High-density Screening Research Articles

Classification of crystallization outcomes using deep convolutional neural networks.

The Machine Recognition of Crystallization Outcomes (MARCO) initiative has assembled roughly half a million annotated images of macromolecular crystallization experiments from various sources and setups. Here, state-of-the-art machine learning algorithms are trained and tested on different parts of this data set. We find that more than 94% of the test images can be correctly labeled, irrespective of their experimental origin. Because crystal recognition is key to high-density screening and the systematic analysis of crystallization experiments, this approach opens the door to both industrial and fundamental research applications.

Embryo Biopsy for Aneuploidy Detection in the General Infertility Population

Since report of the first live birth following preimplantation genetic screening (PGS) in 1995, the procedure and available technologies for aneuploidy detection have rapidly evolved. Through these efforts, the biology of meiotic and mitotic segregation errors has been partially elucidated. A process that began with polar body biopsy and four-color fluorescence in situ hybridization to detect copy number in a limited number of chromosomes is now hardly recognizable: current molecular methods permit high-density screening of the entire human genome for copy number variants, structural rearrangements, microdeletions, and polyploids to a resolution of 35 kilobases in less than 48 hours. Indeed, with the advent of real-time quantitative analyses of ploidy status that allow same-day trophectoderm biopsy with fresh transfer of a euploid blastocyst, the future is bright for PGS. Questions remain about how best to safely offer this technology to patients, and which patients, if any, will benefit from routine biopsy. Herein, we will review the limited available evidence for application of PGS in the general infertility population as an adjunct method to optimize live birth rates.

Screening for Mevalonate Biosynthetic Pathway Inhibitors Using Sensitized Bacterial Strains

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.

Autoassembly Protein Arrays for Analyzing Antibody Cross-Reactivity

We report an autoassembly protein array capable of rapidly screening for aberrant antibody−antigen binding events. Our technique combines magnetic nanoparticle technology with proximity-based, magnetically responsive nanosensors for rapid (under 15 min) and high-density screening of antibody cross-reactivity at sensitivities down to 50 fM in a homogeneous assay. This method will enable the identification of the precise cause of aberrant or cross-reactive binding events in an easy-to-use, rapid, and high-throughput manner.

A novel light‐dependent selection marker system in plants

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.

High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors

The existence of multiple subsets of chronic lymphocytic leukemia expressing 'stereotyped' B-cell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that 'stereotypy' may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets. We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients. Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and non-subset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy. Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.

An oligonucleotide microarray to characterize multidrug resistant plasmids

Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replicon type Inc A/C and Inc H1. It is important to understand the transmission of these MDR plasmids because the genes they carry can affect the outcome of antimicrobial therapy. The aim of this study was to design a microarray with oligonucleotide probes for every gene in the six Inc A/C and one Inc H1 plasmids of interest while representing all redundant sequences only once. The microarray is printed in triplicate with 493 unique oligonucleotide probes 70 nucleotides in length. Salmonella enterica and Escherichia coli control strains and test plasmids (in the parent strain and transformed into a known E. coli background strain) were hybridized to the plasmid microarray. This hybridization arrays presents a rapid and cost effective method for high-density screening of isolates to evaluate the gene content of Inc A/C and H1 plasmids and will show how plasmids can change content with transmission.

Developing Specific Markers and Improving Genetic Mapping for a Major Locus <em>Tyr1</em> of Citrus Nematode Resistance

The NBS-LRR class resistance-gene candidate sequences (Pt8a and Pt9a) were used to develop new specific markers to the citrus nematode resistance gene locus Tyr1 . By high-density colony screening, over 200 positive clones were pulled out from the BAC library. A few of the clones were found to be closely linked with the Tyr1 region, because the primers from these clones insert sequence produced polymorphism which matched up with the phenotype after bulked segregant analysis. By primer walking approach, three integrate NBS-LRR class resistance-gene sequences were tagged and identified separately in three clones (7A4, 4L17 and 29F20). More specific markers were developed from these tagged sequences and relatively high-density genetic maps were constructed by incorporating the newly developed markers and previously developed markers in the ‘9145 family’. New markers were applied in ‘9401 family’ trying to estimate roughly the genetic distance between the Ctv and Tyr1 region.

Integrated patch-clamp biosensor for high-density screening of cell conductance

The first fully integrated implementation of a patch-clamp measurement system is presented. The system was implemented in a 0.5 µm silicon-on-sapphire process. The system can record cell membrane currents up to ±20 nA, with an rms noise of 5 pA at 10 kHz bandwidth. The system can compensate for the capacitance and resistance of the pipette electrode, up to 20 pF and 4 MΩ, respectively. The die size is 1150 by 700 µm. The power consumption is 3.3 mW at 3.3 V.

Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants

The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

Quantifying Bioactivity on a Large Scale: Quality Assurance and Analysis of Multiparametric Ultra-HTS Data

There is a growing need to precisely quantify the selectivity of large compound sets in high throughput screening, directing investment in lead optimization towards compounds with a high chance of success. High-content, high-density screening technologies such as multiparametric ultra-HTS provide a basis for highly precise screening with unprecedented scope for delineating process artifacts from reliable signals. However, the full potential of these technologies can only be realized with suitable experimental design and sophisticated data analysis tools. We present two advanced analysis workflows demonstrating how multiparametric readouts from a high throughput primary screen can improve decision quality in the hit identification process. The first involves discrete thresholding and the application of multiple selection criteria. The second uses machine learning algorithms and allows an unbiased consideration of all measured parameters.

High-density screening of the Zbtb7gene in breast cancer patients

It has been proposed that the excess of the familiar risk associated with breast cancer could be explaining by multiple weakly predisposing alleles. Hence there is much interest in the search for low-penetrance genes/variants for breast cancer, which exist with high prevalence in the general population. Maeda and colleagues [1] recently identified the transcriptional repressor FBI1, which they called Pokemon (POK erythroid myeloid ontogenic factor), as a critical factor in oncogenesis. This protein is codified by the ZBTB7 gene ('zing finger and BTB domain containing 7'; GeneID, 51341). Mouse embyronic fibroblasts lacking Zbtb7 were completely refractory to oncogene-mediated cellular transformation. Conversely, FBI1 overexpression led to overt oncogenic transformation both in vitro and in vivo in transgenic mice. FBI1 can specifically repress the transcription of the tumor suppressor gene ARF (600160). Maeda and colleagues [1] found that FBI1 is aberrantly overexpressed in human cancers, and its expression levels predict biologic behavior and clinical outcome. On the other hand, tissue microarray analysis (TMA) in breast carcinomas has revealed high levels of Pokemon expression in a subset of these tumors. In addition, the genomic region where the ZBTB7 gene resides (19p13.3) is a hotspot for chromosomal translocations [2]. ZBTB7 is therefore a good candidate for a breast cancer low-penetrance gene. We aim to determine whether common polymorphisms (frequency ≥ 10%) in the ZBTB7 gene are associated with breast cancer risk in Spanish patients. A set of 22 validated binary SNP polymorphisms were selected from public databases (e.g. HapMap) and private databases (e.g. Celera and RealSNP) according to criteria of coverage (average 1 SNP/1.7 kb). These SNPs cover the flanking regions (10,000 bp), including the promoter region, introns, and coding non-synonymous SNPs. About 550 cases and 550 controls have been genotyped. Genotyping was performed using the MassARRAY SNP genotyping system (Sequenom Inc., San Diego, CA, USA). To our knowledge, this is the first time that the ZBTB7 gene has been analyzed in breast cancer patients. The results will be presented at the time of the congress and will be interpreted in the light of the worldwide population study shown in another presentation from the same authors.

Nonlinear screening and stopping power in two-dimensional electron gases

We have used density-functional theory to study the nonlinear screening properties of a two-dimensional (2D) electron gas. In particular, we consider the screening of an external static point charge of magnitude $Z$ as a function of the distance of the charge from the plane of the gas. The self-consistent screening potentials are then used to determine the 2D stopping power in the low-velocity limit based on the momentum transfer cross section. Calculations as a function of $Z$ establish the limits of validity of linear and quadratic response theory calculations, and show that nonlinear screening theory already provides significant corrections in the case of protons. In contrast to the 3D situation, we find that the nonlinearly screened potential supports a bound state even in the high-density limit. This behavior is elucidated with the derivation of a high-density screening theorem which proves that the screening charge can be calculated perturbatively in the high-density limit for arbitrary dimensions. However, the theorem has particularly interesting implications in 2D where, contrary to expectations, we find that perturbation theory remains valid even when the perturbing potential supports bound states.

Fluorescent peptide probes for high-throughput measurement of protein phosphatases.

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.

Use of phage display and high-density screening for the isolation of an antibody against the 51-kDa subunit of complex I

Monoclonal antibodies play an increasingly important role in structural biology. In this report, we develop the use of phage display technology for the isolation of an antibody that binds to a specific subunit of a macromolecular assembly. Antibodies that bind to the intact complex are selected from a phage display library and screened with a high-density Western blot to identify a subunit-specific binder. Conventional Western blotting and competition ELISA are then used to confirm the identity of the target subunit and that the antibody binds to the native protein complex and not to an epitope that is only revealed when the antibody is immobilized for phage selection. Using this technique, monoclonal scFv and Fab fragments have been produced that bind to the 51-kDa subunit of bovine complex I, a large integral membrane protein complex from mitochondria.

