A High-Throughput Platform for Screening Milligram Quantities of Plant Biomass for Lignocellulose Digestibility

The development of a viable lignocellulosic ethanol industry requires multiple improvements in the process of converting biomass to ethanol. A key step is the improvement of the plants that are to be used as biomass feedstocks. To facilitate the identification and evaluation of feedstock plants, it would be useful to have a method to screen large numbers of individual plants for enhanced digestibility in response to combinations of specific pretreatments and enzymes. This paper describes a high-throughput digestibility platform (HTDP) for screening collections of germplasm for improved digestibility, which was developed under the auspices of the Department of Energy-Great Lakes Bioenergy Research Center (DOE-GLBRC). A key component of this platform is a custom-designed workstation that can grind and dispense 1–5 mg quantities of more than 250 different plant tissue samples in 16 h. The other steps in the processing (pretreatment, enzyme digestion, and sugar analysis) have also been largely automated and require 36 h. The process is adaptable to diverse acidic and basic, low-temperature pretreatments. Total throughput of the HTDP is 972 independent biomass samples per week. Validation of the platform was performed on brown midrib mutants of maize, which are known to have enhanced digestibility. Additional validation was performed by screening approximately 1,200 Arabidopsis mutant lines with T-DNA insertions in genes known or suspected to be involved in cell wall biosynthesis. Several lines showed highly significant (p?<?0.01) increases in glucose and xylose release (20–40% above the mean). The platform should be useful for screening populations of plants to identify superior germplasm for lignocellulosic ethanol applications and also for screening populations of mutant model plants to identify specific genes affecting digestibility.     

Cell-Based Assays for High-Throughput Screening

Cell-based assays represent approximately half of all high-throughput screens currently performed. Here, we review in brief the history and status of high-throughput screening (HTS), and summarize some of the challenges and benefits associated with the use of cell-based assays in HTS. Approaches for successful experimental design and execution of cell-based screens are introduced, including strategies for assay development, implementation of primary and secondary screens, and target identification. In doing so, we hope to provide a comprehensive review of the cell-based HTS process and an introduction to the methodologies and techniques used.     

Novel Sensitive High-Throughput Screening Strategy for Nitrilase-Producing Strains

Nitrilases have found wide use in the pharmaceutical industry for the production of fine chemicals, and it is important to have a method by which to screen libraries of isolated or engineered nitrilase variants (including bacteria and fungi). The conventional methods, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, or gas chromatography, are tedious and time-consuming. Therefore, a direct and sensitive readout of the nitrilase's activity has to be considered. In this paper, we report a novel time-resolved luminescent probe: o-hydroxybenzonitrile derivatives could be applied to detect the activity of the nitrilases. By the action of nitrilases, o-hydroxybenzonitrile derivatives can be transformed to the corresponding salicylic acid derivatives, which, upon binding Tb³⁺, serve as a photon antenna and sensitize Tb³⁺ luminescence. Because of the time-resolved property of the luminescence, the background from the other proteins (especially in the fermentation system) in the assay could be reduced and, therefore, the sensitivity was increased. Moreover, because the detection was performed on a 96- or 384-well plate, the activity of the nitrilases from microorganisms could be determined quickly. Based on this strategy, the best fermentation conditions for nitrilase-producing strains were obtained.     

