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Saliou, Adrien, Pierre Delobel, Martine Dubois, Florence Nicot, Stéphanie Raymond, Vincent Calvez, Bernard Masquelier, and Jacques Izopet. "Concordance between Two Phenotypic Assays and Ultradeep Pyrosequencing for Determining HIV-1 Tropism." Antimicrobial Agents and Chemotherapy 55, no. 6 (April 4, 2011): 2831–36. http://dx.doi.org/10.1128/aac.00091-11.
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ABSTRACTThere have been few studies on the concordance between phenotypic assays for predicting human immunodeficiency virus type 1 (HIV-1) coreceptor usage. The sensitivity of ultradeep pyrosequencing combined with genotyping tools is similar to that of phenotypic assays for detecting minor CXCR4-using variants. We evaluated the agreement between two phenotypic assays, the Toulouse tropism test (TTT) and the Trofile assay, and ultradeep pyrosequencing for determining the tropism of HIV-1 quasispecies. The concordance between the TTT and Trofile assays was assessed for 181 samples successfully phenotyped by both assays. The TTT was 86% concordant with the standard Trofile assay and 91.7% with its enhanced-sensitivity version. The concordance between phenotypic characterization of HIV-1 tropism and ultradeep pyrosequencing genotypic prediction was further studied in selected samples. The HIV-1 tropism inferred from ultradeep pyrosequencing of 11 samples phenotyped as X4 and dualtropic and 12 phenotyped as R5-tropic agreed closely with the results of phenotyping. However, ultradeep pyrosequencing detected minor CXCR4-using variants in 3 of 12 samples phenotyped as R5-tropic. Ultradeep pyrosequencing also detected minor CXCR4-using variants that had been missed by direct sequencing in 6 of 9 samples phenotyped as X4-tropic but genotyped as R5-tropic by direct sequencing. Ultradeep pyrosequencing was 87% concordant with the Trofile and TTT phenotypic assays and was in the same range of sensitivity (0.4%) than these two phenotypic assays (0.3 to 0.5%) for detecting minor CXCR4-using variants. Ultradeep pyrosequencing provides a new way to improve the performance of genotypic prediction of HIV-1 tropism to match that of the phenotypic assays.
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Steigele, Stephan, Daniel Siegismund, Matthias Fassler, Marusa Kustec, Bernd Kappler, Tom Hasaka, Ada Yee, Annette Brodte, and Stephan Heyse. "Deep Learning-Based HCS Image Analysis for the Enterprise." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 7 (May 20, 2020): 812–21. http://dx.doi.org/10.1177/2472555220918837.
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Drug discovery programs are moving increasingly toward phenotypic imaging assays to model disease-relevant pathways and phenotypes in vitro. These assays offer richer information than target-optimized assays by investigating multiple cellular pathways simultaneously and producing multiplexed readouts. However, extracting the desired information from complex image data poses significant challenges, preventing broad adoption of more sophisticated phenotypic assays. Deep learning-based image analysis can address these challenges by reducing the effort required to analyze large volumes of complex image data at a quality and speed adequate for routine phenotypic screening in pharmaceutical research. However, while general purpose deep learning frameworks are readily available, they are not readily applicable to images from automated microscopy. During the past 3 years, we have optimized deep learning networks for this type of data and validated the approach across diverse assays with several industry partners. From this work, we have extracted five essential design principles that we believe should guide deep learning-based analysis of high-content images and multiparameter data: (1) insightful data representation, (2) automation of training, (3) multilevel quality control, (4) knowledge embedding and transfer to new assays, and (5) enterprise integration. We report a new deep learning-based software that embodies these principles, Genedata Imagence, which allows screening scientists to reliably detect stable endpoints for primary drug response, assess toxicity and safety-relevant effects, and discover new phenotypes and compound classes. Furthermore, we show how the software retains expert knowledge from its training on a particular assay and successfully reapplies it to different, novel assays in an automated fashion.
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Kus, Anna, Alice Wiedeman, and Alice Long. "Distinct phenotypes of rare autoreactive and virus-reactive CD8+ T cells revealed through novel clustering algorithm." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 78.07. http://dx.doi.org/10.4049/jimmunol.210.supp.78.07.
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Abstract Antigen-specific cells are implicated in the development and progression of disease in autoimmunity, infectious disease, and cancer, and are an attractive target for therapies. While robust detection of antigen-specific cells is possible with tetramer and activation-induced marker assays, extensive characterization of these cells remains challenging due to their rarity. Here we present a novel computational tool, DISCOV-R, that enables deep phenotyping of rare antigen-specific cells through unbiased clustering of a parent population into subsets defined by expression of phenotyping markers and overlaying the antigen-specific population. Using this tool with 35-parameter mass cytometry (CyTOF) along with class I MHC-tetramer assays, we identified phenotypes enriched among rare islet autoantigen-reactive and chronic virus-reactive CD8+ T cells when compared to total CD8+ T cells from 46 type 1 diabetics. CD8+ T cells were clustered based on expression of 24 phenotypic markers, revealing 12 CD8+ T cell phenotypes shared across subjects. Overlaying antigen-specific tetramer-positive cells on this phenotypic landscape revealed that both islet- and virus-reactive cells were enriched for an exhausted-like memory phenotype (p=1.7E-07 and 1.6E-05, respectively) consistent with chronic antigen exposure, while islet-reactive cells were enriched for two transitional memory phenotypes (p=0.01 and 0.03). The characterization of these rare antigen-specific cells using DISCOV-R reveals unique phenotypes associated with their specificity that may indicate different function, and enables comparison over time and between individuals to identify biomarkers and potential pathways to disease development and progression.
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Yamanishi, Cameron, Stephen Robinson, and Shuichi Takayama. "Biofabrication of phenotypic pulmonary fibrosis assays." Biofabrication 11, no. 3 (June 19, 2019): 032005. http://dx.doi.org/10.1088/1758-5090/ab2286.
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Song, Ok-Ryul, Nathalie Deboosere, Vincent Delorme, Christophe J. Queval, Gaspard Deloison, Elisabeth Werkmeister, Frank Lafont, Alain Baulard, Raffaella Iantomasi, and Priscille Brodin. "Phenotypic assays for Mycobacterium tuberculosis infection." Cytometry Part A 91, no. 10 (May 19, 2017): 983–94. http://dx.doi.org/10.1002/cyto.a.23129.
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Zeng, Min, Yulin Luo, Chunrong Xu, Rong Li, Ni Chen, Xin Deng, Dan Fang, Liqun Wang, Jianbo Wu, and Mao Luo. "Platelet-endothelial cell interactions modulate smooth muscle cell phenotype in an in vitro model of type 2 diabetes mellitus." American Journal of Physiology-Cell Physiology 316, no. 2 (February 1, 2019): C186—C197. http://dx.doi.org/10.1152/ajpcell.00428.2018.
