Academic literature on the topic 'Phenotypic assays'
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Journal articles on the topic "Phenotypic assays"
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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.
Dissertations / Theses on the topic "Phenotypic assays"
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Giannini, Alessia. "HIV drug discovery and resistance testing through cell based assays." Doctoral thesis, Università di Siena, 2019. http://hdl.handle.net/11365/1069117.
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Anti-HIV therapy has dramatically progressed from palliative care to high effectiveness, with virus replication and disease progression halted in most patients yet not eradicated. However, the need for lifelong therapy has kept anti-HIV drug development at high pace and novel strategies, drugs and drug classes have been regularly introduced over time. Issues to be tackled during prolonged antiretroviral therapy include adherence, toxicity, drug-drug interactions and the development of drug resistance. During my PhD course at the HIV Monitoring Laboratory (HML) of the Department of Medical Biotechnology at the University of Siena, I have been given the opportunity to focus my efforts in this area along two different lines.
The first activity has been done in the field of drug discovery within the EU FP7 funded THINPAD project. THINPAD is the acronym of “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance”, thus the objective of this project was to discover and develop a novel class of anti-HIV agents targeting NCp7, a small nucleocapsid protein that plays a number of different roles in HIV replication. The project used a funnel strategy from virtual screening to biochemical testing, then cell based assays and finally humanized mice as the animal model to validate the candidate NCp7 inhibitors (NCIs). My unit measured the antiviral activity of the candidate NCIs through cell based assays developed at the HML. The compounds with the highest selectivity indexes were next demonstrated to retain comparable efficacy against wild-type virus and virus resistant to licensed drug classes, thus confirming NCp7 as a specific target suitable to block the replication of currently circulating drug resistant viruses. The same compounds were used to perform experiments aiming at the definition of the mechanism of action through the measurement of viral nucleic acids intermediates produced in infected cells in presence of inhibitory concentration of NCIs at different time points. The results of this analysis were in agreement with antiviral activity exerted at the early and/or late steps of viral replication, suggesting that NCIs are able to interfere with variable impact on the different functions of NCp7 during HIV life cycle. While preliminary efficacy tests in the humanized mouse model were not successful, in vitro data support further NCI development.
The second activity has investigated the role of natural HIV-1 variability or specific HIV-1 mutants in the susceptibility or genetic barrier to resistance to licensed or upcoming antiretrovirals. Completed studies of this kind have shown (i) a role for the natural polymorphisms E138A in the reverse transcriptase in lowering the genetic barrier to resistance to the non-nucleoside reverse transcriptase inhibitor etravirine, (ii) a minimal impact of the integrase E157Q polymorphism in resistance to integrase inhibitors and (iii) the infrequent occurrence of natural resistance to the novel entry inhibitor fostemsavir in the HIV-1 CRF02_AG, an evolutionary lineage of HIV-1 originated in Africa and substantially represented in Italy. Although different, the three topics shared the need to clarify uncertain areas and derive indications for optimal drug use in the clinic.
Both activities have been using cell based systems developed at the HML and further refined for the specific application. Phenotypic drug susceptibility testing is a fairly complex task, usually assigned to specialized companies in collaborative research projects and not used at all in the clinic. Among academic systems proposed over time, our assay has been uniquely validated through comparison with the de facto reference commercially available Phenosense assay from Monogram Biosciences. Overall, contributing to expanding and using the laboratory portfolio of systems required for advanced investigation of anti-HIV drug was perfectly in line with my expectations as a biotechnologist.
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Clark, Stephen Andrew. "Genotypic and phenotypic assays to improve strain coverage assessments of sub-capsular meningococcal vaccines." Thesis, Manchester Metropolitan University, 2018. http://e-space.mmu.ac.uk/621128/.
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The licensure of recombinant protein-based meningococcal vaccines has increased the complexity of strain coverage assessments. In 2015, the 4CMenB vaccine was introduced into the UK national infant immunisation schedule and an Enhanced Surveillance programme was launched by Public Health England’s Meningococcal Reference Unit. Meningococcal isolates, representing ~50% of laboratory-confirmed cases, are comprehensively characterised using whole genome sequencing and 4CMenB strain coverage is assessed phenotypically using the Meningococcal Antigen Typing System. For the remaining cases, which are confirmed using PCR only, strain characterisation was until recently restricted to geno-grouping and geno-subtyping. The purpose of this research was to establish new genotypic assays to improve strain coverage assessment among non-culture cases, as well as introduce the MEASURE assay to predict coverage of a second sub-capsular vaccine, rLP2086, among isolates. A PCR sequencing assay targeting the Factor H-Binding Protein antigen gene (fHbp) was developed and had an estimated analytical sensitivity limit of between 600ag/μL and 6fg/μL. Using this assay, fHbp was successfully sequenced from 1510 of the 1661 PCR-positive clinical samples tested (91%). The distributions of fHbp peptide variants among culture and non-culture strains were compared and, whilst differences were observed for a small number of predominant variants, the distribution was very similar within each capsular group. The prospect of performing WGS directly from non-culture specimens was investigated using the Agilent SureSelectXT system. Eight of ten clinical specimens yielded genomes of acceptable quality. It was estimated that up to 54% of non-culture cases could be sequenced using this technique, however, the financial cost is currently prohibitive. Finally, the MEASURE assay was risk assessed and overnight formaldehyde incubation was introduced to ensure cells were fully fixed. The assay results were similar to those generated in a collaborating laboratory, however, further standardisation may be required. These assays will help to increase the accuracy of strain coverage predictions of the currently-licenced and future sub-capsular meningococcal vaccines.
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Rehbach, Kristina [Verfasser]. "Rapid semi-automated phenotypic assays for compound testing in patient-derived SPG4 neurons / Kristina Rehbach." Bonn : Universitäts- und Landesbibliothek Bonn, 2018. http://d-nb.info/1173789707/34.
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Blank, Carrine E., Hong Cui, Lisa R. Moore, and Ramona L. Walls. "MicrO: an ontology of phenotypic and metabolic characters, assays, and culture media found in prokaryotic taxonomic descriptions." BIOMED CENTRAL LTD, 2016. http://hdl.handle.net/10150/614758.
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Background: MicrO is an ontology of microbiological terms, including prokaryotic qualities and processes, material entities (such as cell components), chemical entities (such as microbiological culture media and medium ingredients), and assays. The ontology was built to support the ongoing development of a natural language processing algorithm, MicroPIE (or, Microbial Phenomics Information Extractor). During the MicroPIE design process, we realized there was a need for a prokaryotic ontology which would capture the evolutionary diversity of phenotypes and metabolic processes across the tree of life, capture the diversity of synonyms and information contained in the taxonomic literature, and relate microbiological entities and processes to terms in a large number of other ontologies, most particularly the Gene Ontology (GO), the Phenotypic Quality Ontology (PATO), and the Chemical Entities of Biological Interest (ChEBI). We thus constructed MicrO to be rich in logical axioms and synonyms gathered from the taxonomic literature. Results: MicrO currently has similar to 14550 classes (similar to 2550 of which are new, the remainder being microbiologically-relevant classes imported from other ontologies), connected by similar to 24,130 logical axioms (5,446 of which are new), and is available at (http://purl.obolibrary.org/obo/MicrO.owl) and on the project website at https://github.com/carrineblank/MicrO. MicrO has been integrated into the OBO Foundry Library (http://www.obofoundry.org/ontology/micro.html), so that other ontologies can borrow and re-use classes. Term requests and user feedback can be made using MicrO's Issue Tracker in GitHub. We designed MicrO such that it can support the ongoing and future development of algorithms that can leverage the controlled vocabulary and logical inference power provided by the ontology. Conclusions: By connecting microbial classes with large numbers of chemical entities, material entities, biological processes, molecular functions, and qualities using a dense array of logical axioms, we intend MicrO to be a powerful new tool to increase the computing power of bioinformatics tools such as the automated text mining of prokaryotic taxonomic descriptions using natural language processing. We also intend MicrO to support the development of new bioinformatics tools that aim to develop new connections between microbial phenotypes and genotypes (i.e., the gene content in genomes). Future ontology development will include incorporation of pathogenic phenotypes and prokaryotic habitats.
