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SERS platforms for high density DNA arrays.

Surface Enhanced Raman Scattering (SERS) gives rise to analytical applications with much promise. In our approach three steps are necessary. We require a SERS platform of high enhancement. This has been achieved using the special technique of Island Lithography, combined with Ag deposition by galvanic exchange, yielding an enhancement factor of 10(8). Probe oligonucleotide molecules are attached to a specific area on the platform, at the optimized surface concentration, using thiolated single stranded (ss) DNA molecules. The optimum surface concentration has been determined and interpreted in the light of the polyelectrolyte behaviour of ssDNA. Finally the change in SERS produced by hybridisation of the probe molecules to a target DNA molecule is measured. Highly discernible changes have been obtained. No change in probe signal is seen when presented with one base mismatched target. From this work it is concluded that the prospects for label-free DNA detection in high-density arrays is now close to achievement.

Adsorption↗

Ribosome structure: localization of 3' end of RNA in small subunit by immunoelectronmicroscopy.

The 3' end of the RNA in the 30S ribosomal subunit of Escherichia coli has been modified by oxidation with sodium periodate and conjugation with the (mono) dinitrophenyl derivative of ethylenediamine. Antibodies, induced with dinitrophenyl-bovine serum albumin, interact with the modified ribosomal subunits. Electron micrographs of negatively stained antibody-subunit complexes show individual ribosomal subunits to which a single antibody molecule is bound and subunit dimers cross-linked by an IgG molecule. The modified 3' terminus has been localized to a single site on the upper portion of the platform region of the 30S subunit. This location is consistent with earlier placements of proteins that react with the 3' end of the RNA.

Dinitrobenzenes↗

A simple height-specific and rate-specific step test for children.

Recently an anatomic model was reported for adults that standardized the platform height for step tests using an individual's stature and a specified hip angle. In order to determine if the model could be used to predict the platform height for children, platform heights were calculated and hip angles were measured in 146 boys and 140 girls ages 6 to 18 years who were divided into four age groups (6 to 8, 9 to 11, 12 to 15, and 16 to 18 years old). There were no statistical differences between measured and calculated hip angles in any of the age groups. In order to determine the validity for predicting maximal oxygen consumption from stepping using a calculated platform height, three step tests were employed using one platform height and stepping frequencies of 22, 26, or 30 ascents/min. Each of the tests was administered to 93 6- to 18-year-olds. Correlation coefficients between the 15-second recovery heart rate after stepping and maximal oxygen consumption measured on a treadmill were .80 at 30 ascents/min, .79 at 26 ascents/min, and .81 at 22 ascents/min. Each of the correlation coefficients was significant at the P less than .01 level. It can be concluded that the model is valid for standardizing the platform height for children for use with a single stage step test for estimating maximal oxygen consumption.

Adolescent↗

HyperCas12a enables multiplexed CRISPRi screens.

Interactions between multiple genes or cis-regulatory elements (CREs) underlie a wide range of biological processes in both health and disease. High-throughput screens using dCas9 fused to epigenome editing domains have allowed researchers to assess the impact of activation or repression of both coding and non-coding genomic regions on a phenotype of interest, but assessment of genetic interactions between those elements has been limited to pairs. Here, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays to enable efficient highly-multiplexed epigenome editing. We demonstrate that this system is compatible with several activation and repression domains, including the P300 histone acetyltransferase domain and SIN3A interacting domain (SID). We further show that the system can be used in cultured primary immune cells and to drive differentiation of induced pluripotent stem cells. We also developed new approaches to use the dCas12a platform for simultaneous activation and repression from a single crRNA array via co-expression of multiple dCas12a orthologues. Lastly, we demonstrate that the dHyperLbCas12a effectors are highly effective for multiple modalities of high-throughput screens, namely proliferation screens and screens to dissect the independent and combinatorial contributions of CREs on gene expression. The tools and methods introduced here create new possibilities for highly multiplexed control of gene expression in a wide variety of biological systems.

