Greater Dynamic Range Research Articles

A genome‐wide, promoter tiling design scheme for high quality real‐time PCR assays to measure genomic DNA in chromatin immunoprecipitation (ChIP) samples

PCR is the standard for measuring DNA from ChIP because of its ability to simultaneously amplify and detect from limited samples. Compared to end‐point PCR, real‐time PCR (qPCR) provides faster data acquisition, greater dynamic range and increased sensitivity. However, the traditional “targeted” assay design scheme (primers flanking a binding site) hinders the design of amplicons meeting the performance criteria of specificity and amplification efficiency. To avoid this issue, we have applied an alternative “tiling” assay design scheme that allows greater flexibility in positioning the primers away from poor PCR sequences. Using this approach on a genome‐wide scale, 30 kb regions of promoters have been successfully “tiled” at a 1 kb interval with assays that display consistent, high‐quality qPCR performance. When used to measure p53 binding interactions in the CDKN1A promoter of HCT116 cells after treatment with 5‐FU, the ChIP‐qPCR assays showed that p53 binding increased from a few fold to 50 fold above background. Tiling assays corresponding to p53 binding sites in other genes show similar enrichments. The functional consequences of the p53 binding was also confirmed by RT‐qPCR measurement of mRNA. The genome‐wide availability of promoter tiling ChIP‐qPCR assays allows an increased number of binding sites to be surveyed simultaneously and facilitates ChIP‐chip microarray validation.

High resolution GPR and its experimental study

We develop a high resolution ground penetrating radar system (LANRCS-GPR) based on the E5071B Vector Network Analyzer (VNA). This system takes advantage of a wideband and adjustable frequency domain ground penetrating radar system and adds the characteristics of a network analyzer with ultra-wideband and high precision measurement. It adopts the LAN mode to concatenate system control that reduces construction cost and makes the system easy to expand. The high resolution ground penetrating radar system carries out real time imaging using F-K migration with high calculation efficiency. The experiment results of the system indicate that the LANRCS-GPR system provides high resolution and precision, high signal-to-noise ratio, and great dynamic range. Furthermore, the LANRCSGPR system is flexible and reliable to operate with easy to expand system functions. The research and development of the LANRCS-GPR provide the theoretical and experimental foundation for future frequency domain ground penetrating radar production and also can serve as an experimental platform with high data gathering precision, enormous information capability, wide application, and convenient operation for electromagnetic wave research and electromagnetic exploration

Focus on materials

Focus on materials

Fluorometric Assay for the Determination of Glutathione Reductase Activity

The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.

The ON–OFF dichotomy in visual processing: From receptors to perception

Vision scientists long ago pointed to black and white as separate sensations and saw confirmation in the fact that in the absence of light, one perceives the visual field as gray against which the negative after-image of a bright light appeared blacker. The first recordings from optic nerve fibers in vertebrates revealed ON and OFF signals, later associated with separate streams, arising already at the synapse between receptors and bipolar cells. These can be identified anatomically and physiologically and remain distinct all the way to the lateral geniculate nucleus, whose fibers form the input to the primary visual cortex. The dichotomy has been probed by electroretinography and analyzed by means of pharmacological agents and dysfunction due to genetic causes. The bi- rather than a unidirectional nature of the retinal output has advantages in allowing small signals to remain prominent over a greater dynamic range. The two streams innervate cortical neurons in a push-pull manner, generating receptive fields with spatial sensitivity profiles featuring ON and OFF subregions. Manifestations of the dichotomy appear in a variety of simple visual discriminations where there are often profound threshold differences in patterns with same polarity as compared with mixed contrast-polarity components. But even at levels in which the spatial, contrast and color attributes have already been securely established and black and white elements participate equally, a categorical difference between blackness and whiteness of a percept persists. It is an opponency, akin to the ones in the color domain, derived from the original ON and OFF signals and subsequently bound with the other attributes to yield a feature's unitary percept.