Microarray Technology and Applications: An All-Language Literature Survey Including Books and Patents

The development and use of microarrays are expanding rapidly, making it difficult to find comprehensive sources of information about them. This all-language literature survey categorizes and lists papers, abstracts, reviews, books, and patents on the topic of microarrays, published up to the end of the year 2000. It has been compiled from searches of OVID Medline, INSPEC, Biosis, PubMed, and various patent databases (http://www.uspto.gov/; http://www.jpo.go.jp/; http://gb.espacenet.com/). The listing of references for each of the 11 sections can be found at Clinical Chemistry Online (www.clinchem.org/content/vol47/issue8/). A microarray is an analytical device that comprises an array of molecules (oligonucleotides, cDNAs, clones, PCR products, polypeptides, antibodies, and others) or tissue sections immobilized at discrete ordered or nonordered micrometer-to-millimeter-sized locations on the surface of a porous or nonporous insoluble solid support. These devices have been highly effective for the simultaneous detection of large numbers of analytes in a sample, and microarrays have quickly emerged as important analytical tools in many branches of the biological sciences. A microarray-based analytical strategy is quicker and more convenient than serial testing for each analyte, and it has been successfully applied to both immunoassays and DNA-based assays. The current scope of microarray applications includes sequencing by hybridization, resequencing, mutation detection, assessment of gene copy number, comparative genome hybridization, drug discovery, expression analysis, and immunoassay (protein microarrays). In addition, oligonucleotide microarrays have been used for a nonbiological application: computing. The task of compiling this survey has been complicated by the diverse and nonstandardized nomenclature for microarrays. Arrays have been named for their application (hybridization chip, high-density screening filters, genosensor), the identity of the arrayed molecules (cDNA library array, high-density gridded cDNA library, “printed circuit for proteins”, colony array, tissue array), the physical attributes of the array or the substrate on which the array is formed (matrix, two-dimensional …

A high-density differential screening strategy for identification of fovea genes with altered expression in the retina during aging

A human fovea cDNA library was arrayed at high-density and reacted with age-specific (4 yr & 80 yr) retinal probes to identify genes with altered expression during aging. Using this approach, we screened 55,368 genes by Southern analysis and determined that ∼0.7% are differentially expressed in young and old tissues. Northern analysis of total RNA from retina of humans aged 2–94 yr show that expression of one of the clones identified (clone dd112) decreases with aging.

Robotics, computing, and biology An interdisciplinary approach to the analysis of complex genomes

A staggering amount of genetic information is contained within the DNA of complex organisms such as humans. Of these vast tracts of DNA code, only very small regions actually constitute the 'genes'; with the information ultimately translated into proteins, the basic machinery of cells. However, a complex organism has a cell or tissue type and developmental specific pattern of gene expression which interact in complex networks within and between these cells or tissues. Such a combination of vast tracts of genomic DNA, tissue and developmental specific transcription of small subsets of this information, plus complex interactions between gene products provides a daunting scientific and logistical challenge if a global picture of gene function within complex organisms is to be obtained. This objective can be achieved only through an interdisciplinary combination of biology, computer image analysis, and robotic engineering. The development of a global approach to the understanding of complex genomes is not just an academic challenge. For example, the ability to understand the complex network of gene interactions, or to be able rapidly to screen many genetic samples in parallel, would have enormous consequences for the pharmaceutical and diagnostic industries. The systems described are used in several genetic screening or drug discovery centres that require rapid, reliable, high density screening of small quantities of biological or biologically active material.

A High-Density Screening Format for Encoded Combinatorial Libraries: Assay Miniaturization and Its Application to Enzymatic Reactions

A novel, miniaturized high-throughput screening format is described for assay of combinatorial libraries generated on beads. This approach, which is ideally suited to encoded libraries synthesized on beads, utilizes the photolytic cleavage of individual compounds into a high-density well array (>6500 wells within a standard 96-well microtiter plate footprint) with well volumes as low as 0.37 μl. As a model study, an encoded dipeptide library (324 members) acylated with isobutyl succinate was assayed using this format to search for potential inhibitors of matrilysin, a member of the matrix metalloproteinase superfamily.In siturelease of compounds from solid support was accomplished by photochemical cleavage after beads and enzyme were distributed to the wells. After the addition of a fluorogenic substrate to the array, the extent of enzyme inhibition and identification of active compounds was quantitated by imaging of the fluorescence emission upon uv irradiation. The structure–activity relationship data generated from the identified inhibitors in this study corroborate previous findings, thus validating the utility of this approach as a means of high-throughput screening of bead-based libraries.

Generation and fluorescent in situ hybridization mapping of yeast artificial chromosomes of 1p, 17p, 17q, and 19q from a hybrid cell line by high-density screening of an amplified library

A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe ( Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.