BackgroundThe interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.Methodology/Principal FindingsWe present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.Conclusions/SignificanceWe describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.
A method is described for using a limited number (typically 10-50) of low-dose radiographs to reconstruct the three-dimensional (3D) distribution of x-ray attenuation in the breast. The method uses x-ray cone-beam imaging, an electronic digital detector, and constrained nonlinear iterative computational techniques. Images are reconstructed with high resolution in two dimensions and lower resolution in the third dimension. The 3D distribution of attenuation that is projected into one image in conventional mammography can be separated into many layers (typically 30-80 1-mm-thick layers, depending on breast thickness), increasing the conspicuity of features that are often obscured by overlapping structure in a single-projection view. Schemes that record breast images at nonuniform angular increments, nonuniform image exposure, and nonuniform detector resolution are investigated in order to reduce the total x-ray exposure necessary to obtain diagnostically useful 3D reconstructions, and to improve the quality of the reconstructed images for a given exposure. The total patient radiation dose can be comparable to that used for a standard two-view mammogram. The method is illustrated with images from mastectomy specimens, a phantom, and human volunteers. The results show how image quality is affected by various data-collection protocols.
Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.
Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides. In addition to the catalytic domain, IMPDH contains a subdomain of unknown function composed of two cystathione beta-synthase domains. Our results, using three different assays, show that IMPDHs from Tritrichomonas foetus, Escherichia coli, and both human isoforms bind single-stranded nucleic acids with nanomolar affinity via the subdomain. Approx. 100 nucleotides are bound per IMPDH tetramer. Deletion of the subdomain decreases affinity 10-fold and decreases site size to 60 nucleotides, whereas substitution of conserved Arg/Lys residues in the subdomain with Glu decreases affinity by 20-fold. IMPDH is found in the nucleus of human cells, as might be expected for a nucleic-acid-binding protein. Lastly, immunoprecipitation experiments show that IMPDH binds both RNA and DNA in vivo. These experiments indicate that IMPDH has a previously unappreciated role in replication, transcription or translation that is mediated by the subdomain.
We are developing a modular detector for applications in full field digital mammography and for diagnostic breast imaging. The detector is based on a design that has been refined over the past decade for applications in x-ray crystallography [Kalata et al., Proc. SPIE 1345, 270-279 (1990); Phillips et al. ibid. 2009, 133-138 (1993), Phillips et al., Nucl. Instrum. Methods Phys. Rev. A 334, 621-630 (1993)]. The full field mammographic detector, currently undergoing clinical evaluation, is formed from a 19 cm x 28 cm phosphor screen, read out by a 2 x 3 array of butted charge-coupled device (CCD) modules. Each 2k x 2k CCD is optically coupled to the phosphor via a fiber optic taper with dimensions of 9.4 cm x 9.4cm at the phosphor. This paper describes the imaging performance of a two-module prototype, built using a similar design. In this paper we use cascaded linear systems analysis to develop a model for calculating the spatial frequency dependent noise power spectrum (NPS) and detective quantum efficiency (DQE) of the detector using the measured modulation transfer function (MTF). We compare results of the calculation with the measured NPS and DQE of the prototype. Calculated and measured DQEs are compared over a range of clinically relevant x-ray exposures and kVps. We find that for x-ray photon energies between 10 and 28 keV, the detector gain ranges between 2.5 and 3.7 CCD electrons per incident x-ray, or approximately 5-8 electrons per absorbed x ray. Using a Mo/Mo beam and acrylic phantom, over a detector entrance exposure range of approximately 10 to 80 mR, the volume under the measured 2-d NPS of the prototype detector is proportional to the x-ray exposure, indicating quantum limited performance. Substantial agreement between the calculated and measured values was obtained for the frequency and exposure dependent NPS and DQE over a range of tube voltage from 25 to 30 kVp.
The detector is designed for imaging measurements requiring relatively high sensitivity and high spatial resolution. The detector can discriminate single X-ray photons, yet has the wide dynamic range ($ 10 000 : 1) associated with integrating detectors. A GdO 2 S 2 phosphor screen converts the incoming X-ray image into an optical image. The optical image is coupled (without demagni®cation) to the CCD image sensor using a ®ber optic faceplate. The CCD (Philips Semiconductors) has an area of 4.9 Â 8.6 cm with 4000 Â 7000 12 mm pixels. A single 12 keV X-ray photon produces a signal of 100 e À . With 2 Â 2 pixel binning, the total noise per 24 mm pixel in a 100 s image is $ 30 e À , the detective quantum ef®ciency is > 0.6 at 1 X-ray photon per pixel, and the full image can be read out in < 4 s. The spatial resolution is 50 mm. The CCD readout system is fully computer-controlled, allowing¯exible operation in time-resolved experiments. The detector has been characterized using visible-light images, X-ray images and time-resolved muscle diffraction measurements. research papers Figure 11Radiograph of a daffodil recorded at 13 keV with 2 Â 2 pixel binning and 5000 incident xphs per pixel.
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