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Low, Andrew John. "Using genotypic and phenotypic methods to determine the HIV co-receptor phenotype in the clinical setting." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/226.
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Objective: The human immunodeficiency virus type 1 (HIV-1) currently infects over 30 million people worldwide. It uses one of two main co-receptors to infect cells. The primary objective of this thesis is to evaluate genotypic and phenotypic assays for co-receptor usage in the clinical setting and investigate approaches for improvement of these assays.
Methods: The concordance of recombinant co-receptor phenotyping assays and the predictive ability of genotype-based methods including the ‘11/25’ rule, position specific scoring matrices (PSSMs), and support vector machines (SVMs) were evaluated in the clinical setting using patient-derived plasma samples. Samples and patient data were evaluated in cross-sectional analyses from a retrospective population-based cohort of HIV-infected individuals enrolled in the HIV/AIDS Drug Treatment Program in British Columbia, Canada.
Results: Current implementations of HIV V3 region-based predictors for HIV co-receptor usage tested on patient derived samples are inadequate in the clinical setting, primarily due to low sensitivities as a result of difficult to detect minority species. Recombinant phenotype assays also show discordances when tested against each other on the same set of patient derived samples, raising doubts if any of these assays can truly be considered a ‘gold standard’. Significant associations between clinical progression, viral sequence-based predictors of co-receptor usage and the output of recombinant assays are observed, suggesting that sensitivity can be improved by incorporating CD4% into genotype-based predictors. This is verified with a SVM model which showed a 17% increase in sensitivity when CD4% was incorporated into training and testing.
Conclusion: This work in this thesis has exposed the difficulty in determining the co-receptor phenotype in the clinical setting, primarily due to minority species. Although genotypic methods of screening for HIV co-receptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turn-around time over phenotypic methods, they are currently inadequate for use in the clinical setting due to low sensitivities. Although the addition of clinical parameters such as CD4 count significantly increases the predictive ability of genotypic methods, the presence of low-levels of X4 virus continues to reduce the sensitivity of both genotypic and phenotypic methods.
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Aghighi, Saman. "Global coagulation assays in haemophilia : comparison and correlation with conventional assays and clinical phenotype." Thesis, University of Portsmouth, 2011. https://researchportal.port.ac.uk/portal/en/theses/global-coagulation-assays-in-haemophilia(b66cfc62-e38f-4d66-88df-0caa2dfef09a).html.
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Background: The global assays assess the interaction of procoagulants and anticoagulants on the generation of thrombin. These assays, as functional tests, may reflect the bleeding tendency in patients with haemophilia better than the conventional assays. Design and Methods: In this study the use of three global assays including rotational thromboelastometry (ROTEM®), thrombin generation test (TGT), and clot waveform analysis were evaluated on a cohort of haemophilia A (HA) and haemophilia B (HB) patients. The global assay parameters were compared to the conventional assays and the most sensitive parameters correlated with the clinical phenotype of haemophilia patients. Results and discussion: The modified ROTEM® analysis initiated by a minute amount of tissue factor proved to be useful in assessing haemophilia patients. A strong correlation was found between the Maximum Velocity (MaxVel) parameter of ROTEM® and the factor VIII level of haemophilia individuals. The use of corn trypsin inhibitor (CTI) to inhibit contact pathway improved the sensitivity and specificity of the ROTEM® analysis. A significant difference between the TGT parameters of patients groups and the control was noted. The use of CTI improved the sensitivity and specificity of the test, in particular peak height in platelet rich plasma (PRP) samples. The results in PRP+CTI group showed that platelets play an important role in heterogeneity of thrombin generation amongst severe haemophilia patients where the relevant deficient factor is < 1.0 IU/dL compared to conventional clotting assays. In clot waveform analysis, this study showed a better correlation between the velocity indices of the APTT test (Min1 and Min2) and the FVIII activity of HA individuals compared to the correlation between the APTT test itself and the FVIII activity level. The Min1 and Min2 appear to be more sensitive, simple, fast and cost effective in the diagnosis of hypocoagulability, and monitoring of haemophilia patients compared to the conventional tests. The area under the thrombin generation curve (AUC), peak height (PH), and time to peak (TP) parameters of the in-house TGT in PRP+CTI test category showed a strong correlation with patient age at first joint bleed (r = 0.693 p = 0.003, r = 0.718 p = 0.002, and r = -0.703 p = 0.002 respectively). The type of F8 mutation was also correlated with the patient’s thrombin generation capacity. Based on two subgroups of null mutations and non-null mutations, the PH and AUC in PPP+CTI category showed a significant increase in the non-null mutation group. The time to the maximum velocity of ROTEM® (tMaxVel) was also shorter in the non-null mutation group. Conclusion: The use of global assays in diagnosing hypocoagulability, and monitoring haemophilia patients proved to be useful, especially in areas where conventional methods are not responsive. However, the multifactorial dependency of global assays make them difficult to interpret on their own, therefore, these assays should be used in parallel with the conventional tests in order to be more useful.
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Boyd, Joseph. "Deep learning for computational phenotyping in cell-based assays." Thesis, Université Paris sciences et lettres, 2020. https://pastel.archives-ouvertes.fr/tel-02928984.