Journal Article↗

The high-speed Hydra-Plus-One system for automated high-throughput protein crystallography.

An automated high-throughput dispenser has been developed for the setup of protein crystallization trials by vapor diffusion or Microbatch methods. The Hydra-Plus-One is composed of a Hydra-PP system equipped with a motorized XYZ-platform, 96 precision glass syringes and a single-channel microsolenoid dispenser, which transfers 100 nl-50 micro l of protein solution with an accuracy of > 90% at a speed of 60s per 96 wells. Up to 300 micro l of premixed cocktails can be aspirated with the 96-syringe-assembly and dispensed into reservoir and droplet wells within 60s. The Hydra-Plus-One combines high precision, reliability and speed in a cost-effective high-throughput system ideally suited for protein crystallization

Bacterial Proteins↗

Time delay study of a CT simulator in respiratory gated CT scanning.

In respiratory-gated radiotherapy (RGRT), if the time delay in a computed tomography (CT) simulator and that in a linear accelerator (Linac) are different, the simulation and the treatment cannot be synchronized. In this study, we presented a technique to measure the time delay of the AcQSim CT simulator (Philips Medical Systems, Cleveland, OH) using Varian's Real-Time Positioning Management (RPM) system (Varian Medical Systems, Palo Alto, CA). A respiratory gating platform (REF 91150, Standard Imaging, Inc., Middleton, MI) was first set at the position of amplitude maximum (phase 0). Then a ball of 1.3 cm diameter was put on the platform and set at the CT laser. A single axial scan was acquired across the center of the ball without motion. Then the motion was turned on and single axial scans gated at different phases were acquired with a very narrow gating window. The time between the phase giving a good estimate of the ball and phase 0 is the overall delay time. We found that for AcQSim CT, the overall delay for a single axial scan (with 1 s scan time) is 1.75 s. For multiple axial scans, the overall delay is 1.75 s for the first scan and 0.75 s for the subsequent ones. This demonstrated that the CT mechanical startup delay is 1 s. After the first axial scan, the overall delay per scan is less because CT gantry continuously spins and no mechanical delay exists. We call the overall delay without mechanical part the scanning delay, which basically equals half the scan time (0.5 s for 1 s scan time) plus the gating pulse triggering delay (250 ms). The delays were also verified using metal balls of 1.5 mm diameter set at the amplitude minimum (phase 180, initially). We note that it is the scanning delay rather than the triggering delay that should be compensated when doing motion-synchronized radiotherapy. The current interface between the RPM system and the AcQSim CT does not compensate for this scanning delay.

Artifacts↗

High-Throughput GoMiner, an 'industrial-strength' integrative gene ontology tool for interpretation of multiple-microarray experiments, with application to studies of Common Variable Immune Deficiency (CVID).

BACKGROUND: We previously developed GoMiner, an application that organizes lists of 'interesting' genes (for example, under-and overexpressed genes from a microarray experiment) for biological interpretation in the context of the Gene Ontology. The original version of GoMiner was oriented toward visualization and interpretation of the results from a single microarray (or other high-throughput experimental platform), using a graphical user interface. Although that version can be used to examine the results from a number of microarrays one at a time, that is a rather tedious task, and original GoMiner includes no apparatus for obtaining a global picture of results from an experiment that consists of multiple microarrays. We wanted to provide a computational resource that automates the analysis of multiple microarrays and then integrates the results across all of them in useful exportable output files and visualizations. RESULTS: We now introduce a new tool, High-Throughput GoMiner, that has those capabilities and a number of others: It (i) efficiently performs the computationally-intensive task of automated batch processing of an arbitrary number of microarrays, (ii) produces a human-or computer-readable report that rank-orders the multiple microarray results according to the number of significant GO categories, (iii) integrates the multiple microarray results by providing organized, global clustered image map visualizations of the relationships of significant GO categories, (iv) provides a fast form of 'false discovery rate' multiple comparisons calculation, and (v) provides annotations and visualizations for relating transcription factor binding sites to genes and GO categories. CONCLUSION: High-Throughput GoMiner achieves the desired goal of providing a computational resource that automates the analysis of multiple microarrays and integrates results across all of the microarrays. For illustration, we show an application of this new tool to the interpretation of altered gene expression patterns in Common Variable Immune Deficiency (CVID). High-Throughput GoMiner will be useful in a wide range of applications, including the study of time-courses, evaluation of multiple drug treatments, comparison of multiple gene knock-outs or knock-downs, and screening of large numbers of chemical derivatives generated from a promising lead compound.