New Device for High-Throughput Viability Screening of Flow Biofilms

Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.Bacterial biofilms are surface-attached communities that are encased in a polymeric matrix, which exhibit a high degree of resistance to antimicrobial agents and the host immune system (12, 16). This makes them medically important; diseases with a biofilm component are chronic and difficult to eradicate. Examples of such diseases are cystitis (1), endocarditis (31), cystic fibrosis (35), and middle-ear (17) and indwelling medical device-associated (20) infections. Biofilms also play important environmental roles in, for example, wastewater treatment (38), bioremediation (29, 30), biofouling (7), and biocorrosion (2). Better control of biofilms requires elucidation of the molecular basis of their superior resistance (by identifying resistance-compromised mutants) and identification of compounds with antibiofilm activity. While our understanding of these aspects of biofilms has increased (11, 15, 25-27, 36), further work, including development of accurate high-throughput (HTP) methods for screening biofilm viability, is needed.Two major biofilm models are studied in the laboratory, biofilms grown without a continuous flow of fresh medium and biofilms grown with a continuous flow of fresh medium; examples of these two models are microtiter well biofilms and flow cell biofilms, respectively. Methods have been developed for HTP screening of the viability of static biofilms (6, 28, 32, 33), but there are no methods for HTP screening of flow biofilms. The latter biofilms are typically grown in flow cells, which have to be examined individually to determine viability and thus cannot be used for rapid screening. An HTP screening method for flow biofilms is desirable, as these biofilms more closely approximate natural biofilms and can differ from static biofilms evidently due to hydrodynamic influences on cell signaling (22, 34). For example, the ability of rpoS-deficient Escherichia coli (lacking σ^S) to form flow biofilms is impaired, but its capacity to form biofilms under static conditions is enhanced (18).We describe here a new application of a recently developed device (8-10, 13), the “BioFlux” device consisting of microfluidic channels for biofilm growth. Other microfluidic devices have recently been used for biofilm formation (14, 19, 21, 23), but none of them has been used for HTP screening. The BioFlux device permits rapid measurement of the fluorescence of flow biofilms with a plate reader, which permits initial HTP screening of the viability of such biofilms.     

Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm². We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.     

High-Throughput Carrier Screening Using TaqMan Allelic Discrimination

Members of the Ashkenazi Jewish community are at an increased risk for inheritance of numerous genetic diseases such that carrier screening is medically recommended. This paper describes the development and evaluation of 30 TaqMan allelic discrimination qPCR assays for 29 mutations on 2 different high-throughput platforms. Four of these mutations are in the GBA gene and are successfully examined using short amplicons due to the qualitative nature of TaqMan allelic discrimination. Two systems were tested for their reliability (call rate) and consistency with previous diagnoses (diagnostic accuracy) indicating a call rate of 99.04% and a diagnostic accuracy of 100% (+/−0.00%) from one platform, and a call rate of 94.66% and a diagnostic accuracy of 93.35% (+/−0.29%) from a second for 9,216 genotypes. Results for mutations tested at the expected carrier frequency indicated a call rate of 97.87% and a diagnostic accuracy of 99.96% (+/−0.05%). This study demonstrated the ability of a high throughput qPCR methodology to accurately and reliably genotype 29 mutations in parallel. The universally applicable nature of this technology provides an opportunity to increase the number of mutations that can be screened simultaneously, and reduce the cost and turnaround time for accommodating newly identified and clinically relevant mutations.     

High-Throughput Screening for Ligands of the HEPN Domain of Sacsin

Sacsin is a large protein implicated in the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which features the loss of Purkinje neurons in the cerebellum. Although the domain architecture of sacsin suggests that it is a neuronal chaperone assisting in protein quality control, the precise function of sacsin remains elusive. Using fluorescence polarization (FP) assays, we confirmed that the HEPN domain of sacsin binds to nucleotides with low micromolar affinities. FP competition assays with a variety of nucleotides and nucleotide analogs revealed that the binding is primarily mediated by the phosphate groups of nucleotides. A high-throughput screen subsequently identified novel small molecule ligands of HEPN, providing new chemical probes for cell culture studies and drug development. Together, the results are consistent with the HEPN domain contributing to the functional activity of sacsin by binding to nucleotides or other multiply charged anionic compounds in neurons.     

High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite''s infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.     

An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH₃, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more).     

A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs(sg RNAs).Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing(HTS).We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.     

High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.     

A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening

The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish). This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf), knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1), and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant) revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers.     

Mining Chemical Activity Status from High-Throughput Screening Assays

High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.     

High-Throughput Screening for Growth Inhibitors Using a Yeast Model of Familial Paraganglioma

Classical tumor suppressor genes block neoplasia by regulating cell growth and death. A remarkable puzzle is therefore presented by familial paraganglioma (PGL), a neuroendocrine cancer where the tumor suppressor genes encode subunits of succinate dehydrogenase (SDH), an enzyme of the tricarboxylic acid (TCA) cycle of central metabolism. Loss of SDH initiates PGL through mechanisms that remain unclear. Could this metabolic defect provide a novel opportunity for chemotherapy of PGL? We report the results of high throughput screening to identify compounds differentially toxic to SDH mutant cells using a powerful S. cerevisiae (yeast) model of PGL. Screening more than 200,000 compounds identifies 12 compounds that are differentially toxic to SDH-mutant yeast. Interestingly, two of the agents, dequalinium and tetraethylthiuram disulfide (disulfiram), are anti-malarials with the latter reported to be a glycolysis inhibitor. We show that four of the additional hits are potent inhibitors of yeast alcohol dehydrogenase. Because alcohol dehydrogenase regenerates NAD⁺ in glycolytic cells that lack TCA cycle function, this result raises the possibility that lactate dehydrogenase, which plays the equivalent role in human cells, might be a target of interest for PGL therapy. We confirm that human cells deficient in SDH are differentially sensitive to a lactate dehydrogenase inhibitor.     