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Le phénotypage computationnel est un ensemble de technologies émergentes permettant d’étudier systématiquement le rôle du génome dans l’obtention de phénotypes, les caractéristiques observables d’un organisme et de ses sous- systèmes. En particulier, les essais cellulaires permettent de cribler des panels de petites molécules ou de moduler l’expression des gènes, et de quantifier les effets sur les caractéristiques phénotypiques allant de la viabilité à la morphologie cellulaire. Le criblage à haut contenu étend les méthodologies des criblages cellulaires à une lecture à haut contenu basée sur des images, en particulier les canaux multiplexés de la microscopie à fluorescence. Les cribles basés sur de multiples lignées cellulaires sont aptes à différencier les phénotypes de différents sous-types d’une maladie, représentant l’hétérogénéité moléculaire concernée dans la conception de thérapies médicales de précision. Ces modèles biologiques plus riches sous-tendent une approche plus ciblée pour le traitement de maladies mortelles telles que le cancer. Un défi permanent pour le criblage à haut contenu est donc la synthèse des lectures hétérogènes dans les cribles à multiples lignées cellulaires. Parallèlement, l’état de l’art établi en matière d’applications d’analyse d’images et de vision par ordinateur est l’apprentissage profond. Cependant, son rôle dans le criblage à haut contenu ne fait que commencer à être réalisé. Cette thèse aborde deux problématiques de l’analyse à haut contenu des lignées cellulaires cancéreuses. Les contributions sont les suivantes : (i) une démonstration du potentiel d’apprentissage profond et de modèles générateurs dans le criblage à haut contenu ; (ii) une solution basée sur l’apprentissage profond au problème de l’hétérogénéité dans un criblage de médicaments sur plusieurs lignées cellulaires ; et (iii) de nouvelles applications de modèles de traduction d’image à image comme alternative à la microscopie à fluorescence coûteuse actuellement nécessaire pour le criblage à haut contenuComputational phenotyping is an emergent set of technologies for systematically studying the role of the genome in eliciting phenotypes, the observable characteristics of an organism and its subsystems. In particular, cell-based assays screen panels of small compound drugs or otherwise modulations of gene expression, and quantify the effects on phenotypic characteristics ranging from viability to cell morphology. High content screening extends the methodologies of cell-based screens to a high content readout based on images, in particular the multiplexed channels of fluorescence microscopy. Screens based on multiple cell lines are apt to differentiating phenotypes across different subtypes of a disease, representing the molecular heterogeneity concerned in the design of precision medicine therapies. These richer biological models underpin a more targeted approach for treating deadly diseases such as cancer. An ongoing challenge for high content screening is therefore the synthesis of the heterogeneous readouts in multi-cell-line screens. Concurrently, deep learning is the established state-of-the-art image analysis and computer vision applications. However, its role in high content screening is only beginning to be realised. This dissertation spans two problem settings in the high content analysis of cancer cell lines. The contributions are the following: (i) a demonstration of the potential for deep learning and generative models in high content screening; (ii) a deep learning-based solution to the problem of heterogeneity in a multi-cell-line drug screen; and (iii) novel applications of image-to-image translation models as an alternative to the expensive fluorescence microscopy currently required for high content screening
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Nguyen, Duy Thanh. "Identification of Chemical Probes from Macleaya cordata (Willd.) R.Br." Thesis, Griffith University, 2019. http://hdl.handle.net/10072/389089.
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The lack of our understanding about Parkinson’s Disease (PD), the second most
common and incurable neurodegenerative disorder, has caused a great impact in both
treatment and research for this disease. Although not a few intensive studies have been
conducted with the hope of gaining more knowledge about the complex biology
underlying this debilitating condition, it seems that our efforts have not completely
uncovered the molecular mechanisms related to the neurodegeneration, which is critical
in PD.
Phenotypic assay, particularly cytological profiling (CP), has recently emerged as
a powerful unbiased strategy to study the mechanisms of action in many biological
systems. CP is a cell-based and image-based approach that evaluates the effects of small
molecules on several organelles within the cell, enabling the assessment of
multidimensional phenotypic functions instead of a single interaction that is usually seen
in target-based approaches. Previous research in our institute utilizing CP has identified
quite a few compounds that interacted with PD patient-derived cells. It is therefore worth
believing that CP could be a future approach for combating PD.
This project was conducted as part of our ongoing research intending to discover
molecules from nature to study the biology of PD. Our aim was to use 1H NMR guidance
to discover natural products from a traditional Chinese medicinal plant. Cytological
profiling on a patient-derived cell line (human olfactory neurosphere-derived (hONS)
cells) was performed for the isolated compounds to determine if they are potentially
qualified as chemical probes to serve the research of PD. This thesis will demonstrate the
study that integrates chemistry and biology to identify small molecules and the effects they influenced to the cell model, revealing their potential value as molecular tools for a
better interrogation of the disease mechanism.
The thesis was initiated with an introduction that covered several concepts,
including traditional Chinese medicine (TCM), PD, CP, hONS cell model and chemical
probes. Starting with TCM – a multi-century health care system in China, the introduction
acknowledged that TCM herbal plants are invaluable sources of bioactive small
molecules with respect to the traditional experience accumulated for thousands of years.
It then moved on to a brief discussion of Parkinson’s Disease ‒ an irremediable neural
degenerative disorder, and some current therapeutic treatments for this disease, as well as
giving rationale as to why this condition should be researched. The introduction went on
with a review on CP and its application in drug discovery, followed by a description of
current disease models for PD, as well as the hONS cell model used in this project. It was
noted that hONS cells derived from PD patients can recapitulate some functional aspects
of this disease, and thus coupling CP with hONS cell model could shed light on the
biological studies of PD. The chapter continued with a description of chemical probes,
their importance in medicinal research and that these small molecules are valuable tools
for interrogating the pathways of PD. Finally, the durability of NMR fingerprinting in
identify compounds was demonstrated in the last part of this chapter.
Chapter 2 provided details of the equipment and procedures involved in this
project to assist the chemical purification and biological characterization of the
compounds isolated from the selected herbal plant. Detailed spectroscopic data were also
present in this chapter.
Chapter 3 first gave in introduction on the selected biota, Macleaya cordata
(Willd.) R.Br., including how it was initially chosen in the previous PhD project and its
traditional medicinal use. The chapter then moved on to the targeted isolation of natural
product from this species by utilizing the robustness of 1H NMR spectroscopy and mass
spectrometry (MS), which resulted in the isolation of two new compounds and fourteen
known metabolites. The in-depth structural elucidation of the two new compounds, (6R)-
10-methoxybocconoline and 6-(1-Hydroxyethyl)-10-methoxy-5,6-dihydrochelerythrine,
were also mentioned in this chapter, followed by the use of density functional theory
(DFT) to assign the absolute configuration (AC) of one new molecule. The chapter
concluded with discussion on the validity of NMR fingerprinting and future directions on
the remaining work which involved AC assignment for the other new compound.
Chapter 4, the final chapter, presented the intensive data analysis for the CP
screening of the isolated compounds. It was revealed that four out of sixteen metabolites
showed significant perturbations to the hONS cellular parameters. Those compounds
included bocconoline which impacted EEA1- and mitochondria-associated features, 6-
(1-hydroxyethyl)-5,6-dihydrochelerythrine, 3-O-feruloylquinic acid and ferulic acid 4-Oglucoside
which influenced LC3b-related features. These compounds can potentially be
used as molecular tools to probe the biological pathways of PD. The last part of the
chapter was the discussion on the CP platform used to identify the above potential probes,
as well as future directions on the biology cohort. It was concluded that more justifications
should be made in order to verify the potency of the compounds being qualified as useful
probes. Dose dependence, mechanisms of action and drug-like properties for the
candidate probes were among the future follow-up research to have a better understanding
in the exploration of complex molecular mechanisms underlying PD.Thesis (Masters)
Master of Science (MSc)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Pan, Cuiping. "Phosphoproteomics and proteomic phenotyping to assess signal transduction in cancer cells." Diss., kostenfrei, 2008. http://edoc.ub.uni-muenchen.de/9241/.
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Cumagun, Christian Joseph R. "Molecular and phenotypic analyses of pathogenicity, aggressiveness, mycotoxin production, and colonization in the wheat-Gibberella zeae pathosystem." [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11163838.
Full textBooks on the topic "Phenotypic assays"
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Miu, Andrei C., Judith R. Homberg, and Klaus-Peter Lesch, eds. Genes, brain, and emotions. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780198793014.001.0001.