Binding Sites↗

Swelling archives warrant closer look at compression.

Image compression is broadly categorized as lossless or lossy. With lossless compression, a compressed image can be decompressed and displayed as an exact digital replica of the original. With lossy compression, redundant pixel data are discarded during the compression process so that the compressed image is only an approximation of the original, therefore it cannot be returned to an original state. Although both types of compression are commonly used within the medical imaging community, institutions are much more likely to depend upon lossless compression for diagnostic purposes, even though lossy images, saved as a fraction of the original file size, are often diagnostically equivalent. With more and more digital modalities coming online and image studies growing ever larger, institutions failing to take full advantage of lossy compression are missing an opportunity to slow the growth of their image archives and IT infrastructure costs. Today, most PACS vendors include some form of image compression technology within their product offerings. Of 13 vendors sampled during an informal survey in June 2003, eight employ JPEG 2000 compression, which was incorporated into the DICOM standard in 2001, while the remainder use wavelet compression, which is the underlying methodology used in JPEG 2000. JPEG 2000 is an industry standard that enables image sharing across platforms and product lines. It also provides a single mechanism for creating lossless and lossy images, and gives institutions the flexibility to apply unique rates of compression to individual images based on modality, patient history, image size or other factors. Although it can be used judificiously to great advantage, compression is most often applied in an "across the board" manner to all images. The incorporation of JPEG 2000 within the DICOM standard does little to guarantee its longevity, or the quality of every JPEG 2000 implementation. At some point in time, especially as the demand for more compact, higher-quality lossy images grows, institutions will begin experimenting with and employing more advanced compression methods.

Data Compression↗

Balloon alignment T-stenting for bifurcation coronary artery disease using the sirolimus-eluting stent.

OBJECTIVE: The purpose of this study was to evaluate the long-term outcomes of bifurcation coronary lesions treated with a novel technique called "balloon alignment T-stenting". BACKGROUND: The optimal technique for the treatment of bifurcation coronary disease has not yet been established. METHODS: Twenty-six patients with bifurcation coronary lesions were treated with currently available drug-eluting stent (DES) platforms. This was a pilot study to determine the long-term efficacy of a novel technique, "balloon alignment T-stenting". Based on this technique we performed 6-month anniversary angiography to determine restenosis rates for both main and side branch lesions treated. RESULTS: Twenty-six main branches and 27 side branches were treated with this novel technique. Angiographic 6-month follow up was available for all 26 patients treated. There were no postprocedural mortalities and no cases of postprocedural stent thrombosis during the follow-up period. At 6-month anniversary angiography, no evidence of main branch restenosis was seen. However, there were 2 cases of side branch restenosis for a total side branch restenosis rate of 7.4%. Target lesion revascularization for the 26 patients in the study was 7.6%. The total major adverse cardiac event rate for the group as a whole was 15.4%, with 2 periprocedural non-Q-wave myocardial infarctions and 2 target lesions requiring revascularization at 6 months. CONCLUSION: Using currently available DES platforms, the data compiled from this small, single-center pilot study suggest that balloon alignment T-stenting may represent a reasonable approach for the treatment of bifurcation coronary artery disease. However, more comprehensive, larger-scale trials will be required to validate these preliminary findings.

