Development Of High-throughput Screening Methods Research Articles

A High-throughput Response to the SARS-CoV-2 Pandemic

Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.

The highly diverse antiphage defence systems of bacteria.

Bacteria and their viruses have coevolved for billions of years. This ancient and still ongoing arms race hasled bacteria to develop a vast antiphage arsenal. The development of high-throughput screening methods expanded our knowledge of defence systems from a handful to more than a hundred systems, unveiling many different molecular mechanisms. These findings reveal that bacterial immunity is much more complex than previously thought. In this Review, we explore recently discovered bacterial antiphage defence systems, with a particular focus on their molecular diversity, and discuss the ecological and evolutionary drivers and implications of the existing diversity of antiphage defence mechanisms.

Exploring Natural Products as Radioprotective Agents for Cancer Therapy: Mechanisms, Challenges, and Opportunities.

Radiotherapy is an important cancer treatment. However, in addition to killing tumor cells, radiotherapy causes damage to the surrounding cells and is toxic to normal tissues. Therefore, an effective radioprotective agent that prevents the deleterious effects of ionizing radiation is required. Numerous synthetic substances have been shown to have clear radioprotective effects. However, most of these have not been translated for use in clinical applications due to their high toxicity and side effects. Many medicinal plants have been shown to exhibit various biological activities, including antioxidant, anti-inflammatory, and anticancer activities. In recent years, new agents obtained from natural products have been investigated by radioprotection researchers, due to their abundance of sources, high efficiency, and low toxicity. In this review, we summarize the mechanisms underlying the radioprotective effects of natural products, including ROS scavenging, promotion of DNA damage repair, anti-inflammatory effects, and the inhibition of cell death signaling pathways. In addition, we systematically review natural products with radioprotective properties, including polyphenols, polysaccharides, alkaloids, and saponins. Specifically, we discuss the polyphenols apigenin, genistein, epigallocatechin gallate, quercetin, resveratrol, and curcumin; the polysaccharides astragalus, schisandra, and Hohenbuehelia serotina; the saponins ginsenosides and acanthopanax senticosus; and the alkaloids matrine, ligustrazine, and β-carboline. However, further optimization through structural modification, improved extraction and purification methods, and clinical trials are needed before clinical translation. With a deeper understanding of the radioprotective mechanisms involved and the development of high-throughput screening methods, natural products could become promising novel radioprotective agents.

Photoclick reaction for rapid and simple fluorescence detection of itaconic acid and its derivatives in fungal cultures.

Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates thedetection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4°C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.

Arrayed labeling-free cultivation and growth evaluation from a single microorganism.

The development of high-throughput screening methods for microorganisms is desired because microorganisms are useful and sustainable resources with which valuable substances utilized in various industries can be produced. Micro-space-based methods are the best candidates for the efficient screening of microorganisms owing to their low reagent consumption and compact integration. In this study, we developed a picoliter-sized incubator array for quantitative and labeling-free evaluation of the growth process of Escherichia coli (E. coli) by autofluorescence. Because the array with 8464 incubators is able to compartmentalize single E. coli individually utilizing the Poisson distribution, the array can evaluate 100 single E. coli simultaneously. Our incubator array not only realized the high-throughput screening of microorganisms, but also provided an analytical tool for assessing individual differences in E. coli.

Signed, sealed, and delivered: RNA localization and translation at centrosomes.

Protein localization is intrinsic to cellular function and specialized activities, such as migration or proliferation. Many localized proteins enrich at defined organelles, forming subdomains of functional activity further specified by interacting protein assemblies. One well-studied organelle showing dynamic, functional changes in protein composition is the centrosome. Centrosomes are microtubule-organizing centers with diverse cellular functions largely defined by the composition of the pericentriolar material, an ordered matrix of proteins organized around a central pair of centrioles. Also localizing to the pericentriolar material are mRNAs. Although RNA was identified at centrosomes decades ago, the characterization of specific RNA transcripts and their functional contributions to centrosome biology remained largely unstudied. While the identification of RNA localized to centrosomes accelerated with the development of high-throughput screening methods, this discovery still outpaces functional characterization. Recent work indicates RNA localized to centrosomes is biologically significant and further implicates centrosomes as sites for local protein synthesis. Distinct RNA localization and translational activities likely contribute to the diversity of centrosome functions within cells.

Antiviral Compounds for Blocking Arboviral Transmission in Mosquitoes.