Full textAbstract:
With the advent of methods from behavioral genetics, molecular biology, and cognitive neuroscience, affective science has recently started to approach genetic influences on emotion, and the underlying intermediate neural mechanisms through which genes and experience shape emotion. The aim of this volume is to offer a comprehensive account of current research in the genetics of emotion, written by leading researchers, with extensive sections focused on methods, intermediate phenotypes, and clinical and translational work. Major methodological approaches are reviewed in the first section, including the two traditional “workhorses” in the field, twin studies and gene–environment interaction studies, and the more recently developed epigenetic modification assays, genome-wide association studies, and optogenetic methods. Parts 2 and 3 focus on a variety of psychological (e.g. fear conditioning, emotional action control, emotion regulation, emotional memory, decision-making) and biological (e.g. neural activity assessed using functional neuroimaging, electroencephalography, and psychophysiological methods; telomere length) mechanisms, respectively, that may be viewed as intermediate phenotypes in the pathways between genes and emotional experience. Part 4 concentrates on the genetics of emotional dysregulation in neuropsychiatric disorders (e.g. post-traumatic stress disorder, eating disorders, obsessive–compulsive disorder, Tourette’s syndrome), including factors contributing to the risk and persistence of these disorders (e.g. child maltreatment, personality, emotional resilience, impulsivity). In addition, two chapters in Part 4 review genetic influences on the response to psychotherapy (i.e. therapygenetics) and pharmacological interventions (i.e. pharmacogenetics) in anxiety and affective disorders.
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Landau, Ruth, and Clemens Ortner. Genetics. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780198713333.003.0052.
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Phenotyping is key in all genetic association studies and designing clinical studies to assess the genetic contribution to pain and analgesic response in general, and in the context of obstetric pain is even more challenging. In addition, interpreting results, particularly when multiple genes are evaluated, requires large sample sizes and appropriate statistical analysis to avoid misconstrued finding. The genetic contribution to labour pain or even that of pharmacogenetics to explain differences in analgesic response is probably not simple and straightforward and we are at the beginning of our explorations. Firm recommendations to tailor opioid regimens based on patients’ individual genetic profile are not available and are unlikely to become available in the near future other than for the prescription of codeine. To help explore genetic variants that influence the progress of labour and other various obstetric outcomes, the concept of mathematical modelling of labour progress is extremely promising and may in the future allow identification of some important genetic contributions and will perhaps one day predict labour outcome and labour pain perception.
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Schoenen, Jean, Valentin Bohotin, and Alain Maertens De Noordhout. Tms in Migraine. Edited by Charles M. Epstein, Eric M. Wassermann, and Ulf Ziemann. Oxford University Press, 2012. http://dx.doi.org/10.1093/oxfordhb/9780198568926.013.0024.
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Transcranial magnetic stimulation (TMS) has been used to search for cortical dysfunction in migraine. Both, the motor and the visual cortices have been explored in this area. This article reviews and discusses the results of the various studies performed in migraine patients with TMS of motor or visual cortices. The majority of evoked and event-related potential studies in migraine have shown two abnormalities: increased amplitude of grand averaged responses and lack of habituation in successive blocks of averaged responses with decreased amplitude in the first block. These abnormalities suggest that the excitability state of the cerebral cortex, particularly of the visual cortex, is abnormal in migraineurs between attacks. The use of TMS to assess motor and visual cortex excitability has yielded conflicting results, which could be due to methodological differences. Taken together, all studies indicate that the changes in cortical reactivity are more complex in migraineurs than initially thought and suggest that both larger multidisciplinary studies and focused analyses of subgroups of patients with more refined clinical phenotypes are necessary to disentangle the role of the cerebral cortex in migraine pathophysiology.
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Thun, Michael J., Martha S. Linet, James R. Cerhan, Christopher A. Haiman, and David Schottenfeld. Introduction. Oxford University Press, 2017. http://dx.doi.org/10.1093/oso/9780190238667.003.0001.
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This Introduction provides a broad overview of the scientific advances and crosscutting developments that increasingly influence epidemiologic research on the causes and prevention of cancer. High-throughput technologies have identified the molecular “driver” events in tumor tissue that underlie the multistage development of many types of cancer. These somatic (largely acquired) alterations disrupt normal genetic and epigenetic control over cell maintenance, division and survival. Tumor classification is also changing to reflect the genetic and molecular alterations in tumor tissue, as well as the anatomic, morphologic, and histologic phenotype of the cancer. Genome-wide association studies (GWAS) have identified more than 700 germline (inherited) genetic loci associated with susceptibility to various forms of cancer, although the risk estimates for almost all of these are small to modest and their exact location and function remain to identified. Advances in genomic and other “OMIC” technologies are identifying biomarkers that reflect internal exposures, biological processes and intermediate outcomes in large population studies. While research in many of these areas is still in its infancy, mechanistic and molecular assays are increasingly incorporated into etiologic studies and inferences about causation. Other sections of the book discuss the global public health impact of cancer, the growing list of exposures known to affect cancer risk, the epidemiology of over 30 types of cancer by tissue of origin, and preventive interventions that have dramatically reduced the incidence rates of several major cancers.
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Wajdzik, Marek. Zmienność cech fenotypowych samców sarny europejskiej (Capreolus capreolus L.) na tle gospodarowania jej populacją w północno-zachodniej Małopolsce. Publishing House of the University of Agriculture in Krakow, 2019. http://dx.doi.org/10.15576/978-83-66602-45-8.
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The objective of the present work was to evaluate the individual quality of male European roe deer by statistical analysis of antler traits, craniometric characters, and age of hunter-harvested bucks. Those measurements enabled a reliable assessment of antler quality, changes in carcass weight with age and between hunting seasons, as well as phenotypic traits depending on habitat (percentage forest cover, geographic mesoregion). The work also aimed to assess the effectiveness of deer population management in the Cracow Region of the Polish Hunting Association and determine the potential of that population based on analysis of medal-quality roebucks harvested there over the past 10 years. The study involved data concerning roe deer in the 60 hunting districts comprising the Cracow Region for the 2008/2009-2017/2018 hunting seasons. The study material consisted of data concerning antlers from 8132 roebucks taken over that decade, such as the age of hunted roebucks, hunting district, hunt date, carcass weight, as well as the gross weight and form of antlers. A detailed evaluation of antler quality was conducted using records for 2874 individuals, including 284 medal-quality antlers, harvested in the 2014/2015-2016/2017 seasons. The trophies were evaluated in accordance with the criteria of the International Council for Game and Wildlife Conservation (CIC) and were subjected to craniometric analysis. For a comprehensive examination of roe deer quality in the Cracow Region, the study analyzed gamekeeping data, that is, annual hunting plans of the Polish Hunting Association, for the period 2008/2009-2017/2018. Several gamekeeping indicators were calculated based on those data to evaluate the effects of roe deer management and gamekeeping practices. The studied antler traits (mean beam length, antler weight and volume, CIC scores) as well as carcass weight culminated in 6-year-old individuals. In turn cra-niometric traits (skull length and width) increased significantly until the 4th year of life, while the width of pedicles increased throughout the life of the individual. The development of antlers over time was characterized by a declining rate of growth for all the analyzed parameters. The highest growth rate (more than 100%) was found between the second and third years of life in terms of antler weight and volume, as well as front tine length. Antler quality in terms of overall CIC scores was to the greatest extent affected by weight and volume. The combined contribution of these factors increased with age, and ranged from 63.9% in the youngest individuals to 74.6% in the oldest ones. Within the study area, the individual quality of roe deer varied depending on the forest cover and mesoregion. Roebucks with the lowest carcass and antler weight occurred in hunting districts with a forest cover exceeding 40%, while the highest values of those parameters were found in districts with 5% forest cover or less. In open-land areas, the share of medal-quality roebucks in the total number of harvested males was higher, at approx. 5%, as compared to 1.29% in the woodlands. The carcass and antler weight of roebucks taken in the Cracow Region was higher than that of roebucks harvested in western Poland, similar to the Kielce Region, and lower than that for the Lublin and Krosno Regions, which is in keeping with Bergmann's ecogeographical rule. Analysis of carcass weight throughout the hunting season showed that the most pronounced rutting activity was observed for individuals 6 years of age and older, which lost as much as 6% of their weight. Over the ten-year period of study, the roebucks harvested using a uniform set of selection criteria revealed an increase in mean carcass weight as well as antler weight and form, which indicates appropriate management of the roe deer population in the examined hunting region.