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Platelet (PLT)-endothelial cell (EC) interaction appears to contribute to phenotypic transition of vascular smooth muscle cells (VSMCs), which play an important role in the physiological and pathological process of vascular complications in type 2 diabetes mellitus (DM2). However, the precise mechanisms by which interactions between PLTs and ECs affect VSMC phenotype have largely remained unclear. We determined the effect of diabetic PLT-EC interaction to influence VSMC migration, proliferation, and phenotypic transformation in triple-cell coculture models using the quantitative real-time PCR, Western blot, fluorescence microscopy, wound scratch assays, CCK-8 assays, and gelatin zymography assays. Our results revealed DM2 PLT-EC interaction to be associated with a significant downregulation of VSMC-specific contractile phenotypic genes and proteins, including SM22α, smooth muscle actin, Smoothelin-B, and smooth muscle-myosin heavy chain. Inversely, VSMC-specific proliferative phenotype gene and protein levels, including cyclin D1 and 2, nonmuscle myosin heavy chain B, and PCNA were in upregulation. Furthermore, the DM2-originated PLT-EC interaction promoted the expression level of transforming growth factor-β1, and the PI3K/Akt and matrix metalloproteinase 9 signaling pathway was activated subsequently. Finally, these reactions contributed to a synthetic phenotype of VSMCs, including the proliferation, migration, and gelatinolytic activities. These findings suggest that PLT-EC interaction modulates the phenotypic transition of VSMCs between a contractile and proliferative/synthetic phenotype under diabetic conditions, conceivably providing important implications regarding the mechanisms controlling the VSMC phenotypic transition and the development of cardiovascular complications.
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Carlson, Coby, Chad Koonce, Natsuyo Aoyama, Shannon Einhorn, Steve Fiene, Arne Thompson, Brad Swanson, Blake Anson, and Steven Kattman. "Phenotypic Screening with Human iPS Cell–Derived Cardiomyocytes." Journal of Biomolecular Screening 18, no. 10 (September 26, 2013): 1203–11. http://dx.doi.org/10.1177/1087057113500812.
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A major hurdle for cardiovascular disease researchers has been the lack of robust and physiologically relevant cell-based assays for drug discovery. Derivation of cardiomyocytes from human-induced pluripotent stem (iPS) cells at high purity, quality, and quantity enables the development of relevant models of human cardiac disease with source material that meets the demands of high-throughput screening (HTS). Here we demonstrate the utility of iPS cell–derived cardiomyocytes as an in vitro model of cardiac hypertrophy. Exposure of cardiomyocytes to endothelin 1 (ET-1) leads to reactivation of fetal genes, increased cell size, and robust expression of B-type natriuretic peptide (BNP). Using this system, we developed a suite of assays focused on BNP detection, most notably a high-content imaging-based assay designed for phenotypic screening. Miniaturization of this assay to a 384-well format enabled the profiling of a small set of tool compounds known to modulate the hypertrophic response. The assays described here provide consistent and reliable results and have the potential to increase our understanding of the many mechanisms underlying this complex cardiac condition. Moreover, the HTS-compatible workflow allows for the incorporation of human biology into early phases of drug discovery and development.
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Clemons, Paul A. "Complex phenotypic assays in high-throughput screening." Current Opinion in Chemical Biology 8, no. 3 (June 2004): 334–38. http://dx.doi.org/10.1016/j.cbpa.2004.04.002.
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Von Bargen, Jennifer, Christian T. Smith, and John Rueth. "Development of a Chinook Salmon Sex Identification SNP Assay Based on the Growth Hormone Pseudogene." Journal of Fish and Wildlife Management 6, no. 1 (February 1, 2015): 213–19. http://dx.doi.org/10.3996/012014-jfwm-004.
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Abstract Genotypic sex identification assays can provide valuable information about fish populations when phenotypic sex determination is difficult. Here we describe the development of a TaqMan® assay (Ots_SexID) designed to identify the genotypic sex by targeting a region previously examined in the growth hormone pseudogene for winter-run Chinook salmon (Oncorhynchus tshawytscha) collected from the Sacramento River and spawned at the Livingston Stone National Fish Hatchery. Accuracy of the marker was assessed by comparing genotypic sex assignments for Chinook salmon spawned at Livingston Stone National Fish hatchery in 2012 (n = 84) with phenotypic sex recorded during spawning. Genotypic sex was observed to be concordant with phenotypic sex identified using Ots_SexID in 83/84 individuals, suggesting that the assay could be used to predict phenotypic sex with ∼︀99% accuracy. To evaluate the utility of the TaqMan assay in other parts of the species’ range, we examined collections from 29 other populations ranging from Alaska to California. Genotypic sex assignments based on the assay were generally concordant with observed phenotypes, but there were some strong exceptions. These results suggest that the new assay will be very useful for Sacramento River winter-run Chinook salmon, but also highlight the importance of thoroughly testing any genotypic sex identification assay before application in a population of interest.
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Morimont, Laure, Nathalie Donis, Céline Bouvy, François Mullier, Jean-Michel Dogné, and Jonathan Douxfils. "Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance." Seminars in Thrombosis and Hemostasis 48, no. 06 (September 2022): 680–89. http://dx.doi.org/10.1055/s-0042-1758162.
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AbstractActivated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.
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Lee, Jonathan A., Shaoyou Chu, Francis S. Willard, Karen L. Cox, Rachelle J. Sells Galvin, Robert B. Peery, Sarah E. Oliver, et al. "Open Innovation for Phenotypic Drug Discovery: The PD2 Assay Panel." Journal of Biomolecular Screening 16, no. 6 (April 26, 2011): 588–602. http://dx.doi.org/10.1177/1087057111405379.
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Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target “agnostic” fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD2), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD2 assay panel. Analysis of PD2 submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD2 have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD2, may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.
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Roberts, Jonathan C., Patti A. Morateck, Pamela A. Christopherson, Ke Yan, Raymond G. Hoffmann, Joan Cox Gill, and Robert R. Montgomery. "Rapid discrimination of the phenotypic variants of von Willebrand disease." Blood 127, no. 20 (May 19, 2016): 2472–80. http://dx.doi.org/10.1182/blood-2015-11-664680.
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Key Points A novel ELISA-based VWF multiplex activity assay assigns VWD phenotype among a cohort of type 1 and 2 VWD with an overall accuracy of >88%. This assay shows correlation with traditional quantitative clinical VWF assays and may provide a rapid diagnostic method for variant VWD.
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Fluit, Ad C., Maarten R. Visser, and Franz-Josef Schmitz. "Molecular Detection of Antimicrobial Resistance." Clinical Microbiology Reviews 14, no. 4 (October 1, 2001): 836–71. http://dx.doi.org/10.1128/cmr.14.4.836-871.2001.
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SUMMARY The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art.
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Tanabe, Kenji. "Abstract LB238: Image-based phenotypic profiling of a chemogenomic screening library identifies nobel targets of known inhibitors." Cancer Research 83, no. 8_Supplement (April 14, 2023): LB238. http://dx.doi.org/10.1158/1538-7445.am2023-lb238.
