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Biomedical subjects

Darrell P Chandler

Publications and source records attributed to Darrell P Chandler.

18 recordsLinked to original sources
PubMed2025

EZH2-driven immune evasion at disease presentation defines a targetable high-risk subset of acute leukemia exemplified by t(16;21) FUS::ERG AML.

The past 25 years of clinical trials have produced few improvements in pediatric AML (pAML) outcomes. This is acutely evident in patients with t(16;21)(p11;q22), yielding FUS::ERG. Patients with FUS::ERG-positive AML relapse quickly and do not respond to transplantation. Major histocompatibility complex (MHC) class I & II receptors and costimulatory molecules are absent at diagnosis in FUS::ERG-positive AML, mirroring the phenotype and outcomes of post-transplant relapse. We show that this is driven by overexpression of EZH2, in vitro and in multiple clinical cohorts. While FUS::ERG AML is the most extreme example, this phenotype is shared by lethal CBFA2T3::GLIS2-driven AML, and patients with RUNX1::RUNX1T1 have significantly worse outcomes when EZH2 overexpression co-occurs. The FDA-approved EZH2 inhibitor tazemetostat reverses this phenotype, re-establishes MHC presentation, and elicits immune effector cell-mediated elimination. EZH2 inhibitors may provide the first targeted therapeutic frontline option for AML patients with FUS::ERG, with the potential for broader frontline immunostimulatory benefits.

Journal Article↗
PubMed2006

Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation.

Methods to assess the quality and performance of protein microarrays fabricated from undefined protein content are required to elucidate slide-to-slide variability and interpolate resulting signal intensity values after an interaction assay. We therefore developed several simple total- and posttranslational modification-specific, on-chip staining methods to quantitatively assess the quality of gel element protein arrays manufactured with whole-cell lysate in vitro protein fractions derived from two-dimensional liquid-phase fractionation (PF2D) technology. A linear dynamic range of at least 3 logs was observed for protein stains and immobilized protein content, with a lower limit of detection at 8 pg of protein per gel element with Deep Purple protein stain and a field-portable microarray imager. Data demonstrate the successful isolation, separation, transfer, and immobilization of putative transmembrane proteins from Yersinia pestis KIM D27 with the combined PF2D and gel element array method. Internal bovine serum albumin standard curves provided a method to assess on-chip PF2D transfer and quantify total protein immobilized per gel element. The basic PF2D array fabrication and quality assurance/quality control methods described here therefore provide a standard operating procedure and basis for developing whole-proteome arrays for interrogating host-pathogen interactions, independent of sequenced genomes, affinity tags, or a priori knowledge of target cell composition.

Animals↗
PubMed2006

Technology development to explore the relationship between oral health and the oral microbial community.

The human oral cavity contains a complex microbial community that, until recently, has not been well characterized. Studies using molecular tools have begun to enumerate and quantify the species residing in various niches of the oral cavity; yet, virtually every study has revealed additional new species, and little is known about the structural dynamics of the oral microbial community or how it changes with disease. Current estimates of bacterial diversity in the oral cavity range up to 700 species, although in any single individual this number is much lower. Oral microbes are responsible for common chronic diseases and are suggested to be sentinels of systemic human diseases. Microarrays are now being used to study oral microbiota in a systematic and robust manner. Although this technology is still relatively young, improvements have been made in all aspects of the technology, including advances that provide better discrimination between perfect-match hybridizations from non-specific (and closely-related) hybridizations. This review addresses a core technology using gel-based microarrays and the initial integration of this technology into a single device needed for system-wide studies of complex microbial community structure and for the development of oral diagnostic devices.

Journal Article↗
PubMed2006

Suspension array analysis of 16S rRNA from Fe- and SO(4)2- reducing bacteria in uranium-contaminated sediments undergoing bioremediation.