Book chapters on the topic "Phenotypic assays"
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Parkin, Neil. "Viral Phenotypic Resistance Assays." In Antimicrobial Drug Resistance, 1187–99. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-595-8_37.
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Reeves, Jacqueline D., and Neil T. Parkin. "Viral Phenotypic Resistance Assays." In Antimicrobial Drug Resistance, 1389–407. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-47266-9_35.
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Fava, Eugenio, Eberhard Krausz, Rico Barsacchi, Ivan Baines, and Marino Zerial. "High-Content Phenotypic Cell-Based Assays." In Imaging Cellular and Molecular Biological Functions, 423–42. Berlin, Heidelberg: Springer Berlin Heidelberg, 2007. http://dx.doi.org/10.1007/978-3-540-71331-9_16.
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Meirelles, Lindolfo da Silva, and Dimas Tadeu Covas. "Phenotypic Analysis and Differentiation of Murine Mesenchymal Stem Cells." In Mesenchymal Stem Cell Assays and Applications, 331–50. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-60761-999-4_25.
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Brumfield, Kyle D., Bailey M. Carignan, and Mike S. Son. "Genotypic and Phenotypic Assays to Distinguish Vibrio cholerae Biotype." In Methods in Molecular Biology, 11–28. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8685-9_2.
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Parkin, Neil T., Eoin Coakley, and Christos J. Petropoulos. "Phenotypic Susceptibility Assays for Human Immunodeficiency Virus Type 1." In Antiviral Research, 283–99. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555815493.ch16.
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Berg, Ellen L., Sheryl P. Denker, and Alison O'Mahony. "Chapter 2. Development and Validation of Disease Assays for Phenotypic Screening." In Drug Discovery, 20–36. Cambridge: Royal Society of Chemistry, 2020. http://dx.doi.org/10.1039/9781839160721-00020.
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Delorme, Vincent, Ok-Ryul Song, Alain Baulard, and Priscille Brodin. "Testing Chemical and Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic Assays." In Methods in Molecular Biology, 387–411. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2450-9_24.
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McNamee, Niamh, and Lorraine O’Driscoll. "Miniaturized In Vitro Assays to Study Cellular Phenotypic Characteristics: Proliferation, Migration, Invasion, and Anoikis-Resistance." In Methods in Molecular Biology, 225–32. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1302-3_19.
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Schneider, Henning. "Zebrafish Neurobehavioral Assays for Drug Addiction Research." In The rights and wrongs of zebrafish: Behavioral phenotyping of zebrafish, 171–205. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-33774-6_8.
Full textConference papers on the topic "Phenotypic assays"
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Kapo, Naida, Jasmin Omeragić, Šejla Goletić, Adis Softić, Emina Šabić, and Teufik Goletić. "Phenotypic and Genotypic Analysis of Anthelmintic Resistance." In Socratic Lectures 8. University of Lubljana Press, 2023. http://dx.doi.org/10.55295/psl.2023.i3.
Full textAbstract:
A growing issue on a global scale is the emergence of helminth species and populations that are resistant to one or more anthelmintics. The majority of currently available anthelmintics used to control parasitic nematodes of cattle and sheep belong to only three main groups, benzimidazoles, imidazothiazoles and avermectins/milbemycins. The availability of reliable and precise techniques for its identification and monitoring is a critical component of the success of helminth control programs intended to prevent the spread of resistance in nematode populations. In vivo method like fecal egg count reduction test and in vitro methods such as egg hatch assays, larval motility test, larval development test and polymerase chain reaction (PCR) can be used for the detection of anthelmintic resistance although each has some reliability, repeatability, sensitivity, and ease of interpretation issues. The genetic basis of resistance to the majority of anthelmintics are still not well understood. Thanks to recent developments in high-throughput sequencing, it is now possible to define features such as drug resistance using genome-wide techniques. Keywords: Anthelmintics; Helminths; Resistance; Detection assays; Molecular diagnostics; Parasite control
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Hay, Carl, Steven Rust, Erin Sult, Lori Clarke, Kim Rosenthal, Sandrine Guillard, David Lowne, et al. "Abstract 4328: Phenotypic selection for identification of functional antibodies and development of high throughput screening assays." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-4328.
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Roy, Samrat, Debabani Roy Chowdhury, Manoj Pandre, Sundarajan Kannan, Rajesh Kumar RK, Amit K. Sharma, John W. Ellingboe, and Arnab Roy Chowdhury. "Abstract 2841: Dissecting tumorigenesis and metastatic properties of cell lines by phenotypic functional assays and plasticity ratio (PR)." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-2841.
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Zahra, R., A. A. Khan, and M. Sajid. "Hydrodynamic Evaluation of Microtiter Plate Assay Using Computational Fluid Dynamics for Biofilm Formation." In ASME-JSME-KSME 2019 8th Joint Fluids Engineering Conference. American Society of Mechanical Engineers, 2019. http://dx.doi.org/10.1115/ajkfluids2019-5425.
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Abstract Biofilms are complex surface associated communities where bacterial cells are enclosed by self-produced extra cellular polymeric substances (EPS), mainly consisting of exopolysaccharides, proteins and extracellular DNA. Treatment of biofilm associated persistent infections is an emerging issue for clinicians as bacterial cells adhere with human epithelial cells or indwelling medical devices such as implants and catheters, used in urinary tract and respiratory infections. Several methods are in practice to assess the biofilm formation of bacterial strains. Most of these are phenotypic methods which include Congo red assay (CRA), Air liquid interface (ALI), tissue culture plate method and Microtiter plate assay (MTPA). MTPA is considered as a standard screening method for comparing adherence pattern and is the most widely used quantitative method for detection of biofilm formation. Generally, the assay is performed under standard static conditions and little is known about the hydrodynamics in the microtiter plates. A few studies have applied computational fluid dynamics (CFD) simulations to describe flow pattern in microtiter plates during biofilm production and optimized the suitable conditions to detect the biofilm formation which have proven to be efficient. In this work the dependencies of biofilm formation on the hydrodynamics in microtiter plate assays were evaluated using OpenFOAM® an open-source toolbox for numerical simulation. It was found that higher flow rates increase the nutrient availability, promote cell growth, and attachment pattern with increased production of exopolymer, while the increase in flow velocity increases the shear rate causing erosion and disassembly of biofilm production because of detachment from the surface.
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Prosniak, R., Q. Yang, H. Wijerathne, N. Marchetti, M. Kiani, and L. Kilpatrick. "Identification of Sepsis Patient Immune Phenotypes Using a Microfluidic Assay." In American Thoracic Society 2021 International Conference, May 14-19, 2021 - San Diego, CA. American Thoracic Society, 2021. http://dx.doi.org/10.1164/ajrccm-conference.2021.203.1_meetingabstracts.a2725.