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Abstract The gene encoding epidermal growth factor receptor (EGFR) is a major driver gene in cancer. Many drugs targeting EGFR-associated molecules have been developed, yet many have failed in clinical trials due to a lack of efficacy and/or unexpected side effects. In this study, I used image-based phenotypic profiling to screen a pharmacologically active compound library with the aim of identifying new druggable targets in the EGFR pathway. As anticipated, the phenotypic screen identified compounds that produce phenotypes resulting from targeting a known specific molecule or pathway. The assay also showed that compounds with diverse known mechanisms of action produced similar, EGFR-related cellular phenotypes. Biochemical assays revealed that those compounds share a previously unappreciated common target/pathway, showing that the image-based assay can identify new target molecules that are independent of the compound’s known target. Citation Format: Kenji Tanabe. Image-based phenotypic profiling of a chemogenomic screening library identifies nobel targets of known inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB238.
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Phillipou, Alexander N., Charles S. Lay, Charlotte E. Carver, Cassie Messenger, John P. Evans, Antonia J. Lewis, Laurie J. Gordon, et al. "Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 2 (December 25, 2019): 163–75. http://dx.doi.org/10.1177/2472555219896278.
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Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound–target engagement within native cellular environments to drive high-throughput, routine structure–activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein–protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound–target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.
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Joslin, John, James Gilligan, Paul Anderson, Catherine Garcia, Orzala Sharif, Janice Hampton, Steven Cohen, et al. "A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery." SLAS DISCOVERY: Advancing the Science of Drug Discovery 23, no. 7 (May 29, 2018): 697–707. http://dx.doi.org/10.1177/2472555218773086.
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The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.
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McSharry, James J., Ann C. McDonough, Betty A. Olson, and George L. Drusano. "Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors." Clinical Diagnostic Laboratory Immunology 11, no. 1 (January 2004): 21–28. http://dx.doi.org/10.1128/cdli.11.1.21-28.2004.
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ABSTRACT A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.
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Coz, Clementine Le, John R. Christin, Talal Syed, Zejian Wang, Caroline J. Laplaca, Andrew T. Lenis, and Michael M. Shen. "Abstract B025: Luminal-to-basal phenotypic plasticity promotes invasive phenotypes in a live-imaging assay using patient-derived bladder tumor organoids." Clinical Cancer Research 30, no. 10_Supplement (May 17, 2024): B025. http://dx.doi.org/10.1158/1557-3265.bladder24-b025.
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Abstract Lineage plasticity in cancer is characterized by changes in cell state that are often associated with tumor progression and treatment resistance. While nearly all non-muscle invasive bladder cancer (NMIBC) tumors have a luminal epithelial phenotype, many muscle invasive bladder cancers (MIBC) display a basal identity. NMIBC to MIBC progression may therefore be related to a phenotypic switch from luminal-to-basal subtypes, which in turn may be associated with poorer clinical outcomes. However, current understanding of the molecular processes linking cell plasticity and tumor invasion is limited, due in part to a lack of physiologically relevant in vitro models and assays. To address these questions, we are utilizing a living biobank of 82 patient-derived bladder tumor organoid lines that our group has established. These organoids represent a 3-dimensional, heterogeneous model system for studying bladder cancer, as they can be genetically manipulated and recapitulate the molecular and histopathological features of the parental tumor. Notably, in previous work, we described luminal-to-basal phenotypic plasticity in a subset of organoid lines derived from luminal tumors, which display a shift to basal/squamous identity in culture that is reversible by orthotopic xenografting. In current studies, we are investigating cell migration and invasiveness using a live-imaging assay that we have developed to examine bladder tumor organoid outgrowth in vitro. Importantly, this assay allows longitudinal visualization as well as quantitation of invasiveness over a four-day time span. Analysis of over 25 organoid lines with plastic, stable luminal, and stable basal properties has revealed a broad spectrum of organoid outgrowth phenotypes that do not correlate with cell proliferation or clinical parameters. Instead, the degree of outgrowth correlates precisely with luminal-to-basal phenotypic plasticity, as assessed by transcriptome analyses, immunofluorescence microscopy, and in vivo xenografting. Organoid lines with minimal outgrowth have stable luminal phenotypes, whereas lines that produce their own extracellular matrix to create a leading edge of migrating cells have plastic phenotypes. Furthermore, movies of the invading organoids reveal distinct cell migratory patterns, as several organoid lines display collective migration that generates an invasive front at the leading edge. This collective migration phenotype is distinct from phenotypic plasticity, is at least partially independent from organoid outgrowth, and is undergoing further analysis. Thus, our organoid outgrowth assay identifies two different parameters of invasive potential, one of which correlates with phenotypic plasticity. Our findings characterize bladder tumor invasion patterns for the first time and suggest that lineage plasticity in NMIBC represents a key mechanism that promotes tumor invasion in MIBC. Citation Format: Clementine Le Coz, John R. Christin, Talal Syed, Zejian Wang, Caroline J. Laplaca, Andrew T. Lenis, Michael M. Shen. Luminal-to-basal phenotypic plasticity promotes invasive phenotypes in a live-imaging assay using patient-derived bladder tumor organoids [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B025.
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Gierahn, Todd M., Ayca Yalcin, Denis Loginov, and J. Christopher Love. "Deep phenotypic profiling of small clinical samples through MultiSpectral Imaging Cytometry (MuSIC) in nanowell arrays." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 138.6. http://dx.doi.org/10.4049/jimmunol.196.supp.138.6.
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Abstract Many disease relevant immune processes occur within tissues, making them challenging to monitor clinically using standard blood based assays. Comprehensive phenotyping of the scarce cells acquired directly from tissue samples is limited by inefficiencies in sample handling and data collection inherent in current flow-based profiling methods. To overcome this barrier, we developed Multi-Spectral Imaging Cytometry (MuSIC), a method that combines an array of sub-nanoliter wells (nanowells) for efficient cell capture with multi-spectral imaging to resolve up to 16 fluorescent markers on ~103–105 cells. Importantly, the cells remain viable for further functional characterization, enabling linkage of deep cell surface phenotypes with functional activity. Using MuSIC in combination with a single-cell protein secretion assay, we were able to identify unique T cell populations present in human cervical biopsies. MuSIC provides an effective and accessible means to acquire deep phenotypic profiles of leukocytes from small clinical samples.
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Koroska, Florian, Stephan Göttig, Martin Kaase, Jörg Steinmann, Sören Gatermann, Julian Sommer, Thorsten Wille, Georg Plum, and Axel Hamprecht. "Comparison of Phenotypic Tests and an Immunochromatographic Assay and Development of a New Algorithm for Detection of OXA-48-like Carbapenemases." Journal of Clinical Microbiology 55, no. 3 (December 28, 2016): 877–83. http://dx.doi.org/10.1128/jcm.01929-16.