Aged↗

Fluorescence labeling methods for microchannel plate capillary electrophoresis DNA sizing.

DNA analysis on microchannel plate (MCP) capillary electrophoresis platforms will be advanced by the development of new methods for the fluorescence labeling of analytes and standard sizing ladders. Here we evaluate end-labeling of commercially available DNA ladders and polymerase chain reaction (PCR) amplicons with terminal deoxynucleotidyl transferase (TdT) as a method for fluorescent double-stranded DNA sizing analysis. A PCR-based procedure for the facile construction of custom energy-transfer-labeled DNA sizing ladders is also presented. High-resolution sizing of single-stranded DNA fragments is demonstrated with this energy-transfer-labeled ladder. These DNA labeling procedures will be useful for double-and single-stranded DNA analyses on microdevices and other electrophoretic platforms.

Base Sequence↗

Enzymatic activation of second-generation dendritic prodrugs: Conjugation of self-immolative dendrimers with poly(ethylene glycol) via click chemistry.

Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer's core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.

Azides↗

Possible limitation of growth in human fibroblast cultures by diffusion.

Secondary cultures of human diploid fibroblasts which demonstrated density dependent inhibition of cell growth (DDI) were used to study the possible limitation of growth in cell cultures by diffusion. An oscillating platform system is described which insures constant mixing of the medium during the culturing period. Using this system, it was found that a greater number of cells in density inhibited cultures, grown to confluence for four days after initial seeding, could be stimulated to resume growth by a fresh medium change if the cultures were incubated on the oscillating platform than if the cultures were left undisturbed. This greater stimulation on the platform was probably not due to mechanical alterations on the surface of the cells due to motion of the medium as judged by TCA precipitable material released into the medium from cells prelabeled with glucosamine-3H. In spite of this greater stimulation after a single treatment with fresh medium, refeeding the cells on the platform every other day was a 12-day period did not affect the final saturation density achieved in the cultures. The results indicate that diffusion limitation of growth might occur under certain circumstances but that it cannot account entirely for the phenomenon of DDI.

Blood↗

The emergence of multiplexed technologies as diagnostic platforms in systemic autoimmune diseases.

In the last decade, there has been a rapid proliferation of new technologies that are capable of identifying an increasing spectrum of autoantibodies and other biomarkers in autoimmune diseases. These newer diagnostic technologies include line immunoassays, addressable laser bead immunoassays, microarrays in microfluidics platforms and nanobarcode particles. Multiplexed bead assays are a particularly robust platform because they are adaptable to the detection of a variety of disease specific biomarkers, such as autoantibodies, cytokines, adipokines, drugs, oligonucleotides and single nucleotide polymorphisms, Although many laboratories have adopted a variety of these diagnostic platforms to improve turn around times and meet budget constraints, there is an urgent need to ensure that the rapid adoption of new technologies is attended by an appropriate balance of assay sensitivity and specificity.

Autoimmune Diseases↗

Human and mouse oligonucleotide-based array CGH.

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using 'off-the-shelf' oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

Animals↗

A personalized multi-platform assessment of somatic mosaicism in the human frontal cortex.

Somatic mutations in individual cells create genomic mosaicism, influencing genetic disorders and cancers. While clonal mutations in cancers are well-studied, rarer somatic variants in normal tissues remain poorly characterized. This study systematically evaluates detection methods using a personalized donor-specific assembly (DSA) from a neurotypical individual's dorsolateral prefrontal cortex assessed with Oxford Nanopore, NovaSeq, linked-read sequencing, Cas9-targeted long-read sequencing (TEnCATS), and single-neuron MALBAC amplification. The haplotype-resolved DSA improved cross-platform analysis, dramatically increasing phasing rates. Germline SNVs, structural variations (SVs), and transposable elements (TEs) were recalled with 99.4%-99.7% accuracy in bulk tissue, and phased haplotype analysis reduced false positives by 15.4%-75.1% for putative somatic candidates. Long-read single-neuron sequencing detected nine somatic SV candidates, demonstrating enhanced sensitivity for rare variants, while TEnCATS identified eight low-frequency somatic TE candidates. These findings highlight advanced methodologies for precise somatic variant detection, critical for understanding mosaicism's role in health and disease.