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Gerckens, Michael, Katharina Heinzelmann, Oliver Eickelberg, and Gerald Burgstaller. "A phenotypic cell-based extracellular matrix deposition assay for target validation and drug discovery." In ERS International Congress 2017 abstracts. European Respiratory Society, 2017. http://dx.doi.org/10.1183/1393003.congress-2017.pa912.
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Mukhopadhyay, Asima, Nicola Curtin, and Richard Edmondson. "Evaluation of different methods to assess homologous recombination status and sensitivity to PARP inhibitors in ovarian cancer." In 16th Annual International Conference RGCON. Thieme Medical and Scientific Publishers Private Ltd., 2016. http://dx.doi.org/10.1055/s-0039-1685289.
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Methods: Matched samples of ascites and tumor tissue were taken from patients undergoing surgery for epithelial ovarian cancer. Tumor samples were formalin fixed and paraffin embedded (FFPE); ascites samples were used to generate primary cultures (PC). HR status was determined in PCs as previously described.[1] IC50 for the PARP inhibitor Rucaparib was estimated using SRB assays. DNA was extracted from the FFPE tissue. The following techniques were evaluated in PCs or paired FFPE samples: DR-GFP reporter assay, PARP activity assay, BRCA1 expression on immunohistochemistry, BRCA1 methylation status and BRCA1/2 mutation analysis. A next generation sequencing based assay was used to detect mutations and other genomic alterations in a large panel of cancer-associated genes, including BRCA1/2. Results: Paired samples were collected from 64 patients and characterized for HR function. 47/64 (76%) were high grade serous. 44% (28/64)) were HR defective (HRD) by Rad51 assay and correlated with Rucaparib sensitivity (PPV-92%, NPV-100%). Molecular analysis revealed that all mutations and other genomic alterations detected in ascites derived PCs were also found in matched FFPE tumor tissues. All tumors with serous histology contained p53 mutations, whilst the remaining tumors without p53 mutations were non-serous in histology. DR-GFP assay was unreliable in PC due to poor transfection. In a subset of 50 cancers there was reduced BRCA1 expression in the HRD vs. HRC tumours (34.8% vs. 22.7%, ns) whilst in a further subset of 30 cases there was no difference in endogenous or stimulated PARP activity between HRD and HRC tumours. Deleterious BRCA2 mutations were identified in 7 tumors, 6 of which were HRD. Only 1 deleterious BRCA1 mutation was detected but methylation of BRCA1 was identified in 13 of 64 (20%) tumors, 7 of which were HRD. Mutation of BRCA2 was mutually exclusive to methylation of BRCA1. HRD vs. HRC tumours showed BRCA1 methylation (25% vs. 17%) and BRCA1/2 mutation (21% vs. 0.3%). 14/28 (50%) HR defective tumors do not have BRCA1/2 mutations or BRCA1 methylation, suggesting other mechanisms can also result in a HR defective phenotype. 28/64 (43%) of samples demonstrated the HR defective phenotype. In all cases HR status correlated with sensitivity to Rucaparib. Conclusion: As expected, deleterious BRCA2 mutations conferred a HRD phenotype in cells but methylation of BRCA1 was not universally associated with HRD. This may be as a result of only partial silencing of the gene by methylation and further work is required to identify thresholds of methylation which may predict HR status. The use of BRCA1/2 mutation testing alone is unlikely to identify the majority of HR defective ovarian tumors. Assessment of functional status of HRD is the preferred option and further technologies should be developed to simplify the Rad51 assay.
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Chowdhury, Arnab Roy, Debabani Roy Chowdhury, Manoj Pandre, Samrat Roy, Sundarajan Kannan, and John W. Ellingboe. "Abstract 2868: A cell based phenotypic assay platform for cancer metastasis drug discovery and diagnostics." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-2868.
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Santiago, Mayhara Rosany da Silva, Renata Amaral Andrade, Ana Caroline Paiva Simeão, and Heloisy Maria Nunes Galvão. "Congenital myasthenic syndrome due rapsyn mutation presenting with predominant ocular symptoms and good therapeutic response with salbutamol." In XIV Congresso Paulista de Neurologia. Zeppelini Editorial e Comunicação, 2023. http://dx.doi.org/10.5327/1516-3180.141s1.622.
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Introduction: Rapsyn mutations are found in Congenital Myasthenic Syndromes (CMS), usually described with early onset, hypotonia and respiratory insufficiency. Late onset phenotypes had already been described, with mild limb weakness and semiptosis. Episodic respiratory symptons and lack of ophthalmoparesis are considered hallmarks of rapsyn – CMS. The aim is to describe an unusual phenotype of a commom mutation of Rapsyn CMS and therapeutic response with salbutamol. Case report: A 29-year-old female patient was referred complaining of diplopia and fatigable assymmetrical semiptosis during the last six years. She pratices physical exercise, with no fatigue or weakness in limbs. Her past medical history was unremarkable except for drop neck episodes triggered by viral infectons during childhood, with first episode taking place around two years of age. Her parents were consaguinous, howeve no similar clinical picture was reported in family members. In clinical examination besides assymetrical semiptosis and ophthalmoparesis, was noted winged scapula, with normal muscle trofism and strength in four limbs. Laboratory investigation showed negative acetylcholine receptor antibodies (anti-AChR), and negative muscle-specific kinase antibodies (anti-MuSK). Single fiber electromyography revealed abnormal jitter. Genetic panel found a pathogenic homozygous mutation in the RAPSN gene (chr11:47.448.079G>T; p.Asn88Lys). She was treated with pyridostigmine and showed poor response. We opted to start salbutamol with marked clinical improvement. Conclusion: Rapsyn-CMS may present as late episodic diplopia, with semiptosis and ophthalmoparesis, and no limb or respiratory muscle weakness. That diagnosis possibility should be also considered in patients with late onset ocular symptons, incomplete response to pyridostigmine and negative assays for auto antibodies.
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Wilkerson, Stephen, S. A. Gadsden, J. Cerreta, and M. Al-Shabi. "Drone-Based technologies used to assess modern farming practices in undergraduate research." In Autonomous Air and Ground Sensing Systems for Agricultural Optimization and Phenotyping IV, edited by J. Alex Thomasson, Mac McKee, and Robert J. Moorhead. SPIE, 2019. http://dx.doi.org/10.1117/12.2519801.
Full textReports on the topic "Phenotypic assays"
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Weil, Clifford F., Anne B. Britt, and Avraham Levy. Nonhomologous DNA End-Joining in Plants: Genes and Mechanisms. United States Department of Agriculture, July 2001. http://dx.doi.org/10.32747/2001.7585194.bard.