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ABSTRACT OXA-48 is the most prevalent carbapenemase in Enterobacteriaceae in Europe and the Middle East, but it is frequently missed because many isolates display low MICs for carbapenems. Furthermore, in contrast to metallo-β-lactamases or Klebsiella pneumoniae carbapenemases (KPC), no specific inhibitor is available for the phenotypic detection of OXA-48. Molecular detection of bla OXA-48 is the “gold standard” but is not available in many laboratories. A few phenotypic assays have been described but have not been independently evaluated. The aim of this study was the systematic comparison of phenotypic tests and an immunochromatographic assay (ICT) for the detection of OXA-48/OXA-48-like carbapenemases and the development of an algorithm for reliable phenotypic detection of OXA-48. Four phenotypic tests (temocillin disk test, faropenem disk test, OXA-48 disk test, and high-inoculum [HI] OXA-48 disk test) and a new ICT (OXA-48 K -SeT) were compared by using a set of 166 Enterobacteriaceae isolates, including isolates producing OXA-48/OXA-48-like carbapenemases ( n = 84) or Ambler class A and B carbapenemases ( n = 41) and carbapenemase-negative isolates ( n = 41). The sensitivity and specificity for the different assays were 100% and 43.9% for temocillin, 57.1% and 98.8% for faropenem, 53.6% and 100% for the OXA-48 disk test, 98.8% and 97.6% for the HI OXA-48 disk test, and 100% and 100% for the ICT, respectively. The ICT displayed the highest sensitivity and specificity and was the most rapid assay, but it is more costly than phenotypic assays. Based on these results, a new algorithm incorporating temocillin, faropenem, and ICT which allows cost-effective detection of OXA-48 with 100% sensitivity and specificity was developed.
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Efstratiou, Androulla, Kathryn H. Engler, Charlotte S. Dawes, and Dorothea Sesardic. "Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria." Journal of Clinical Microbiology 36, no. 11 (1998): 3173–77. http://dx.doi.org/10.1128/jcm.36.11.3173-3177.1998.
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We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates ofCorynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the “gold standard” in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.
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Johnson, Abigail, Bonnie P. Weber, Divek T. Nair, Randall S. Singer, Anup Kollanoor Johny, and Timothy J. Johnson. "Evaluating Turkey-Derived Lactic-Acid-Producing Bacteria as Potential Probiotics for Use in Commercial Turkeys." Applied Sciences 14, no. 5 (February 29, 2024): 2010. http://dx.doi.org/10.3390/app14052010.
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Lactic-acid-producing bacteria (LAB) are widely used in the poultry industry, and they are positively associated with gut health and growth performance. Despite their wide use in poultry production, LAB appear to be highly variable in their ability to modulate poultry gut health and growth performance. Furthermore, most commercially available LAB probiotics are not host specific; thus, few poultry-specific and even fewer turkey-specific probiotics exist. The objective of this study was to use probiotic screening assays to compare relevant phenotypic differences amongst different species of turkey-derived LAB, in an effort to identify potential probiotics for use in turkey production. Different in vitro assays were used to compare the probiotic potential (phenotype) of each turkey-derived LAB isolate. Twenty-four isolates representing eight different species and five different genera were used for our experiments. These assays included acid tolerance, bile tolerance, and adhesion ability. There was variability in assay performance across many individual strains in every assay performed. Isolates between species and, in some cases, isolates within the same species, differed in their performance between the assays. Some isolates that were identified performed favorably in all the assays in this study. In conclusion, high-performing isolates were identified in this study, which hold potential for influencing turkey health and productivity.
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Larsen, Ray A., Gregory J. Chen, and Kathleen Postle. "Performance of Standard Phenotypic Assays for TonB Activity, as Evaluated by Varying the Level of Functional, Wild-Type TonB." Journal of Bacteriology 185, no. 16 (August 15, 2003): 4699–706. http://dx.doi.org/10.1128/jb.185.16.4699-4706.2003.
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ABSTRACT The ability of gram-negative bacterial cells to transport cobalamin and iron-siderophore complexes and their susceptibility to killing by some bacteriophages and colicins are characteristics routinely used to assay mutations of proteins in the TonB-dependent energy transduction system. These assays vary greatly in sensitivity and are subject to perturbation by overexpression of TonB and, perhaps, other proteins that contribute to the process. Thus, the choice of assay and the means by which a potential mutant is expressed can greatly influence the interpretation and recognition of a given mutant. In the present study, we expressed TonB at several different quantified levels in cells that were then subjected to a panel of assays. Our results suggest that it is reasonable to regard the assays as having windows of sensitivity. Thus, while no single assay satisfactorily spans the potential range of TonB activity, it is evident that certain assays are better suited for resolving small deviations from wild-type levels of activity, with others most useful when activity levels are very low. It is apparent from the results that the application of all possible assays to the characterization of new mutants will yield the most meaningful results.
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Simner, Patricia J., Belita N. A. Opene, Krizia K. Chambers, Matthew E. Naumann, Karen C. Carroll, and Pranita D. Tamma. "Carbapenemase Detection among Carbapenem-Resistant Glucose-Nonfermenting Gram-Negative Bacilli." Journal of Clinical Microbiology 55, no. 9 (July 12, 2017): 2858–64. http://dx.doi.org/10.1128/jcm.00775-17.
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ABSTRACT Accurate detection of carbapenemase-producing glucose-nonfermenting Gram-negative bacilli (CPNFs), including Pseudomonas aeruginosa and Acinetobacter baumannii , is necessary to prevent their dissemination within health care settings. We performed a method comparison study of 11 phenotypic carbapenemase detection assays to evaluate their accuracy for the detection of CPNFs. A total of 96 carbapenem-resistant glucose-nonfermenting isolates were included, of which 29% produced carbapenemases. All CPNFs were molecularly characterized to identify β-lactamase genes. A total of 86% of the carbapenemase-producing P. aeruginosa isolates produced class B carbapenemases. Several assays performed with a sensitivity of >90% for the detection of carbapenemase-producing P. aeruginosa , including all rapid chromogenic assays and the modified carbapenem inactivation method. Most included assays, with the exception of the Manual Blue Carba assay, the Modified Carba NP assay, the boronic acid synergy test, and the metallo-β-lactamase Etest, had specificities of >90% for detecting carbapenemase-producing P. aeruginosa . Class D carbapenemases were the most prevalent carbapenemases among the carbapenemase-producing A. baumannii strains, with 60% of the carbapenemase-producing A. baumannii isolates producing acquired OXA-type carbapenemases. Although several assays achieved >90% specificity in identifying carbapenemase-producing A. baumannii , no assays achieved a sensitivity of greater than 90%. Our findings suggest that the available phenotypic tests generally appear to have excellent sensitivity and specificity for detecting carbapenemase-producing P. aeruginosa isolates. However, further modifications to existing assays or novel assays may be necessary to accurately detect carbapenemase-producing A. baumannii .
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Qi, Yan, Xiuying Liang, Haijing Guan, Jingwen Sun, and Wenjuan Yao. "RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation." Biomedicines 9, no. 9 (September 6, 2021): 1169. http://dx.doi.org/10.3390/biomedicines9091169.