Multi-platform Sequencing↗

A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution LC-MS.

MOTIVATION: Comparing two or more complex protein mixtures using liquid chromatography mass spectrometry (LC-MS) requires multiple analysis steps to locate and quantitate natural peptides within a single experiment and to align and normalize findings across multiple experiments. RESULTS: We describe msInspect, an open-source application comprising algorithms and visualization tools for the analysis of multiple LC-MS experimental measurements. The platform integrates novel algorithms for detecting signatures of natural peptides within a single LC-MS measurement and combines multiple experimental measurements into a peptide array, which may then be mined using analysis tools traditionally applied to genomic array analysis. The platform supports quantitation by both label-free and isotopic labeling approaches. The software implementation has been designed so that many key components may be easily replaced, making it useful as a workbench for integrating other novel algorithms developed by a growing research community. AVAILABILITY: The msInspect software is distributed freely under an Apache 2.0 license. The software as well as a Zip file with all peptide feature files and scripts needed to generate the tables and figures in this article are available at http://proteomics.fhcrc.org/.

Algorithms↗

Multiplex diagnostic platforms for detection of biothreat agents.

The availability of rapid, sensitive and cost-effective diagnostic methods is paramount to the success of a comprehensive national health security system in the USA. The national networks that were established to safeguard US infrastructures (e.g., public health, livestock, agriculture and water supply) have developed sufficient capability and capacity for monitoring. However, additional advanced methods will be required to maintain operational readiness. Currently available methods, although sensitive and specific, are generally costly and not amenable to high-throughput analyses. Critical to the success of biothreat surveillance is the ability to screen for and detect multiple agents rapidly in a single reaction and with minimal sample processing. This review will examine currently available diagnostic platforms (i.e., PCR-, immuno- and array-based) and biosensors that can detect multiple biothreat analytes in a single reaction (i.e., multiplex assays). The maturity, benefits and limitations of each platform will be described and a prospective view, from current to future state of the art, will be proposed.

Bioterrorism↗

Homogeneous real-time detection of single-nucleotide polymorphisms by strand displacement amplification on the BD ProbeTec ET system.

BACKGROUND: The BD ProbeTec ET System is based on isothermal strand displacement amplification (SDA) of target nucleic acid coupled with homogeneous real-time detection using fluorescent probes. We have developed a novel, rapid method using this platform that incorporates a universal detection format for identification of single-nucleotide polymorphisms (SNPs) and other genotypic variations. METHOD: The system uses a common pair of fluorescent Detector Probes in conjunction with unlabeled allele-specific Adapter Primers and a universal buffer chemistry to permit analysis of multiple SNP loci under generic assay conditions. We used Detector Probes labeled with different dyes to facilitate differentiation of two alternative alleles in a single reaction with no postamplification manipulation. We analyzed six SNPs within the human beta(2)-adrenergic receptor (beta(2)AR) gene, using whole blood, buccal swabs, and urine samples, and compared results with those obtained by DNA sequencing. RESULTS: Unprocessed whole blood was successfully genotyped with as little as 0.1-1 micro L of sample per reaction. All six beta(2)AR assays were able to accommodate >/==" BORDER="0">20 micro L of unprocessed whole blood. For the 14 individuals tested, genotypes determined with the six beta(2)AR assays agreed with DNA sequencing results. CONCLUSION: SDA-based allelic differentiation on the BD ProbeTec ET System can detect SNPs rapidly, using whole blood, buccal swabs, or urine.

Alleles↗