High-Throughput Profiling of the Fiber and Sugar Composition of Sugarcane Biomass

Lignocellulosic biomass from sugarcane (Saccharum spp. hybrids) could potentially be a major feedstock for second-generation biofuel production. Consequently, selecting sugarcane varieties with favorable biomass characteristics, typically less enzymatic recalcitrance and better saccharification yield without sugar-yield penalty, will be important in sugarcane breeding. Economical and high-throughput techniques for profiling the major biomass components of this complex system will facilitate selection of clones with ideal lignocellulosic composition from large numbers of genotypes in breeding programs. We used a combined high-throughput profiling approach to evaluate the biomass composition of samples from a sugarcane germplasm collection. This employed near-infrared (NIR) spectroscopy for fiber characterization and high-performance liquid chromatography (HPLC) for determining the sugar content in juice. The results for 331 samples, from a diverse sugarcane population of 186 genotypes, derived from 143 parents of different genetic backgrounds, showed that high-quality NIR spectroscopic predictions were feasible for cellulose, hemicellulose, lignin, and extractives values in fiber, and sugars in juice were suitably analyzed by HPLC. The analysis of total biomass indicated that this NIR- and HPLC-based high-throughput method allowed a robust phenotypic assessment of a large number of samples for the key biomass traits in the sugarcane system, including total dry biomass, fiber, sugar content, and theoretical ethanol yields, and could potentially become the method of choice for sugarcane germplasm screening in breeding programs targeting the support of biofuel production.     

Improving Enzymes for Biomass Conversion: A Basic Research Perspective

The cost of enzymes for converting plant biomass materials to fermentable sugars is a major impediment to the development of a practical lignocellulosic ethanol industry. Research on enzyme optimization with the goal of reducing the cost of converting biomass materials such as corn stover into glucose, xylose, and other sugars is being actively pursued in private industry, academia, and government laboratories. Under the auspices of the Department of Energy Great Lakes Bioenergy Research Center, we are taking several approaches to address this problem, including “bioprospecting” for superior key enzymes, protein engineering, and high-level expression in plants. A particular focus is the development of synthetic enzyme mixtures, in order to learn which of the hundreds of known enzymes are important and in what ratios. A core set comprises cellobiohydrolase, endoglucanase, β-glucosidase, endoxylanase, and β-glucosidase. Accessory enzymes include esterases, proteases, nonhydrolytic proteins, and glycosyl hydrolases that cleave the less frequent chemical linkages found in plant cell walls.     

High-Throughput Screening for the Identification of New Therapeutic Options for Metastatic Pheochromocytoma and Paraganglioma

Drug repurposing or repositioning is an important part of drug discovery that has been growing in the last few years for the development of therapeutic options in oncology. We applied this paradigm in a screening of a library of about 3,800 compounds (including FDA-approved drugs and pharmacologically active compounds) employing a model of metastatic pheochromocytoma, the most common tumor of the adrenal medulla in children and adults. The collection of approved drugs was screened in quantitative mode, testing the compounds in compound-titration series (dose-response curves). Analysis of the dose-response screening data facilitated the selection of 50 molecules with potential bioactivity in pheochromocytoma cells. These drugs were classified based on molecular/cellular targets and signaling pathways affected, and selected drugs were further validated in a proliferation assay and by flow cytometric cell death analysis. Using meta-analysis information from molecular targets of the top drugs identified by our screening with gene expression data from human and murine microarrays, we identified potential drugs to be used as single drugs or in combination. An example of a combination with a synergistic effect is presented. Our study exemplifies a promising model to identify potential drugs from a group of clinically approved compounds that can more rapidly be implemented into clinical trials in patients with metastatic pheochromocytoma or paraganglioma.