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RhoGTPase is involved in PDGF-BB-mediated VSMC phenotypic modulation. RhoGDIs are key factors in regulating RhoGTPase activation. In the present study, we investigated the regulatory effect of RhoGDI1 on the activation of RhoGTPase in VSMC transformation and neointima formation. Western blot and co-immunoprecipitation assays showed that the PDGF receptor inhibition by crenolanib promoted RhoGDI1 polyubiquitination and degradation. Inhibition of RhoGDI1 degradation via MG132 reversed the decrease in VSMC phenotypic transformation. In addition, RhoGDI1 knockdown significantly inhibited VSMC phenotypic transformation and neointima formation in vitro and in vivo. These results suggest that PDGF-BB promotes RhoGDI1 stability via the PDGF receptor and induces the VSMC synthetic phenotype. The co-immunoprecipitation assay showed that PDGF-BB enhanced the interaction of RhoGDI1 with Cdc42 and promoted the activation of Cdc42; these enhancements were blocked by crenolanib and RhoGDI1 knockdown. Moreover, RhoGDI1 knockdown and crenolanib pretreatment prevented the localization of Cdc42 to the plasma membrane (PM) to activate and improve the accumulation of Cdc42 on endoplasmic reticulum (ER). Furthermore, Cdc42 inhibition or suppression significantly reduced VSMC phenotypic transformation and neointima formation in vitro and in vivo. This study revealed the novel mechanism by which RhoGDI1 stability promotes the RhoGDI1-Cdc42 interaction and Cdc42 activation, thereby affecting VSMC phenotypic transformation and neointima formation.
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Hadad, Ronza, Michelle Jayne Cole, Samantha Ebeyan, Susanne Jacobsson, Lit Yeen Tan, Daniel Golparian, Simon Erskine, et al. "Evaluation of the SpeeDx ResistancePlus® GC and SpeeDx GC 23S 2611 (beta) molecular assays for prediction of antimicrobial resistance/susceptibility to ciprofloxacin and azithromycin in Neisseria gonorrhoeae." Journal of Antimicrobial Chemotherapy 76, no. 1 (September 15, 2020): 84–90. http://dx.doi.org/10.1093/jac/dkaa381.
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Abstract Background Accurate molecular assays for prediction of antimicrobial resistance (AMR)/susceptibility in Neisseria gonorrhoeae (Ng) can offer individualized treatment of gonorrhoea and enhanced AMR surveillance. Objectives We evaluated the new ResistancePlus® GC assay and the GC 23S 2611 (beta) assay (SpeeDx), for prediction of resistance/susceptibility to ciprofloxacin and azithromycin, respectively. Methods Nine hundred and sixty-seven whole-genome-sequenced Ng isolates from 20 European countries, 143 Ng-positive (37 with paired Ng isolates) and 167 Ng-negative clinical Aptima Combo 2 (AC2) samples, and 143 non-gonococcal Neisseria isolates and closely related species were examined with both SpeeDx assays. Results The sensitivity and specificity of the ResistancePlus® GC assay to detect Ng in AC2 samples were 98.6% and 100%, respectively. ResistancePlus® GC showed 100% sensitivity and specificity for GyrA S91 WT/S91F detection and 99.8% sensitivity and specificity in predicting phenotypic ciprofloxacin resistance. The sensitivity and specificity of the GC 23S 2611 (beta) assay for Ng detection in AC2 samples were 95.8% and 100%, respectively. GC 23S 2611 (beta) showed 100% sensitivity and 99.9% specificity for 23S rRNA C2611 WT/C2611T detection and 64.3% sensitivity and 99.9% specificity for predicting phenotypic azithromycin resistance. Cross-reactions with non-gonococcal Neisseria species were observed with both assays, but the analysis software solved most cross-reactions. Conclusions The new SpeeDx ResistancePlus® GC assay performed well in the detection of Ng and AMR determinants, especially in urogenital samples. The GC 23S 2611 (beta) assay performed relatively well, but its sensitivity, especially for predicting phenotypic azithromycin resistance, was suboptimal and further optimizations are required, including detection of additional macrolide resistance determinant(s).
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Meaza, Abyot, Ephrem Tesfaye, Zemedu Mohamed, Betselot Zerihun, Getachew Seid, Kirubel Eshetu, Miskir Amare, et al. "Diagnostic accuracy of Truenat Tuberculosis and Rifampicin-Resistance assays in Addis Ababa, Ethiopia." PLOS ONE 16, no. 12 (December 28, 2021): e0261084. http://dx.doi.org/10.1371/journal.pone.0261084.
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Background Rapid and sensitive Tuberculosis (TB) diagnosis closer to patients is a key global TB control priority. Truenat assays (MTB, MTB Plus, and MTB-RIF Dx) are new TB molecular diagnostic tools for the detection of TB and Rifampicin (RIF)-resistance from sputum samples. The diagnostic accuracy of the assays is needed prior to implementation in clinical use in Ethiopia. This study aimed to determine the sensitivity and specificity of Truenat assays; and aimed to compare the assays to the Xpert MTB/RIF assay. Methods A prospective evaluation study was conducted among 200 presumptive TB patients in microscopy centers in Addis Ababa, Ethiopia from May 2019 to December 2020. Culture (Solid and Liquid methods) and phenotypic (liquid method) drug susceptibility testing (DST) were used as a reference standard. Results Of 200 adult participants, culture confirmed TB cases were 25 (12.5%), and only one isolate was resistant to RIF by phenotypic DST. The sensitivity of Truenat MTB was 88.0% [95% CI 70.1, 95.8], while 91.7 [95% CI 74.2, 97.7] for Truenat MTB Plus at the microscopy centers. The specificity of Truenat MTB was 97.2% [95% CI 93.1, 98.9], while for Truenat MTB Plus was 97.2% [95% CI 93.0, 99.0]. The sensitivity of Truenat MTB was 90.5% while for MTB Plus, 100% compared to the Xpert MTB/RIF assay. Conclusion Truenat assays were found to have high diagnostic accuracy. The assays have the potential to be used as a point of care (POC) TB diagnostic tests.
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Eltringham, I. J., S. M. Wilson, and F. A. Drobniewski. "Evaluation of a Bacteriophage-Based Assay (Phage Amplified Biologically Assay) as a Rapid Screen for Resistance to Isoniazid, Ethambutol, Streptomycin, Pyrazinamide, and Ciprofloxacin among Clinical Isolates of Mycobacterium tuberculosis." Journal of Clinical Microbiology 37, no. 11 (1999): 3528–32. http://dx.doi.org/10.1128/jcm.37.11.3528-3532.1999.
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Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries.
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Swinney, David C. "The Contribution of Mechanistic Understanding to Phenotypic Screening for First-in-Class Medicines." Journal of Biomolecular Screening 18, no. 10 (August 27, 2013): 1186–92. http://dx.doi.org/10.1177/1087057113501199.