A 16S rRNA-targeted tunable bead array was developed and used in a retrospective analysis of metal- and sulfate-reducing bacteria in contaminated subsurface sediments undergoing in situ U(VI) bioremediation. Total RNA was extracted from subsurface sediments and interrogated directly, without a PCR step. Bead array validation studies with total RNA derived from 24 isolates indicated that the behavior and response of the 16S rRNA-targeted oligonucleotide probes could not be predicted based on the primary nucleic acid sequence. Likewise, signal intensity (absolute or normalized) could not be used to assess the abundance of one organism (or rRNA) relative to the abundance of another organism (or rRNA). Nevertheless, the microbial community structure and dynamics through time and space and as measured by the rRNA-targeted bead array were consistent with previous data acquired at the site, where indigenous sulfate- and iron-reducing bacteria and near neighbors of Desulfotomaculum were the organisms that were most responsive to a change in injected acetate concentrations. Bead array data were best interpreted by analyzing the relative changes in the probe responses for spatially and temporally related samples and by considering only the response of one probe to itself in relation to a background (reference) environmental sample. By limiting the interpretation of the data in this manner and placing it in the context of supporting geochemical and microbiological analyses, we concluded that ecologically relevant and meaningful information can be derived from direct microarray analysis of rRNA in uncharacterized environmental samples, even with the current analytical uncertainty surrounding the behavior of individual probes on tunable bead arrays.

Bacteria↗
PubMed2006

Diagnostic oligonucleotide microarray fingerprinting of Bacillus isolates.

A genome-independent microarray and new statistical techniques were used to genotype Bacillus strains and quantitatively compare DNA fingerprints with the known taxonomy of the genus. A synthetic DNA standard was used to understand process level variability and lead to recommended standard operating procedures for microbial forensics and clinical diagnostics.

Bacillus↗
PubMed2005

Comparing bacterial DNA microarray fingerprints.

Epidemiologic and forensic investigations often require assays to detect subtle genetic differences between closely related microorganisms. Typically, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial samples, where the patterns of DNA fragment sizes are viewed as genotype 'fingerprints'. The limited genomic sample captured on a gel, however, is not always sufficient to discriminate closely related strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if enough independent oligonucleotide probes are used on the microarray, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate a statistical measurement error model to fingerprint 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

Journal Article↗
PubMed2005

Automated Microarray Image Analysis Toolbox for MATLAB.

UNLABELLED: The Automated Microarray Image Analysis (AMIA) Toolbox for MATLAB is a flexible, open-source, microarray image analysis tool that allows the user to customize analyses of microarray image sets. This tool provides several methods to identify and quantify spot statistics, as well as extensive diagnostic statistics and images to evaluate data quality and array processing. The open, modular nature of AMIA provides access to implementation details and encourages modification and extension of AMIA's capabilities. AVAILABILITY: The AMIA Toolbox is freely available at http://www.pnl.gov/statistics/amia. The AMIA Toolbox requires MATLAB 6.5 (R13) (MathWorks, Inc. Natick, MA), as well as the Statistics Toolbox 4.1 and Image Processing Toolbox 4.1 for MATLAB or more recent versions. CONTACT: amanda.white@pnl.gov

Algorithms↗
PubMed2005

Taking arrays from the lab to the field: trying to make sense of the unknown.

With the rapid pace of nucleic acid microarray technology development and a renewed national emphasis on detecting and characterizing microorganisms in environmental samples, there is a rush to operationalize existing microarray technologies and apply them to uncharacterized environmental backgrounds. The purpose of this article is to pause and ask a basic question: what do microarray data actually mean in the face of uncharacterized sample backgrounds? In attempting to answer this question, we draw a clear distinction between hypothesis-driven fundamental science and operational uses of microarray technology; assess microarray technology assumptions in the face of uncharacterized environments; offer an environmental microbiologist's perspective on technology needs and requirements for quantitatively analyzing microbial communities; and hopefully stimulate a scientific and technical dialogue around the concept of analytical environmental microbiology and future technology development.

Bacteria↗
PubMed2005

Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array.

Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.

