Beyond the Usual Suspects: Examining the Role of Understudied Histone Variants in Breast Cancer.

The incorporation of histone variants has structural ramifications on nucleosome dynamics and stability. Due to their unique sequences, histone variants can alter histone-histone or histone-DNA interactions, impacting the folding of DNA around the histone octamer and the overall higher-order structure of chromatin fibers. These structural modifications alter chromatin compaction and accessibility of DNA by transcription factors and other regulatory proteins to influence gene regulatory processes such as DNA damage and repair, as well as transcriptional activation or repression. Histone variants can also generate a unique interactome composed of histone chaperones and chromatin remodeling complexes. Any of these perturbations can contribute to cellular plasticity and the progression of human diseases. Here, we focus on a frequently overlooked group of histone variants lying within the four human histone gene clusters and their contribution to breast cancer.

A Benchtop Automated Sputum-to-Genotype System Using a Lab-on-a-Film Assembly for Detection of Multidrug-Resistant Mycobacterium tuberculosis.

Automated genotyping of drug-resistant Mycobacterium tuberculosis (MTB) directly from sputum is challenging for three primary reasons. First, the sample matrix, sputum, is highly viscous and heterogeneous, posing a challenge for sample processing. Second, acid-fast MTB bacilli are difficult to lyse. And third, there are hundreds of MTB mutations that confer drug resistance. An additional constraint is that MTB is most prevalent where test affordability is paramount. We address the challenge of sample homogenization and cell lysis using magnetic rotation of an external magnet, at high (5000) rpm, to induce the rotation of a disposable stir disc that causes chaotic mixing of glass beads ("MagVor"). Nucleic acid is purified using a pipet tip with an embedded matrix that isolates nucleic acid ("TruTip"). We address the challenge of cost and genotyping multiple mutations using 203 porous three-dimensional gel elements printed on a film substrate and enclosed in a microfluidic laminate assembly ("Lab-on-a-Film"). This Lab-on-a-Film assembly (LFA) serves as a platform for amplification, hybridization, washing, and fluorescent imaging, while maintaining a closed format to prevent amplicon contamination of the workspace. We integrated and automated MagVor homogenization, TruTip purification, and LFA amplification in a multisample, sputum-to-genotype system. Using this system, we report detection down to 43 cfu/mL of MTB bacilli from raw sputum.

Genotyping Multidrug-Resistant Mycobacterium tuberculosis from Primary Sputum and Decontaminated Sediment with an Integrated Microfluidic Amplification Microarray Test.

There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) Mycobacterium tuberculosis into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with ∼25 M. tuberculosis genomes (100 fg of M. tuberculosis DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C. The test detected M. tuberculosis in 99.1% of sputum extracts and 100% of sediment extracts and showed 100% concordance with the results of real-time PCR. The levels of concordance between M. tuberculosis and resistance-associated gene detection were 99.1% and 98.4% for sputum and sediment extracts, respectively. Genotyping results were 100% concordant between sputum and sediment extracts. Relative to the results of culture-based drug susceptibility testing, the test was 97.1% specific and 75.0% sensitive for the detection of rifampin resistance in both sputum and sediment extracts. The specificity for the detection of isoniazid (INH) resistance was 98.4% and 96.8% for sputum and sediment extracts, respectively, and the sensitivity for the detection of INH resistance was 63.6%. The amplification microarray reported the correct genotype for all discordant phenotype/genotype results. On the basis of these data, primary sputum may be considered a preferred specimen for the test. The amplification microarray design, shelf life, and analytical performance metrics are well aligned with consensus product profiles for next-generation drug-resistant M. tuberculosis diagnostics and represent a significant ease-of-use advantage over other hybridization-based tests for diagnosing MDR tuberculosis.

Simplified microarray system for simultaneously detecting rifampin, isoniazid, ethambutol, and streptomycin resistance markers in Mycobacterium tuberculosis.

We developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. In a blinded analysis of 153 clinical isolates, microarray sensitivity for first-line drugs relative to phenotypic DST (true resistance) was 100% for rifampin (RIF) (14/14), 90.0% for isoniazid (INH) (36/40), 70% for ethambutol (EMB) (7/10), and 89.1% (57/64) combined. Microarray specificity (true susceptibility) for first-line agents was 95.0% for RIF (132/139), 98.2% for INH (111/113), and 98.6% for EMB (141/143). Overall microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395). The overall positive and negative predictive values for RIF, INH, and EMB combined were 84.9% and 98.3%, respectively. For the second-line drug streptomycin (STR), overall concordance between the agar proportion method and microarray analysis was 89.5% (137/153). Sensitivity was 34.8% (8/23) because of limited microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130). All false-susceptible discrepant results were a consequence of DNA mutations that are not represented by a specific microarray probe. There were zero invalid results from 220 total tests. The simplified microarray system is suitable for detecting resistance-conferring mutations in clinical M. tuberculosis isolates and can now be used for prospective trials or integrated into an all-in-one, closed-amplicon consumable.