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The level of mechanistic understanding required for drug discovery is a central feature of most strategies. However, an understanding of mechanism is not required for regulatory approval. This paradox is particularly relevant to the role of phenotypic assays in drug discovery. A recent analysis revealed that phenotypic drug discovery strategies were more successful for first-in-class medicines, whereas target-based molecular strategies were more successful for followers ( Nat. Rev. Drug Discov.2011, 10, 507–519). The rationale for the success of phenotypic screening was the unbiased identification of the molecular mechanism of action. In this follow-up analysis, the format and mechanistic information used to establish the phenotypic assays that led to the first-in-class small-molecule new molecular entities approved by the U.S. Food and Drug Administration between 1999 and 2008 were analyzed and compared with those approved in 2012. Not surprisingly, some level of mechanistic understanding was used to select the assay formats and chemicals screened. It is concluded that mechanism takes on different connotations depending on context and perspective and that a target need not always be the exclusive definition of mechanism.
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Lurain, Nell S., and Sunwen Chou. "Antiviral Drug Resistance of Human Cytomegalovirus." Clinical Microbiology Reviews 23, no. 4 (October 2010): 689–712. http://dx.doi.org/10.1128/cmr.00009-10.
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SUMMARY The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.
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Wang, Jixia, Xiuli Zhang, Ye Fang, and Xinmiao Liang. "Label-Free Cell Phenotypic Assays for Assessing Drug Polypharmacology." Current Pharmaceutical Design 22, no. 21 (May 30, 2016): 3190–200. http://dx.doi.org/10.2174/1381612822666160224142048.
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Marks, Denese C., and Lacey Johnson. "Assays for phenotypic and functional characterization of cryopreserved platelets." Platelets 30, no. 1 (September 25, 2018): 48–55. http://dx.doi.org/10.1080/09537104.2018.1514108.
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Vincent, Fabien, Paula Loria, Marko Pregel, Robert Stanton, Linda Kitching, Karl Nocka, Regis Doyonnas, et al. "Developing predictive assays: The phenotypic screening “rule of 3”." Science Translational Medicine 7, no. 293 (June 24, 2015): 293ps15. http://dx.doi.org/10.1126/scitranslmed.aab1201.
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Demeter, Lisa, and Richard Haubrich. "Phenotypic and Genotypic Resistance Assays: Methodology, Reliability, and Interpretations." JAIDS Journal of Acquired Immune Deficiency Syndromes 26 (March 2001): S3—S9. http://dx.doi.org/10.1097/00126334-200103011-00002.
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Jeong, Euna, Sung Ung Moon, Mee Song, and Sukjoon Yoon. "Transcriptome modeling and phenotypic assays for cancer precision medicine." Archives of Pharmacal Research 40, no. 8 (August 2017): 906–14. http://dx.doi.org/10.1007/s12272-017-0940-z.
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TOHSATO, YUKAKO, TOMOYA BABA, YUSAKU MAZAKI, MASAHIRO ITO, BARRY L. WANNER, and HIROTADA MORI. "ENVIRONMENTAL DEPENDENCY OF GENE KNOCKOUTS ON PHENOTYPE MICROARRAY ANALYSIS IN ESCHERICHIA COLI." Journal of Bioinformatics and Computational Biology 08, supp01 (December 2010): 83–99. http://dx.doi.org/10.1142/s021972001000521x.
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Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray™ (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.
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Gkoutos, G. V., E. C. J. Green, A. M. Mallon, A. Blake, S. Greenaway, J. M. Hancock, and D. Davidson. "Ontologies for the Description of Mouse Phenotypes." Comparative and Functional Genomics 5, no. 6-7 (2004): 545–51. http://dx.doi.org/10.1002/cfg.430.
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Ontologies are becoming increasingly important for the efficient storage, retrieval and mining of biological data. The description of phenotypes using ontologies is a particularly complex problem. We outline a schema that can be used to describe phenotypes by combining orthologous axiomatic ontologies. We also describe tools for storing, browsing and searching such complex ontologies. Central to this approach is that assays (protocols for measuring phenotypic characters) describe what has been measured as well as how this was done, allowing assays to link individual organisms to ontologies describing phenotypes. We have evaluated this approach by automatically annotating data on 600 000 mutant mice phenotypes using the SHIRPA protocol. We believe this approach will enable the flexible, extensible and detailed description of phenotypes from any organism.
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Weinberg, Adriana, Lin-Ye Song, Cynthia Wilkening, Anne Sevin, Bruce Blais, Raul Louzao, Dana Stein, et al. "Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization." Clinical and Vaccine Immunology 16, no. 8 (June 10, 2009): 1176–86. http://dx.doi.org/10.1128/cvi.00342-08.
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ABSTRACT The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.
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Roy, Ruchi, Sunil Kumar Singh, Nashrah Ahmad, and Sweta Misra. "Challenges and advancements in high-throughput screening strategies for cancer therapeutics." Global Translational Medicine 3, no. 1 (March 12, 2024): 2448. http://dx.doi.org/10.36922/gtm.2448.
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Drug discovery relies on high-throughput screening (HTS) methods incorporating both target- and cell-based assays. This comprehensive review delves into the challenges and benefits associated with these assays within the context of HTS. The strategies for developing screening assays, spanning both primary and secondary screens for target identification, are discussed. Furthermore, we review the methods of identifying the most efficacious drugs through these approaches for the treatment of cancer in detail. While various drugs have been identified for cancer treatment, there remains a pressing need for more relevant phenotypic assays. These assays aim to produce the desired disease phenotype, with a specific emphasis on highlighting targets rather than off-targets. The ultimate goal is to pave the way for innovative drug development strategies that can effectively treat cancer patients, thereby reducing the mortality rate.
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Bhattacharyya, Roby, Jamin Liu, Peijun Ma, Nirmalya Bandyopadhyay, Jonathan Livny, and Deborah Hung. "Rapid Phenotypic Antibiotic Susceptibility Testing Through RNA Detection." Open Forum Infectious Diseases 4, suppl_1 (2017): S33. http://dx.doi.org/10.1093/ofid/ofx162.082.