Equipment Design↗
PubMed2004

Quantitative oligonucleotide microarray fingerprinting of Salmonella enterica isolates.

We report on a genome-independent microbial fingerprinting method using nucleic acid microarrays for microbial forensics and epidemiology applications and demonstrate that the microarray method provides high resolution differentiation between closely related microorganisms, using Salmonella enterica strains as the test case. In replicate trials we used a simple 192 probe nonamer array to construct a fingerprint library of 25 closely related Salmonella isolates. Controlling false discovery rate for multiple testing at alpha = 0.05, at least 295 of 300 pairs of S.enterica isolate fingerprints were found to be statistically distinct using a modified Hotelling T2 test. Although most pairs of Salmonella fingerprints are found to be distinct, forensic applications will also require a protocol for library construction and reliable microbial classification against a fingerprint library. We outline additional steps required to produce such a protocol.

Bayes Theorem↗
PubMed2004

Automated purification and suspension array detection of 16S rRNA from soil and sediment extracts by using tunable surface microparticles.

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA ( approximately 10(4) cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA ( approximately 10(5) cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.

Automation↗
PubMed2003

Enhanced nucleic acid capture and flow cytometry detection with peptide nucleic acid probes and tunable-surface microparticles.

New methods for automated, direct nucleic acid purification and detection are required for the next generation of unattended environmental monitoring devices. In this study we investigated whether tunable-surface bead chemistry and peptide nucleic acids (PNA) could enhance the recovery and detection of intact rRNA in both test tube and automated suspension array hybridization formats. Intact rRNA was easily captured and detected on PNA-coated Lumavidin beads from 0.1 ng total RNA with a 15-min hybridization in pH 7 buffer, representing 1.7 x 10(3) cell equivalents of total RNA. DNA-conjugated beads in pH 5 hybridization buffer required an overnight hybridization to achieve a detectable signal at 0.1 ng target RNA. Standard DNA hybridization conditions (pH 7) were one order of magnitude less sensitive than the tunable-surface (pH 5) condition. The PNA-conjugated particles were 100x more sensitive than the tunable-surface DNA particles in the automated format, with a detection limit of 0.1 ng total RNA. The detection limits for total RNA on PNA-conjugated microparticles is immediately conducive to the detection and characterization of microorganisms in low-biomass environments or to the identification of rare sequences in a complex sample mixture, without using PCR.

Bacillus↗
PubMed2003

Towards a unified system for detecting waterborne pathogens.

Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. This review discusses some of the challenges in developing a universal pathogen detection method, current technology that may be employed to overcome these challenges, and the remaining needs for developing an integrated pathogen detection and monitoring system for source or finished water.

Animals↗
PubMed2003

Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays.

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.

Base Sequence↗
PubMed2002

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays.

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.

DNA Fingerprinting↗
PubMed2002

Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray.

We investigated the application of an oligonucleotide microarray to (i) specifically detect Cryptosporidium spp., (ii) differentiate between closely related C. parvum isolates and Cryptosporidium species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the hsp70 gene of Cryptosporidium parvum was constructed. Labeled hsp70 targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I C. parvum isolates (TU502 and UG502), two C. parvum genotype II isolates (Iowa and GCH1), and DNAs from 22 non-Cryptosporidium sp. organisms were used to test method specificity. Only DNAs from C. parvum isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.

Animals↗
PubMed2002

Advances towards integrated biodetection systems for environmental molecular microbiology.

To overcome many of the limitations associated with indirect detection methods, new techniques for the sensitive, specific, and direct detection of nucleic acids are required in order to accurately and quantitatively ascribe phenotype/function to uncultivated microorganisms. However, if advanced diagnostic and detection systems are going to be applied in environmental microbiology, future "biodetection" technologies and systems must be developed not from the point of view of the detector, but from the unique aspects of the environmental sample and the entire analytical process. This article highlights recent advances in nucleic acid-based technologies, and looks towards future advances that may address the broad needs and conditions imposed by environmental molecular microbiology.

Bacteria↗