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Repair of DNA breaks is an essential function in plant cells as well as a crucial step in addition of modified DNA to plant cells. In addition, our inability to introduce modified DNA to its appropriate locus in the plant genome remains an important hurdle in genetically engineering crop species.We have taken a combined forward and reverse genetics approach to examining DNA double strand break repair in plants, focusing primarily on nonhomologous DNA end-joining. The forward approach utilizes a gamma-plantlet assay (miniature plants that are metabolically active but do not undergo cell division, due to cell cycle arrest) and has resulted in identification of five Arabidopsis mutants, including a new one defective in the homolog of the yeast RAD10 gene. The reverse genetics approach has identified knockouts of the Arabidopsis homologs for Ku80, DNA ligase 4 and Rad54 (one gene in what proves to be a gene family involved in DNA repair as well as chromatin remodeling and gene silencing)). All these mutants have phenotypic defects in DNA repair but are otherwise healthy and fertile. Additional PCR based screens are in progress to find knockouts of Ku70, Rad50, and Mre11, among others. Two DNA end-joining assays have been developed to further our screens and our ability to test candidate genes. One of these involves recovering linearized plasmids that have been added to and then rejoined in plant cells; plasmids are either recovered directly or transformed into E. coli and recovered. The products recovered from various mutant lines are then compared. The other assay involves using plant transposon excision to create DNA breaks in yeast cells and then uses the yeast cell as a system to examine those genes involved in the repair and to screen plant genes that might be involved as well. This award supported three graduate students, one in Israel and two in the U.S., as well as a technician in the U.S., and is ultimately expected to result directly in five publications and one Masters thesis.
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Alfano, James, Isaac Barash, Thomas Clemente, Paul E. Staswick, Guido Sessa, and Shulamit Manulis. Elucidating the Functions of Type III Effectors from Necrogenic and Tumorigenic Bacterial Pathogens. United States Department of Agriculture, January 2010. http://dx.doi.org/10.32747/2010.7592638.bard.
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Many phytopathogenic bacteria use a type III protein secretion system (T3SS) to inject type III effectors into plant cells. In the experiments supported by this one-year feasibility study we investigated type III effector function in plants by using two contrasting bacterial pathogens: Pseudomonas syringae pv. tomato, a necrotrophic pathogen and Pantoea agglomerans, a tumorigenic pathogen. The objectives are listed below along with our major conclusions, achievements, and implications for science and agriculture. Objective 1: Compare Pseudomonas syringae and Pantoea agglomerans type III effectors in established assays to test the extent that they can suppress innate immunity and incite tumorigenesis. We tested P. agglomerans type III effectors in several innate immunity suppression assays and in several instances these effectors were capable of suppressing plant immunity, outputs that are suppressed by P. syringae effectors. Interestingly, several P. syringae effectors were able to complement gall production to a P. agglomerans pthGmutant. These results suggest that even though the disease symptoms of these pathogens are dramatically different, their type III effectors may function similarly. Objective 2: Construct P. syringae mutants in different combinations of type III-related DNA clusters to reduce type III effector redundancy. To determine their involvement in pathogenicity we constructed mutants that lack individual and multiple type III-related DNA clusters using a Flprecombinase-mediated mutagenesis strategy. The majority of single effector mutants in DC3000 have weak pathogenicity phenotypes most likely due to functional redundancy of effectors. Supporting this idea, Poly-DNAcluster deletion mutants were more significantly reduced in their ability to cause disease. Because these mutants have less functional redundancy of type III effectors, they should help identify P. syringae and P. agglomerans effectors that contribute more significantly to virulence. Objective 3: Determine the extent that P. syringae and P. agglomerans type III effectors alter hormone levels in plants. Inhibition of auxin polar transport by 2,3,5-triiodobenzoic acid (TIBA) completely prevented gall formation by P. agglomerans pv. gypsophilae in gypsophila cuttings. This result supported the hypothesis that auxin and presumably cytokinins of plant origin, rather than the IAA and cytokinins secreted by the pathogen, are mandatory for gall formation. Transgenic tobacco with pthGshowed various phenotypic traits that suggest manipulation of auxin metabolism. Moreover, the auxin levels in pthGtransgenic tobacco lines was 2-4 times higher than the control plants. External addition of auxin or cytokinins could modify the gall size in gypsophila cuttings inoculated with pthGmutant (PagMx27), but not with other type III effectors. We are currently determining hormone levels in transgenic plants expressing different type III effectors. Objective 4: Determine whether the P. agglomerans effectors HsvG/B act as transcriptional activators in plants. The P. agglomerans type III effectors HsvG and HsvB localize to the nucleus of host and nonhost plants and act as transcription activators in yeast. Three sites of adjacent arginine and lysine in HsvG and HsvB were suspected to act as Nuclear localization signals (NLS) domains. A nuclear import assay indicated two of the three putative NLS domains were functional NLSs in yeast. These were shown to be active in plants by fusing HsvG and HsvB to YFP. localization to the nucleus was dependent on these NLS domains. These achievements indicate that our research plan is feasible and suggest that type III effectors suppress innate immunity and modulate plant hormones. This information has the potential to be exploited to improve disease resistance in agricultural crops.
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Mendoza, Jonathan Alberto, Carolina Mazo, Lina Margarita Conn, Álvaro Rincón Castillo, Daniel Rojas Tapias, and Ruth Bonilla Buitrago. Evaluation of phosphate-solubilizing bacteria associated to pastures of Bracharia from acid soils. Corporación Colombiana de Investigación Agropecuaria - AGROSAVIA, 2015. http://dx.doi.org/10.21930/agrosavia.informe.2015.5.
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Rhizobia have been widely known by their capacity to form a symbiotic relationship with legumes and fix atmospheric nitrogen. Recently, however, rhizobia have shown to associate with plants in different botanical families. In this study, we aimed at elucidating the diversity of rhizobia associated to grasses, and determine their capabilities to solubilize phosphate in both lab and greenhouse experiments. Isolation of rhizobia was performed using rhizosphere from Brachiaria brizantha and B. decumbens and a promiscuous legume trap plant (i.e. Vigna unguiculata). Thirty days after inoculation of the trap plant, rhizobia were isolated from nodules using the conventional protocol, classified in basis on their phenotypic features, and molecularly grouped using Amplified Ribosomal DNA Restriction Analysis (ARDRA). Finally, phosphate solubilization assays and greenhouse experiments were carried out on representatives of each ARDRA cluster. The results showed that the diversity of rhizobia varied between both plant species, which suggests that plant exudates significantly determine the composition of the plant microbiome. Surprisingly, most of the isolated associated to B. brizantha rhizosphere exhibited typical attributes of slow-growing rhizobia, whereas rhizobia from B. decumbens displayed a mixed diversity including slow-, intermediate-, and fast-growing rhizobia. Sequencing of 16S rRNA of ARDRA representatives showed that most of the rhizobia isolated from B. brizantha belonged to the Mesorhizobium and Bradyrhizobium genera, while those isolated from B. decumbens were phylogenetically clustered into Rhizobium and Bradyrhizobium. The capability of the isolates to solubilize phosphate was studied using iron and calcium phosphate. We observed that overall Bradyrhizobium exhibited the highest ability to solubilize iron phosphate; by contrast, calcium phosphate was similarly solubilized within representatives of the three genera. In greenhouse experiments, we found that plants inoculated with isolated BT53, BD17 and BD21 exhibited a significantly higher content of phosphorus (p≤0.05). Additionally, dry weight was significantly higher in the treatment inoculated with BT16 isolate (p≤0.05). We conclude that 1) rhizobia is found associated with grasses, 2) plant genotype determines rhizobia diversity 3) rhizobia are able to solubilize phosphorus, and 4) they might be used to promote plant in different plant families. We further believe that further studies will reveal the true role of those old-known legume symbionts in development and growth of other important crops.
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Ori, Naomi, and Mark Estelle. Specific mediators of auxin activity during tomato leaf and fruit development. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597921.bard.