A dynamic model of inorganic arsenic-induced carcinogenesis reveals an epigenetic mechanism for epithelial-mesenchymal plasticity.

Inorganic arsenic (iAs) causes cancer by initiating dynamic transitions between epithelial and mesenchymal cell phenotypes. These transitions transform normal cells into cancerous cells, and cancerous cells into metastatic cells. Most in vitro models assume that transitions between states are binary and complete, and do not consider the possibility that intermediate, stable cellular states might exist. In this paper, we describe a new, two-hit in vitro model of iAs-induced carcinogenesis that extends to 28 weeks of iAs exposure. Through week 17, the model faithfully recapitulates known and expected phenotypic, genetic, and epigenetic characteristics of iAs-induced carcinogenesis. By 28 weeks, however, exposed cells exhibit stable, intermediate phenotypes and epigenetic properties, and key transcription factor promoters (SNAI1, ZEB1) enter an epigenetically poised or bivalent state. These data suggest that key epigenetic transitions and cellular states exist during iAs-induced epithelial-to-mesenchymal transition (EMT), and that it is important for our in vitro models to encapsulate all aspects of EMT and the mesenchymal-to-epithelial transition (MET). In so doing, and by understanding the epigenetic systems controlling these transitions, we might find new, unexpected opportunities for developing targeted, cell state-specific therapeutics.

EZH2-driven immune evasion defines high-risk pediatric AML with t(16;21) FUS::ERG gene fusion.

Minimal improvement in outcomes for high-risk pediatric acute myeloid leukemia (pAML) patients has been made in the past decades. Nowhere is this more evident than in patients carrying a t(16;21)(p11;q22) FUS::ERG translocation; quick time to relapse and universal failure of hematopoietic stem cell transplant contribute to one of the lowest survival rates in childhood leukemia. Here, we have identified a unique, defining immune-evasion phenotype in FUS::ERG pAML driven by EZH2 and characterized by loss of MHC class I and II molecules and immune co-stimulatory receptors. This loss of immune engagement, present at diagnosis, allows pervasiveness of blasts that prove resistant to standard treatment. We demonstrate that treatment with the FDA-approved EZH2 inhibitor tazemetostat, in combination with IFN-γ, reverses the phenotype, re-expresses MHC receptor expression, and reduces blast viability. EZH2 inhibitors provide a novel therapeutic option for this high-risk population and may prove a beneficial supplemental treatment for FUS::ERG pAML.

Profiling in situ microbial community structure with an amplification microarray.

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO(3)(-)) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO(3), but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

Developmental priming of cancer susceptibility.

DNA mutations are necessary drivers of cancer, yet only a small subset of mutated cells go on to cause the disease. To date, the mechanisms that determine which rare subset of cells transform and initiate tumorigenesis remain unclear. Here, we take advantage of a unique model of intrinsic developmental heterogeneity (Trim28+/D9) and demonstrate that stochastic early life epigenetic variation can trigger distinct cancer-susceptibility 'states' in adulthood. We show that these developmentally primed states are characterized by differential methylation patterns at typically silenced heterochromatin, and that these epigenetic signatures are detectable as early as 10 days of age. The differentially methylated loci are enriched for genes with known oncogenic potential. These same genes are frequently mutated in human cancers, and their dysregulation correlates with poor prognosis. These results provide proof-of-concept that intrinsic developmental heterogeneity can prime individual, life-long cancer risk.

Nonvolatile copolymer compositions for fabricating gel element microarrays.

By modifying polymer compositions and cross-linking reagents, we have developed a simple yet effective manufacturing strategy for copolymerized three-dimensional gel element arrays. A new gel-forming monomer, 2-(hydroxyethyl) methacrylamide (HEMAA), was used. HEMAA possesses low volatility and improves the stability of copolymerized gel element arrays to on-chip thermal cycling procedures relative to previously used monomers. Probe immobilization efficiency within the new polymer was 55%, equivalent to that obtained with acrylamide (AA) and methacrylamide (MA) monomers. Nonspecific binding of single-stranded targets was equivalent for all monomers. Increasing cross-linker chain length improved hybridization kinetics and end-point signal intensities relative to N,N-methylenebisacrylamide (Bis). The new copolymer formulation was successfully applied to a model orthopox array. Because HEMAA greatly simplifies gel element array manufacture, we expect it (in combination with new cross-linkers described here) to find widespread application in microarray science.

Independent phenotypic plasticity axes define distinct obesity sub-types.

Studies in genetically 'identical' individuals indicate that as much as 50% of complex trait variation cannot be traced to genetics or to the environment. The mechanisms that generate this 'unexplained' phenotypic variation (UPV) remain largely unknown. Here, we identify neuronatin (NNAT) as a conserved factor that buffers against UPV. We find that Nnat deficiency in isogenic mice triggers the emergence of a bi-stable polyphenism, where littermates emerge into adulthood either 'normal' or 'overgrown'. Mechanistically, this is mediated by an insulin-dependent overgrowth that arises from histone deacetylase (HDAC)-dependent ß-cell hyperproliferation. A multi-dimensional analysis of monozygotic twin discordance reveals the existence of two patterns of human UPV, one of which (Type B) phenocopies the NNAT-buffered polyphenism identified in mice. Specifically, Type-B monozygotic co-twins exhibit coordinated increases in fat and lean mass across the body; decreased NNAT expression; increased HDAC-responsive gene signatures; and clinical outcomes linked to insulinemia. Critically, the Type-B UPV signature stratifies both childhood and adult cohorts into four metabolic states, including two phenotypically and molecularly distinct types of obesity.