Shortcomings in methodology complicate measurements of serum retinol binding protein (RBP4) in insulin-resistant human subjects

Levels of retinol binding protein (RBP4) are increased in the serum of insulin-resistant human subjects even before overt diabetes develops. RBP4 levels correlate with insulin resistance, BMI, WHR, dyslipidaemia and hypertension. Improvement of insulin sensitivity with exercise training is associated with reduction in serum RBP4 levels. Therefore serum RBP4 may be useful for early diagnosis of insulin resistance and for monitoring improvements in insulin sensitivity. We sought to determine the performance of assays for this application. We compared quantitative western blotting and three commercially available multiwell immunoassays in parallel measurements of RBP4 concentrations in serum from insulin-sensitive subjects and from insulin-resistant subjects with impaired glucose tolerance or type 2 diabetes. The assays yielded different absolute values and magnitudes of elevation of serum RBP4. Western blotting and a sandwich ELISA reported RBP4 concentrations that highly inversely correlated with insulin sensitivity measured by euglycaemic-hyperinsulinaemic clamp. However, western blotting yielded concentrations with a greater dynamic range and less overlap between control and insulin-resistant subjects. Two competitive enzyme-linked immunoassays undervalued serum RBP4 concentrations in insulin-resistant subjects, possibly due to assay saturation. Poor linearity of dilution also limited assay utility. All assays tested exhibited greater immunoreactivity with urinary (C-terminal proteolysed) RBP4 than with full-length RBP4, the predominant form in serum. These findings support the use of quantitative western blotting standardised to full-length RBP4 protein as a 'gold standard' method for measuring serum RBP4 in insulin-resistant states. Other assays should use full-length RBP4 and be extensively cross-validated using other methods.

A combined dataset of human cerebrospinal fluid proteins identified by multi‐dimensional chromatography and tandem mass spectrometry

Human cerebrospinal fluid (CSF) is an important source for studying protein biomarkers of age-related neurodegenerative diseases. Before characterizing biomarkers unique to each disease, it is necessary to categorize CSF proteins systematically and extensively. However, the enormous complexity, great dynamic range of protein concentrations, and tremendous protein heterogeneity due to post-translational modification of CSF create significant challenges to the existing proteomics technologies for an in-depth, nonbiased profiling of the human CSF proteome. To circumvent these difficulties, in the last few years, we have utilized several different separation methodologies and mass spectrometric platforms that greatly enhanced the identification coverage and the depth of protein profiling of CSF to characterize CSF proteome. In total, 2594 proteins were identified in well-characterized pooled human CSF samples using stringent proteomics criteria. This report summarizes our efforts to comprehensively characterize the human CSF proteome to date.

Analysis of molecular inversion probe performance for allele copy number determination

A new protocol for using molecular inversion probes to specifically and accurately measure allele copy numbers.

Polycomb Complexes and the Propagation of the Methylation Mark at the Drosophila Ubx Gene

Polycomb group proteins are transcriptional repressors that control many developmental genes. The Polycomb group protein Enhancer of Zeste has been shown in vitro to methylate specifically lysine 27 and lysine 9 of histone H3 but the role of this modification in Polycomb silencing is unknown. We show that H3 trimethylated at lysine 27 is found on the entire Ubx gene silenced by Polycomb. However, Enhancer of Zeste and other Polycomb group proteins stay primarily localized at their response elements, which appear to be the least methylated parts of the silenced gene. Our results suggest that, contrary to the prevailing view, the Polycomb group proteins and methyltransferase complexes are recruited to the Polycomb response elements independently of histone methylation and then loop over to scan the entire region, methylating all accessible nucleosomes. We propose that the Polycomb chromodomain is required for the looping mechanism that spreads methylation over a broad domain, which in turn is required for the stability of the Polycomb group protein complex. Both the spread of methylation from the Polycomb response elements, and the silencing effect can be blocked by the gypsy insulator.

Development of a Fluorescent Multiwell Assay for Evaluating the Capacity of the Ciliated Protozoan Tetrahymena for Bacterivory in Water Samples

Abstract Bacterivory by ciliates in various water ecosystems, both natural and artificial, plays a significant role on the microbial population composition and consequently affects water quality. A convenient, rapid and inexpensive methodology to evaluate the capacity of the ciliate protozoan Tetrahymena thermophila for bacterivory was developed utilizing fluorescent protein expressing bacteria (FPEB) in a microtitre plate fluorimeter. Bacterivory was correlated with a loss in fluorescence measured in the fluorimeter and confirmed by fluorescence microscopy showing that the FPEB were engulfed during the assay and subsequently lost their fluorescence, whereas Cytochalasin B, a known inhibitor of phagocytosis, prevented a decrease in relative fluorescence units (RFUs). The ciliate bacterivory (CB) assay has a great dynamic range allowing the assay to be performed with a variety of predator:prey concentrations. A model toxicant, CuCl2, known to have a toxicological impact on protozoa and often present in different types of wastewater, resulted in measurable decreases in bacterivory. As well, starvation of Tetrahymena for 24 h resulted in reduced bacterivory. In the future, the CB assay could be developed for water monitoring purposes to rapidly assess water samples for the capacity to support bacterivory as an indicator of ecosystem health.