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Abstract Background Culture-based antibiotic susceptibility testing, the gold standard, is too slow to guide early antibiotic selection, while newer genotypic methods require comprehensive knowledge of resistance mechanisms to predict phenotype. Quantitative measurement of key antibiotic-responsive transcripts offers a rapid, phenotypic assay for assessing antibiotic susceptibility, agnostic to the genetic basis for resistance. Methods We performed RNA-Seq on Klebsiella pneumoniae and Acinetobacter baumanii treated with ciprofloxacin, gentamicin, or meropenem for 0, 10, 30, and 60 minutes. For each, we identified 50 responsive transcripts whose expression levels differ most between susceptible and resistant organisms upon antibiotic exposure. We measured their expression using a multiplexed fluorescent RNA hybridization assay (NanoString) in 69 clinical isolates, including a “test set” of multidrug-resistant strains from the CDC, in an 8-hour assay. Gene expression data from test strains were compared against known susceptible and resistant isolates to generate a transcriptional susceptibility metric. We also designed NanoString probes to detect 5 carbapenemase genes (KPC-2, KPC-3, NDM-1, OXA-48, and CTX-M15). Results Across all bacteria-antibiotic pairs tested, a susceptibility metric derived from these transcriptional assays correctly grouped isolates in 167 of 173 tests (Table 1), with only 1 of 88 resistant isolates misclassified as susceptible. Five of six incorrectly grouped isolates were within one dilution of the breakpoint MIC, including the misclassified resistant isolate. Conclusion We demonstrate phenotypic antibiotic resistance detection based on fluorescent RNA detection in an 8-hour assay. We have previously published proof-of-concept studies that this assay may be run on a positive blood culture bottle with minimal sample processing. By coupling this phenotypic assay with detection of genetic resistance determinants (demonstrated for carbapenemases) in a single assay, strains with unexplained resistance can be prioritized for further study. Disclosures All authors: No reported disclosures.
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Chamchoy, Kamonwan, Sirapapha Sudsumrit, Jutamas Wongwigkan, Songsak Petmitr, Duantida Songdej, Emily R. Adams, Thomas Edwards, Ubolsree Leartsakulpanich, and Usa Boonyuen. "Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females." PLOS ONE 18, no. 11 (November 15, 2023): e0294200. http://dx.doi.org/10.1371/journal.pone.0294200.
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Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%–100%] with a specificity of 100% (95% CI: 88.78%–100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.
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Bar, Haim, and Adam Zweifach. "Z’ Does Not Need to Be > 0.5." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 9 (August 4, 2020): 1000–1008. http://dx.doi.org/10.1177/2472555220942764.
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The assay metric Z’ has come to play a critical gatekeeping role in determining whether high-throughput assays can be performed. While Z’ is commonly required to be > 0.5, this expectation is not well supported. Requiring Z’ > 0.5 likely prevents many potentially useful phenotypic and cell-based screens from being conducted, and causes other assays to be conducted under extreme conditions that may prevent activity from being found. We used power analysis and a novel numerical simulation approach to determine how Z’ reflects assay performance under a variety of conditions. Our results show that assays with Z’ > 0.5 perform better than assays with lower Z’, but when an appropriate threshold is selected, assays with Z’ < 0.5 can almost always find useful compounds without generating too many false positives. We provide a method that will allow researchers to estimate how to set an appropriate threshold for their assay. We suggest that instead of always requiring Z’ > 0.5, assays with Z’ < 0.5 should be performed when they can be justified in terms of the importance of the target and the limitations of alternate assay formats.
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Gergen, J. Peter. "Dosage Compensation in Drosophila: Evidence That daughterless and Sex-lethal Control X Chromosome Activity at the Blastoderm Stage of Embryogenesis." Genetics 117, no. 3 (November 1, 1987): 477–85. http://dx.doi.org/10.1093/genetics/117.3.477.
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ABSTRACT Dosage compensation is a mechanism that equalizes the expression of X chromosome linked genes in males, who have one X chromosome, with that in females, who have two. In Drosophila, this is achieved by the relative hyperactivation of X-linked genes in males, as was first shown by Muller using a phenotypic assay based on adult eye color. Several genes involved in regulating dosage compensation have been identified through the isolation of mutations that are sex-specific lethals. However, because of this lethality it is not straightforward to assay the relative roles of these genes using assays based on adult phenotypes. Here this problem is circumvented using an assay based on embryonic phenotypes. These experiments indicate that dosage compensation is established early in development and demonstrate that the daughterless and Sex-lethal gene products are involved in regulating X chromosome activity at the blastoderm stage of embryogenesis.
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McDiarmid, Troy A., Manuel Belmadani, Joseph Liang, Fabian Meili, Eleanor A. Mathews, Gregory P. Mullen, Ardalan Hendi, et al. "Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation." Proceedings of the National Academy of Sciences 117, no. 1 (November 21, 2019): 656–67. http://dx.doi.org/10.1073/pnas.1912049116.
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A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we usedCaenorhabditis elegansto systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype–phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered onCHD8•chd-7andNLGN3•nlg-1that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 innlg-1mutantC. elegansrescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.
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Margot, Nicolas, Laurie Vanderveen, Vidula Naik, Renee Ram, PC Parvangada, Ross Martin, Martin Rhee, and Christian Callebaut. "Phenotypic resistance to lenacapavir and monotherapy efficacy in a proof-of-concept clinical study." Journal of Antimicrobial Chemotherapy 77, no. 4 (January 13, 2022): 989–95. http://dx.doi.org/10.1093/jac/dkab503.
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Abstract Background Lenacapavir in vitro resistance selections identified seven mutations in HIV-1 capsid protein (CA) associated with reduced susceptibility. Objectives To analyse lenacapavir activity against lenacapavir-associated resistance mutations in multiple assays. We also report Day 10 resistance analyses conducted in a Phase 1b study of lenacapavir (Study 4072) in people with HIV (PWH). Methods Mutations were inserted in a proviral DNA clone by site-directed mutagenesis, and viruses (n = 12) were generated by transfection. Sequences were used to generate single-cycle (SC) test vectors that were evaluated in a Gag-Pro assay, and replicative viruses were tested in a multicycle (MC) MT-2 assay to determine lenacapavir susceptibility. Study 4072 was a Phase 1b, double-blinded, placebo-controlled, dose-ranging, randomized study of lenacapavir in untreated PWH. Participants received a single dose of lenacapavir (up to 750 mg) or placebo (10 day monotherapy). CA resistance was characterized using genotypic and/or phenotypic assays. Results Lenacapavir susceptibility in the SC assay showed an inverse relationship between replication capacity and resistance. In Study 4072, all 29 participants receiving lenacapavir showed a robust virological response with no rebound. At baseline, no participant had resistance mutations to lenacapavir, and all had WT susceptibility to lenacapavir. Post-monotherapy analyses revealed the emergence of CA mutation Q67H at Day 10 in two participants. Conclusions In vitro assays confirmed that increased resistance to lenacapavir was associated with decreased replication capacity of mutant viruses. In the clinical study no pre-existing lenacapavir resistance was detected. Emergence of Q67H occurred at exposures below the dose used in current Phase 2/3 studies. These results support development of lenacapavir as an antiretroviral agent.
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Wheeler, Nicolas J., Kendra J. Gallo, Elena J. G. Rehborg, Kaetlyn T. Ryan, John D. Chan, and Mostafa Zamanian. "wrmXpress: A modular package for high-throughput image analysis of parasitic and free-living worms." PLOS Neglected Tropical Diseases 16, no. 11 (November 18, 2022): e0010937. http://dx.doi.org/10.1371/journal.pntd.0010937.