Lab-on-a-Film disposable for genotyping multidrug-resistant Mycobacterium tuberculosis from sputum extracts.

We describe a Lab-on-a-Film disposable that detects multidrug-resistant tuberculosis (MDR-TB) from sputum extracts. The Lab-on-a-Film disposable consists of 203 gel elements that include DNA sequences (probes) for 37 mutations, deletions, or insertion elements across 5 genes (including an internal control). These gel elements are printed on a flexible film, which costs approximately 500 times less than microarray glass. The film with printed gel elements is then laminated to additional rollable materials (films) to form a microfluidic flow cell. We combined multiplex amplification and hybridization steps in a single microfluidic chamber, without buffer exchanges or other manipulations up to and throughout hybridization. This flow cell also incorporates post hybridization wash steps while retaining an entirely closed-amplicon system, thus minimizing the potential for sample or amplicon cross-contamination. We report analytical sensitivity of 32 cfu mL-1 across all MDR-TB markers and detection of MDR-TB positive clinical specimens using an automated TruTip workstation for extraction and the Lab-on-a-Film disposable for amplification and detection of the extracts.

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray.

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.

Automated TruTip nucleic acid extraction and purification from raw sputum.

Automated nucleic acid extraction from primary (raw) sputum continues to be a significant technical challenge for molecular diagnostics. In this work, we developed a prototype open-architecture, automated nucleic acid workstation that includes a mechanical homogenization and lysis function integrated with heating and TruTip purification; optimized an extraction protocol for raw sputum; and evaluated system performance on primary clinical specimens. Eight samples could be processed within 70 min. The system efficiently homogenized primary sputa and doubled nucleic acid recovery relative to an automated protocol that did not incorporate sample homogenization. Nucleic acid recovery was at least five times higher from raw sputum as compared to that of matched sediments regardless of smear or culture grade, and the automated workstation reproducibly recovered PCR-detectable DNA to at least 80 CFU mL-1 raw sputum. M. tuberculosis DNA was recovered and detected from 122/123 (99.2%) and 124/124 (100%) primary sputum and sediment extracts, respectively. There was no detectable cross-contamination across 53 automated system runs and amplification or fluorescent inhibitors (if present) were not detectable. The open fluidic architecture of the prototype automated workstation yields purified sputum DNA that can be used for any molecular diagnostic test. The ability to transfer TruTip protocols between personalized, on-demand pipetting tools and the fully automated workstation also affords public health agencies an opportunity to standardize sputum nucleic acid sample preparation procedures, reagents, and quality control across multiple levels of the health care system.

A bench-top automated workstation for nucleic acid isolation from clinical sample types.

Systems that automate extraction of nucleic acid from cells or viruses in complex clinical matrices have tremendous value even in the absence of an integrated downstream detector. We describe our bench-top automated workstation that integrates our previously-reported extraction method - TruTip - with our newly-developed mechanical lysis method. This is the first report of this method for homogenizing viscous and heterogeneous samples and lysing difficult-to-disrupt cells using "MagVor": a rotating magnet that rotates a miniature stir disk amidst glass beads confined inside of a disposable tube. Using this system, we demonstrate automated nucleic acid extraction from methicillin-resistant Staphylococcus aureus (MRSA) in nasopharyngeal aspirate (NPA), influenza A in nasopharyngeal swabs (NPS), human genomic DNA from whole blood, and Mycobacterium tuberculosis in NPA. The automated workstation yields nucleic acid with comparable extraction efficiency to manual protocols, which include commercially-available Qiagen spin column kits, across each of these sample types. This work expands the scope of applications beyond previous reports of TruTip to include difficult-to-disrupt cell types and automates the process, including a method for removal of organics, inside a compact bench-top workstation.

Saliva-based diagnostics using 16S rRNA microarrays and microfluidics.

The development of a diagnostic system based on DNA microarrays for rapid identification and enumeration of microbial species in the oral cavity is described. This system uses gel-based microarrays with immobilized probes designed within a phylogenetic framework that provides for comprehensive microbial monitoring. Understanding the community structure in the oral cavity is a necessary foundation on which to understand the breadth and depth of different microbial communities in the oral cavity and their role in acute and systemic disease. Our ultimate goal is to develop a diagnostic device to identify individuals at high risk for oral disease, and thereby reduce its prevalence and therefore the economic burden associated with treatment. This article discusses recent improvements of our system in reducing diffusional constraints in order to provide more rapid and accurate measurements of the microbial composition of saliva.

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.

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.

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.

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.

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.