem. As an oxidized surface seems to facilitate implant integration and to result in a stronger fixation to bone, it was hypothesized that this surface would improve the performance of wide implants in posterior mandibles. As high initial stability most often is achieved in this region, it was hypothesized that the load per se is not a problem. PURPOSE: The purpose of the present study is to evaluate immediate loading of Brånemark System TiUnite Wide-Platform implants (Nobel Biocare AB, Gothenburg, Sweden) supporting single molar crowns in the lower jaw. MATERIALS AND METHODS: The study includes 44 patients treated in three separate private dental offices between March 2001 and June 2002. A total of 50 implants were placed, and all implants have passed the 6-month follow-up; 24 have been followed up for 1 year. All implants were provided with provisional crowns in centric occlusion at the time of surgery. RESULTS: No implant has been lost. Marginal bone levels were found in accordance with normal biologic width requirements. Resonance frequency analysis showed high and consistent implant stability. No biomechanical problems were found associated with the use of wide implants, and the potential physiologic problem in dense mandibular bone was eliminated. CONCLUSION: Although limited by being a short-term study, the result encourages immediate loading of single Brånemark System TiUnite Wide-Platform implants placed in the molar regions in the lower jaw.

Adult↗

PubMed2025

Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.

Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.

Humans↗

PubMed2026

High-throughput single-cell proteomics and transcriptomics from same cells with a nanoliter-scale, spin-transfer approach.

Single-cell multiomic platforms provide a comprehensive snapshot of cellular states and cell types by offering critical insights into the spatiotemporal regulation of biomolecular networks at a systems level, thereby defining the basis of multicellularity. Here, we introduce nanoSPINS, an advanced platform that enables high-throughput profiling and integrative analysis of the transcriptome and proteome from the same single cells using RNA sequencing and isobaric labeling LC-MS-based proteomics, respectively. NanoSPINS can efficiently transfer mRNA-containing droplets across two microarrays via a centrifugation-based approach, while proteins are retained on the initial platform. Benchmarking of nanoSPINS on two cell lines demonstrates its ability to generate global proteomic and transcriptomic profiles that align well with previously established methodologies/platforms. The incorporation of isobaric TMTpro labeling into this single-cell multiomics platform significantly enhances the throughput of single-cell proteomic analyses. Through the high-throughput quantification of the proteome and transcriptome, nanoSPINS not only facilitates the identification of molecular features at both mRNA and protein level but also provides larger sample sizes for improved statistical power in clustering and differential abundance. Given the broad applicability of single-cell multiomics in biological research and clinical settings, we believe nanoSPINS represents a powerful platform for the characterization of heterogeneous cell populations.

Single-Cell Analysis↗

PubMed2006

Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform.

A device based on five individually addressable microelectrodes, fully integrated within a microfluidic system, has been fabricated to enable the real-time measurement of ionic and metabolic fluxes from electrically active, beating single heart cells. The electrode array comprised one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an electrochemical lactate microbiosensor, that were used to measure the amounts of lactate produced by the heart cell. The device also allowed simultaneous in-situ microscopy, enabling optical measurements of cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+. Initial experiments aimed to create a metabolic profile of the beating heart cell, and results show well defined excitation-contraction (EC) coupling at different rates. Ca2+ transients and extracellular pH measurements were obtained from continually paced single myocytes, both as a function of the rate of cell contraction. Finally, the relative amounts of intra- and extra-cellular lactate produced during field stimulation were determined, using cell electroporation where necessary.

Aerobiosis↗