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Advances in high-throughput and high-content imaging technologies require concomitant development of analytical software capable of handling large datasets and generating relevant phenotypic measurements. Several tools have been developed to analyze drug response phenotypes in parasitic and free-living worms, but these are siloed and often limited to specific instrumentation, worm species, and single phenotypes. No unified tool exists to analyze diverse high-content phenotypic imaging data of worms and provide a platform for future extensibility. We have developed wrmXpress, a unified framework for analyzing a variety of phenotypes matched to high-content experimental assays of free-living and parasitic nematodes and flatworms. We demonstrate its utility for analyzing a suite of phenotypes, including motility, development/size, fecundity, and feeding, and establish the package as a platform upon which to build future custom phenotypic modules. We show that wrmXpress can serve as an analytical workhorse for anthelmintic screening efforts across schistosomes, filarial nematodes, and free-living model nematodes and holds promise for enabling collaboration among investigators with diverse interests.
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Matsumoto, Yuji, Tomotsugu Ichikawa, Kazuhiko Kurozumi, Yoshihiro Otani, Atsushi Fujimura, Kentaro Fujii, Yosuke Shimazu, et al. "ANGI-08. AN ANNEXIN A2-REGULATED PHENOTYPIC SHIFT IN GLIOMA." Neuro-Oncology 21, Supplement_6 (November 2019): vi31—vi32. http://dx.doi.org/10.1093/neuonc/noz175.119.
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Abstract BACKGROUND Malignant glioma has a poor prognosis and is characterized by excessive proliferation, invasion, and angiogenesis. Previously, we established subclones of glioma cell lines with different invasive phenotypes to investigate the mechanisms of invasion in this malignancy. That study revealed annexin A2 (ANXA2) as an activator of angiogenesis and perivascular invasion. Here, we investigated the molecular mechanism of the phenotypic shift in glioma that is induced by ANXA2. METHODS We identified ANXA2 target genes using microarray analyses of our four established cell lines and RNA-seq data from the IVY Glioblastoma Atlas Project. Co-expression was then confirmed in human glioma cells. Additionally, qRT-PCR, immunoblotting, invasion assays, proliferation assays, and tube formation assays were performed to uncover the specific roles of these angiogenic invasion-related genes. Furthermore, we examined survival times and phenotypic shifts in vivo. RESULTS We identified oncostatin M receptor (OSMR) as an ANXA2-target gene using microarray and RNA-seq, and confirmed its expression correlated with ANXA2 in glioma cells. ANXA2-overexpressing glioma cells showed enhanced OSMR expression and STAT3 phosphorylation, while STAT3 knockdown reduced OSMR expression. When ANXA2 was overexpressed in glioma cells, invasion, angiogenesis, proliferation, and epithelial-mesenchymal transition were promoted; however, silencing OSMR attenuated the ANXA2-induced phenotypic shift. In vivo, ANXA2-overexpressing glioma cells shortened the survival time of tumor-bearing mice, whereas OSMR knockdown increased survival times. CONCLUSIONS We analyzed genes whose expression was regulated by ANXA2 in glioma using invasive glioma models. Through this analysis, we identified that ANXA2 and OSMR regulate a phenotypic shift, suggesting that OSMR could be a promising target to treat and prevent glioma invasion.
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Wang, Kai, Ram Samudrala, and John E. Mittler. "Antivirogram or Phenosense: A Comparison of their Reproducibility and an Analysis of their Correlation." Antiviral Therapy 9, no. 5 (July 1, 2003): 703–12. http://dx.doi.org/10.1177/135965350400900501.
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The Antivirogram and PhenoSense assays are widely used phenotypic tests for HIV drug resistance. There are limited data on the reproducibility of each assay, and little is known about the correlation between the two. Using data from the Stanford HIV drug resistance database, we performed a comprehensive analysis of the reproducibility of each assay, and calculated the correlation and concordance of the two assays using both general IC50 fold change cutoff values and drug-specific cutoff values. Although the within-assay correlations were high (rank correlation coefficients r=0.94 and r=0.95 for the Antivirogram and PhenoSense assays, respectively), the between-assay correlation was considerably lower ( r=0.36). Using drug-specific cutoff values for viruses classified as resistant by the Antivirogram or PhenoSense assays, respectively, only 71.4% [95% confidence intervals (95% CI): 58.7–82.1%] and 57.0% (95% CI: 45.3–68.1%) of the samples were classified as resistant using the other assay. The poor agreement between the assays was primarily due to the extremely poor correlation between these assays for samples with low resistance values ( r=0.02 and r=0.61 for samples with the Antivirogram measurements lower or higher than 2.0, respectively). Since the cutoff values for both assays are relatively low, our analysis suggests that one should be very careful when interpreting measurements that are near the cutoff values for drug resistance.
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Tamma, Pranita D., Belita N. A. Opene, Andrew Gluck, Krizia K. Chambers, Karen C. Carroll, and Patricia J. Simner. "Comparison of 11 Phenotypic Assays for Accurate Detection of Carbapenemase-Producing Enterobacteriaceae." Journal of Clinical Microbiology 55, no. 4 (January 11, 2017): 1046–55. http://dx.doi.org/10.1128/jcm.02338-16.
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ABSTRACT Early identification of carbapenemase-producing Enterobacteriaceae (CPE) is essential to prevent their dissemination within health care settings. Our objective was to evaluate the accuracy of 11 phenotypic assays for the detection of CPE. Two collections of carbapenem-resistant Enterobacteriaceae (CRE) isolates were evaluated, including 191 retrospective isolates (122 CP-CRE and 69 non-CP isolates) as well as 45 prospective clinical isolates (15 CP-CRE and 30 non-CP-CRE) obtained over a 3-month period. The sensitivity and specificity of each test was determined, with molecular genotype serving as the gold standard. Among the retrospective cohort, sensitivities ranged from 72% for the boronic acid synergy test for the detection of KPC producers to ≥98% for the modified Carba NP, the Rapidec Carba NP, the manual Blue Carba, and the modified carbapenem inactivation method for the detection of any CPE. Sensitivity differed among tests across enzyme classes. All assays had excellent specificity exceeding 95%, with the exception of the boronic acid synergy test (88%) and modified Hodge test (91%). Prospectively, 45 CRE isolates were encountered over a 3-month period, including 15 CPE (33%) and 30 non-CP-CRE (67%). Results from the prospective cohort were similar. However, a decrease in specificity was observed across most tests, likely due to restricted inclusion of non-CP-CRE to assess the specificity of the assays. Overall, accuracy of CPE detection varied across phenotypic tests. Local epidemiology of CP genotypes, turnaround time, and ease of incorporation into the laboratory workflow should be considered when selecting a phenotypic assay for clinical use.
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Jung, Sang-Kyu, Boanerges Aleman-Meza, Celeste Riepe, and Weiwei Zhong. "QuantWorm: A Comprehensive Software Package for Caenorhabditis elegans Phenotypic Assays." PLoS ONE 9, no. 1 (January 8, 2014): e84830. http://dx.doi.org/10.1371/journal.pone.0084830.
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