Mosquito-borne arthropod-borne viruses (arboviruses) such as the dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are important human pathogens that are responsible for significant global morbidity and mortality. The recent emergence and re-emergence of mosquito-borne viral diseases (MBVDs) highlight the urgent need for safe and effective vaccines, therapeutics, and vector-control approaches to prevent MBVD outbreaks. In nature, arboviruses circulate between vertebrate hosts and arthropod vectors; therefore, disrupting the virus lifecycle in mosquitoes is a major approach for combating MBVDs. Several strategies were proposed to render mosquitoes that are refractory to arboviral infection, for example, those involving the generation of genetically modified mosquitoes or infection with the symbiotic bacterium Wolbachia. Due to the recent development of high-throughput screening methods, an increasing number of drugs with inhibitory effects on mosquito-borne arboviruses in mammalian cells were identified. These antivirals are useful resources that can impede the circulation of arboviruses between arthropods and humans by either rendering viruses more vulnerable in humans or suppressing viral infection by reducing the expression of host factors in mosquitoes. In this review, we summarize recent advances in small-molecule antiarboviral drugs in mammalian and mosquito cells, and discuss how to use these antivirals to block the transmission of MBVDs.

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution.

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

Genome-wide analysis of E. coli cell-gene interactions

BackgroundThe pursuit of standardization and reliability in synthetic biology has achieved, in recent years, a number of advances in the design of more predictable genetic parts for biological circuits. However, even with the development of high-throughput screening methods and whole-cell models, it is still not possible to predict reliably how a synthetic genetic construct interacts with all cellular endogenous systems. This study presents a genome-wide analysis of how the expression of synthetic genes is affected by systematic perturbations of cellular functions. We found that most perturbations modulate expression indirectly through an effect on cell size, putting forward the existence of a generic Size-Expression interaction in the model prokaryote Escherichia coli.ResultsThe Size-Expression interaction was quantified by inserting a dual fluorescent reporter gene construct into each of the 3822 single-gene deletion strains comprised in the KEIO collection. Cellular size was measured for single cells via flow cytometry. Regression analyses were used to discriminate between expression-specific and gene-specific effects. Functions of the deleted genes broadly mapped onto three systems with distinct primary influence on the Size-Expression map. Perturbations in the Division and Biosynthesis (DB) system led to a large-cell and high-expression phenotype. In contrast, disruptions of the Membrane and Motility (MM) system caused small-cell and low-expression phenotypes. The Energy, Protein synthesis and Ribosome (EPR) system was predominantly associated with smaller cells and positive feedback on ribosome function.ConclusionsFeedback between cell growth and gene expression is widespread across cell systems. Even though most gene disruptions proximally affect one component of the Size-Expression interaction, the effect therefore ultimately propagates to both. More specifically, we describe the dual impact of growth on cell size and gene expression through cell division and ribosomal content. Finally, we elucidate aspects of the tight control between swarming, gene expression and cell growth. This work provides foundations for a systematic understanding of feedbacks between genetic and physiological systems.

DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods.

Viruses transmitted by small mammals and arthropods serve as global threats to humans. Most emergent and re-emergent viral agents are transmitted by these groups; therefore, the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest. In this study, we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods (SMAvirusChip) with nucleotide sequences that have been deposited in the GenBank. SMAvirusChip was designed with more than 15,000 oligonucleotide probes (60-mers), including viral and control probes. Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses, while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses. SMAvirusChip was evaluated with 20 laboratory reference-strain viruses. These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample. The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus (DENV). The results showed a detection limit as low as 2.6E3 RNA copies/mL. Additionally, the sensitivity was one log10 lower (2.6E2 RNA copies/mL) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a pool of mosquito samples spiked with DENV, the virus was also detectable. SMAvirusChip was able to specifically detect viruses in cell cultures, serum samples, total blood samples and a pool of mosquitoes, confirming that cellular RNA/DNA did not interfere with the assay. Therefore, SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small mammals and arthropods.

Abstract B10: Development of high-throughput screening methods for detecting germline cancer causing mutations in the Hispanic population

Abstract Colorectal and breast cancers are leading causes of cancer morbidity and mortality in Hispanics. Most of the genes predisposing to these common malignancies are yet to be identified. Moreover, the prevalence of mutations in known cancer genes in Hispanic populations remains largely undetermined. Our present study aims to establish the prevalence of mutations that predispose to cancer susceptibility syndromes in Hispanics using a high throughput next-generation sequencing approach. We have developed an amplicon sequencing method that uses microfluidics PCR to simultaneously identify mutations in up to 480 amplicons. This amount of sequence data is enough to cover, at a high depth, the coding sequence of ∼20 different genes in a single reaction. To do so, the samples are barcoded individually using indexing adapters that allows pooling of multiple amplicon libraries for up to 384 samples in a single MiSeq run. The sequence data is analyzed with a locally developed bioinformatics pipeline that uses BWA, VarScan V2.0 and custom shell and Perl scripts. Our preliminary data, in several hundred familial and early-onset colon and breast cancer patients from South America, have identified many individuals with mutations in known cancer genes, such as APC, MSH2, MLH1, BRCA1 and BRCA2, and that have been previously reported in Southern European populations. We have also identified several mutations that have not been reported previously and that appear to have an Amerindian origin. Interestingly, a large fraction of these mutations are shared by multiple patients and suggest that founder effects are common in some of these populations. We have also found that more than over 50% of the patients screened so far are mutation negative. We plan to use this latter set of samples for novel gene discovery by whole exome or genome sequencing in the next phase of our study. In conclusion, we have developed a rapid, cost effective, and efficient method for screening mutations in known cancer genes in the Hispanic population. Hispanics are the youngest, largest and the fastest growing US minority and the largest population group living in the Americas. However, to a large extent, this population remains under-studied and under-screened, contributing to health care disparities. Prevalence of mutations in known cancer genes as well as novel mutations identified in our study can be used during initial steps of genetic testing programs that can improve the detection of early cancers among Hispanic communities. Citation Format: Ruta Sahasrabudhe, Anna-Marie Tuazon, Paul Lott, Ana Estrada, Mabel Bohorquez, Rodrigo Prieto, Angel Criollo, Carolina Ramirez, Magdalena Echeverry, Luis G. Carvajal-Carmona. Development of high-throughput screening methods for detecting germline cancer causing mutations in the Hispanic population. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr B10. doi:10.1158/1538-7755.DISP13-B10