Dynamic range of ultrabroadband terahertz detection using GaAs photoconductors

Electromagnetic transients with frequency components spanning the far and midinfrared are coherently detected by means of the polarization current induced in an electron-hole plasma optically excited in As implanted GaAs. The impulse response is governed by the frequency dependence of the photocarrier mobility and density and the lattice contribution to the dielectric constant. Compared with electro-optic sampling using thin ZnTe crystals, photoconductive detection can yield a comparable or greater dynamic range up to 40THz.

Proteomic analysis in cancer research: potential application in clinical use

The ultimate goal of cancer proteomics is to adapt proteomic technologies for routine use in clinical laboratories for the purpose of diagnostic and prognostic classification of disease states, as well as in evaluating drug toxicity and efficacy. The novel technologies allows researchers to facilitate the comprehensive analyses of genomes, transcriptomes, and proteomes in health and disease. The information that is expected from such technologies may soon exert a dramatic change in cancer research and impact dramatically on the care of cancer patients. Analysis of tumor-specific proteomic profiles may also allow better understanding of tumor development and the identification of novel targets for cancer therapy. The localization of gene products, which is often difficult to deduce from the sequence, can be determined experimentally. Mechanisms, such as regulation of protein function by proteolysis, recycling, and isolation in cell compartments, affect gene products, not genes. Finally, protein-protein interactions and the molecular composition of cellular structures can be determined only at the protein level. The biological variability among patient samples as well as the great dynamic range of biomarker concentrations are currently the main challenges facing efforts to deduce diagnostic patterns that are unique to specific disease states. While several strategies exist to address this problem, we have tried to offer a wide perspective about the current possibilities.

In vivo quantification of human lumbar disc degeneration using T1ρ-weighted magnetic resonance imaging

Diagnostic methods and biomarkers of early disc degeneration are needed as emerging treatment technologies develop (e.g., nucleus replacement, total disc arthroplasty, cell therapy, growth factor therapy) to serve as an alternative to lumbar spine fusion in treatment of low back pain. We have recently demonstrated in cadaveric human discs an MR imaging and analysis technique, spin-lock T(1rho)-weighted MRI, which may provide a quantitative, objective, and non-invasive assessment of disc degeneration. The goal of the present study was to assess the feasibility of using T(1rho) MRI in vivo to detect intervertebral disc degeneration. We evaluated ten asymptomatic 40-60-year-old subjects. Each subject was imaged on a 1.5 T whole-body clinical MR scanner. Mean T(1rho) values from a circular region of interest in the center of the nucleus pulposus were calculated from maps generated from a series of T(1rho)-weighted images. The degenerative grade of each lumbar disc was assessed from conventional T(2)-weighted images according to the Pfirmann classification system. The T(1rho) relaxation correlated significantly with disc degeneration (r=-0.51, P<0.01) and the values were consistent with our previous cadaveric study, in which we demonstrated correlation between T(1rho) and proteoglycan content. The technique allows for spatial measurements on a continuous rather than an integer-based scale, minimizes the potential for observer bias, has a greater dynamic range than T(2)-weighted imaging, and can be implemented on a 1.5 T clinical scanner without significant hardware modifications. Thus, there is a strong potential to use T(1rho) in vivo as a non-invasive biomarker of proteoglycan loss and early disc degeneration.

Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution

The development of a sensitive fluorescence-based assay for the quantitative determination of protein concentration is described. The assay is based on the natural product epicocconone, which produces a large increase in fluorescence quantum yield upon binding to detergent-coated proteins in solution. There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 microg/mL for most proteins. There is little protein-to-protein variation except for iron-containing proteins and the assay can be used so that it is tolerant of chemicals commonly used in 2-D sample buffers. The assay is more sensitive than standard absorption assays such as the Bradford and Lowry assays, and has a greater dynamic range and sensitivity than other fluorescent assays.

Validation of High Dynamic Range Imaging to Luminance Measurement

High dynamic range imaging is a set of techniques that allows for a far greater dynamic range of exposure than normal digital imaging techniques. The intention is to accurately represent the dynamic range of lighting levels found in real scenes, ranging from direct sunlight to deep shadows. Various software packages have emerged that translate these HDR images into high resolution luminance maps. This paper tests the accuracy of one such package, taking into consideration different Munsell hues, values and chroma. It investigates the impact of different light spectra, spatial frequency, vignetting, and thermal noise on the accuracy of luminance measurements and determines the potential errors.