Microarray-Based Prenatal Diagnosis in Developing Countries

Detection of chromosomal alterations in prenatal diagnosis requires speed and precision, imposing the development of high-throughput screening methods. Thus the array CGH technology, used to diagnose genomic rearrangements in children with multiple congenital anomalies, idiopathic mental retardation and developmental delay, caught the attention of specialists worldwide who are now trying to implement it in routine prenatal practice in order to obtain rapidly useful information for a more accurate genetic counseling. Array comparative genomic hybridisation (Array CGH) has the capacity to detect genomic rearrangements at a higher resolution than other methods used in prenatal testing, like standard G banding, multiplex ligation dependent probe amplification (MLPA) or fluorescent in-situ hybridization (FISH), being able to detect chromosomal imbalances smaller than 3 Mb. Despite all these evidences that prove its utility in prenatal diagnosis, this method is not used worldwide either because of lack of experienced specialists in interpreting array results or because, especially in developing countries, the national health insurance systems does not settle the high cost of this analysis and patients cannot afford it. This paper is a summary of recent progresses made using the array CGH technology in prenatal testing to detect if genomic imbalances are involved in the development of congenital malformations. Our aim is to reveal the importance of using advanced technologies like array CGH in routine prenatal practice, and thus to contribute to its implementation in developing countries like ours.

Recent advances in microfluidic technologies for biochemistry and molecular biology

Advances in the fields of proteomics and genomics have necessitated the development of high-throughput screening methods (HTS) for the systematic transformation of large amounts of biological chemical data into an organized database of knowledge. Microfluidic systems are ideally suited for high-throughput biochemical experimentation since they offer high analytical throughput, consume minute quantities of expensive biological reagents, exhibit superior sensitivity and functionality compared to traditional micro-array techniques and can be integrated within complex experimental work flows. A range of basic biochemical and molecular biological operations have been transferred to chip-based microfluidic formats over the last decade, including gene sequencing, emulsion PCR, immunoassays, electrophoresis, cell-based assays, expression cloning and macromolecule blotting. In this review, we highlight some of the recent advances in the application of microfluidics to biochemistry and molecular biology.

Combinatorial materials research applied to the development of new surface coatings: VIII: Overview of the high-throughput measurement systems developed for a marine coating workflow

A combinatorial workflow has been produced for the development of novel, environmental-friendly marine coatings. A particularly challenging aspect of the workflow development was the selection and development of high-throughput screening methods that allow for some degree of prediction of coating performance in the aquatic environment of interest. The high-throughput screening methods currently in place include measurements of surface energy, viscoelastic properties, pseudobarnacle adhesion, and a suite of biological assays based on various marine organisms. An experiment involving a series of fouling-release coatings was used to correlate high-throughput screening data to data obtained from ocean site immersion testing. The results of the experiment showed that both bacterial biofilm surface coverage and storage modulus at 30 °C showed a good correlation with barnacle adhesion strength and a fair correlation with fouling rating, but surface energy and pseudobarnacle adhesion did not correlate with the results from ocean site testing.

Transcreener™: screening enzymes involved in covalent regulation

Enzymes that catalyse group transfer reactions comprise a significant fraction of the human proteome and are a rich source of drug targets because of their role in covalent regulatory cycles. Phosphorylation, glycosylation, sulfonation, methylation and acetylation represent some of the key types of group transfer reactions that modulate the function of diverse biomolecules through covalent modification. Development of high-throughput screening methods for these enzymes has been problematic because of the diversity of acceptor substrates. Recently, the authors developed a novel assay platform called Transcreener™ that relies upon fluorescence detection of the invariant reaction product of a group transfer reaction, usually a nucleotide. This platform enables screening of any isoform in a family of group transfer enzymes, with any acceptor substrate, using the same assay reagents.