Cloud and aerosol measurements from GLAS: Overview and initial results

Global space borne lidar profiling of atmospheric clouds and aerosol began in 2003 following the launch of the Geoscience Laser Altimeter System (GLAS) on the Ice, Cloud and land Elevation Satellite. GLAS obtains nadir profiles through the atmosphere in two wavelength channels, day and night, at a fundamental resolution of 76.8 m vertical and 172 m along track. The 532 nm channel uses photon‐counting detectors and resolves profiles of observed backscatter cross sections to 10−7 1/m‐sr. The 1064 nm channel employs analog detection adequate to 10−6 1/m‐sr and with greater dynamic range. By 2005 approximately seven months of global data are available. Processing algorithms produce data products for the corrected lidar signal, cloud and aerosol layer boundaries and optical thickness and extinction and backscatter cross sections. Operational sensitivity is shown by the frequency distribution for cloud optical thickness peaking at approximately 0.02.

A Combined Yeast/Bacteria Two-hybrid System: Development and Evaluation

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.

Elimination of environmental sensitivity in a cameleon FRET-based calcium sensor via replacement of the acceptor with Venus

Genetically encoded sensors are becoming a powerful tool for investigating cellular signaling pathways and, potentially, signaling in vivo. Many sensors use changes in fluorescence resonance energy transfer (FRET) between donor and acceptor variants of GFP separated by a ligand binding domain sensitive to a particular signaling pathway. Accurate measurements require that sensors be insensitive to extraneous intracellular environmental factors. We have found that the responsiveness of the Ca 2+ sensor, cameleon YC6.1, varies linearly with the resting YFP/CFP emission ratio in the cell. However, cells expressing responsive or non-responsive sensor can easily be segregated by determining a resting YFP/CFP ratio cutoff for the sensor. This environmental sensitivity has been eliminated by replacing EYFP with Venus to produce a new cameleon we have designated VC6.1. Measurements show that VC6.1 has a greater dynamic range than YC6.1 and better environmental resistance. We also show that YC6.1 is inactivated by persistent activation of the IP 3 pathway following expression of constitutively active G q, while VC6.1 is not. The stability of VC6.1 may make it well suited to studies utilizing mixed cell populations such as those encountered in vivo.

High-throughput analysis of mRNA expression: microarrays are not the whole story

Analysis of gene expression has become one of the most widely used techniques in biomedical research. The publication of the human genome, together with the technical ability to automatically detect and process very large numbers of samples, has forced many research groups in academia and industry to shift to the complex and demanding study of genome-wide gene expression. DNA microarrays or ‘gene chips’, have become one of the most popular techniques for this purpose but their use is limited by technical deficiencies and the fact that prior knowledge on the genome of interest is a prerequisite. During the last few years, several approaches have been developed that overcome many of the limitations of DNA microarrays, while yielding greater sensitivity and a greater dynamic range in the absence of prior genomic knowledge. This review is an overview of selected high-throughput methods that provide a viable alternative to the use of DNA microarrays for analysing gene expression. It is a companion paper to one specifically on DNA microarrays, recently published in this journal by the same authors.

Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells

The enumeration of antigen-specific T cell responses has been greatly facilitated in recent years by the development of methods based on the detection of cytokines. In particular, the enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays have become popular. Since both assays are likely to continue to be in widespread use, it is important to evaluate whether their results are comparable. In the current study, we compared the results obtained in the ELISPOT and CFC assays using peptide pools corresponding to CMV and HIV-1 proteins in chronically HIV-1-infected individuals. Analysis of T cell responses to peptide pools indicated that the CMV pp65 and HIV-1 Gag CFC and ELISPOT-derived results were statistically correlated. However, the results obtained with each assay differed in important ways: the magnitude of the response was consistently higher in the CFC assay while the CFC assay was less likely than the ELISPOT assay to detect low-level responses. Furthermore, there was a lack of numeric agreement between ELISPOT and CFC results. For studies that require the detection of low-level responses, or definition of responses as positive or negative, the ELISPOT assay may be preferable. In contrast, the CFC has a greater dynamic range and allows for phenotypic discrimination of responding cells, making it the assay of choice for most other applications.