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The plant hormone auxin is involved in numerous developmental processes, including leaf and fruit development. The tomato (Solanumlycopersicum) gene ENTIRE (E) encodes an auxin-response inhibitor from the Aux/IAA family. While most loss-offunction mutations in Aux/IAA genes are similar to the wild type due to genetic redundancy, entire (e) mutants show specific effects on leaf and fruit development. e mutants have simple leaves, in contrast to the compound leaves of wild type tomatoes. In addition, e plants produce parthenocarpic fruits, in which fruit set occurs independently of fertilization. The aim of this research program was to utilize the e mutation to identify and characterize genes that mediate the specific effect of auxin in leaf and fruit development. The specific objectives of the project were to: 1. Characterize and map modifiers of the e leaf phenotype. 2. Characterize and map suppressors of the e fruit phenotype. 3. Dissect the developmental specificity of the E gene. 4. Examine the effect of fruit-overexpression of identified genes on fruit set and seed production. To identify mediators of auxin in leaf development, we mainly focused on one mutant, crawling elephant (crel, previously called t282), which showed substantial suppression of the e phenotype and other auxin-relatedphenotypes. We have identified the CREL gene as a homolog of the Arabidopsis VRN5 gene, involved in recruiting polycomb silencing complexes to specific targets. We showed that CREL affects auxin sensitivity in tomato. Suppressors of the e fruit phenotype have been further characterized and selected for more profound effects. Expression profiling by RNAseq was used to analyze the effect of e as well as crel on gene expression in leaves and fruits. This analysis has identified putative E and CREL targets. We have initiated studies to assess the role of some of these targets in flower and fruit development. The research has identified potential mediators of auxin response in leaf, flower and fruit development.
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Marchionni, Enrica, Daniele Guadagnolo, Gioia Mastromoro, and Antonio Pizzuti. Diagnostic yield of prenatal Exome Sequencing in fetal Central Nervous System Anomalies: systematic review and meta-analysis. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, May 2023. http://dx.doi.org/10.37766/inplasy2023.5.0003.
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Review question / Objective: The aim of this study is to assess the incremental diagnostic yield of prenatal exome sequencing analysis after inconclusive result of karyotype and Chromosomal Microarray Analysis in Central Nervous System fetal anomalies detected by ultrasound. Eligibility criteria: Inclusion criteria: papers describing fetuses with the indication to perform genome-wide sequencing studies based on prenatal imaging findings who underwent previous inconclusive karyotype and Chromosomal Microarray Analyses. The diagnostic yields of prenatal exome sequencing analysis OR prenatal genome sequencing analysis (with ≥20–30x depth of coverage and including only Single Nucleotide Variants) will be pooled in a meta-analysis. Exclusion Criteria: case reports and papers describing less than 5 cases; papers not describing the application of genome-wide sequencing studies based on prenatal imaging findings; papers describing genome-wide sequencing studies performed after negative targeted panels; papers describing fetuses with recurrent phenotypes as an explicitly selection criterium.
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Enlow, Michelle Bosquet, Richard J. Chung, Melissa A. Parisi, Sharon K. Sagiv, Margaret A. Sheridan, Annemarie Stroustrup, Rosalind J. Wright, et al. Standard Measurement Protocols for Pediatric Development Research in the PhenX Toolkit. RTI Press, September 2022. http://dx.doi.org/10.3768/rtipress.2022.mr.0049.2209.
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A challenge in conducting pediatric research is selecting reliable, valid measurement protocols, across a range of domains, that are appropriate for the developmental level of the study population. The purpose of this report is to introduce the research community to the Pediatric Development Research Domain of the National Institutes of Health (NIH)–supported PhenX Toolkit (consensus measures for Phenotypes and eXposures). The PhenX Toolkit provides a catalog of recommended measurement protocols to address a wide range of research topics that are suitable for inclusion in a variety of study designs. In 2018, the Pediatric Development Working Group of experts identified 18 well-established protocols of pediatric development for inclusion in the Toolkit to complement existing protocols. Collectively, the protocols assess parenting, child care attendance and quality, peer relationships, home environment, neonatal abstinence, emotional and behavioral functioning, and other factors that influence child development. The Toolkit provides detailed data collection protocols, data dictionaries, and worksheets to help investigators incorporate these protocols into their study designs. Using standard protocols in studies with pediatric participants will support consistent data collection, improve data quality, and facilitate cross-study analyses to ultimately improve child health.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas, and Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597929.bard.
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The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Levisohn, Sharon, Mark Jackwood, and Stanley Kleven. New Approaches for Detection of Mycoplasma iowae Infection in Turkeys. United States Department of Agriculture, February 1995. http://dx.doi.org/10.32747/1995.7612834.bard.
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Mycoplasma iowae (Mi) is a pathogenic avian mycoplasma which causes mortality in turkey embryos and as such has clinical and economic significance for the turkey breeder industry. Control of Mi infection is severely hampered by lack of adequate diagnostic tests, together with resistance to most antibiotics and resilience to environment. A markedly high degree of intra-species antigenic variation also contributes to difficulties in detection and control of infection. In this project we have designed an innovative gene-based diagnostic test based on specific amplification of the 16S rRNA gene of Mi. This reaction, designed Multi-species PCR-RFLP test, also amplifies the DNA of the pathogenic avian mycoplasmas M. gallisepticum (Mg) and M. synoviae (Ms). This test detects DNA equivalent to about 300 cfu Mi or either of the other two target mycoplasmas, individually or in mixed infection. It is a quick test, applicable to a wide variety of clinical samples, such as allantoic fluid or tracheal or cloacal swab suspensions. Differential diagnosis is carried out by gel electro-phoresis of the PCR amplicon digested with selected restriction enzymes (Restriction Fragment Length Polymorphism). This can also be readily accomplished by using a simple Dot-Blot hybridization assay with digoxigenin-labeled oligonucleotide probes reacting specifically with unique Mi, Mg or Ms sequences in the PCR amplicon. The PCR/OLIGO test increased sensitivity by at least 10-fold with a capacity for rapid testing of large numbers of samples. Experimental infection trials were carried out to evaluate the diagnostic tools and to study pathogenesis of Mi infection. Field studies and experimental infection of embryonated eggs indicated both synergistic and competitive interaction of mycoplasma pathogens in mixed infection. The value of the PCR diagnostic tests for following the time course of egg transmission was shown. A workable serological test (Dot Immunobinding Assay) was also developed but there was no clear-cut evidence that infected turkeys develop an immune response. Typing of a wide spectrum of Mi field isolates by a variety of gene-based molecular techniques indicated a higher degree of genetic homogeneity than predicted on the basis of the phenotypic variability. All known strains of Mi were detected by the method developed. Together with an M. meleagridis-PCR test based on the same gene, the Multi-species PCR test is a highly valuable tool for diagnosis of pathogenic mycoplasmas in single or mixed infection. The further application of this rapid and specific test as a part of Mi and overall mycoplasma control programs will be dependent on developments in the turkey industry.
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Ohad, Nir, and Robert Fischer. Regulation of Fertilization-Independent Endosperm Development by Polycomb Proteins. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695869.bard.
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Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.
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Freeman, Stanley, and Russell J. Rodriguez. The Interaction Between Nonpathogenic Mutants of Colletotrichum and Fusarium, and the Plant Host Defense System. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7573069.bard.
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The intent of this proposal was to study the interaction between nonpathogenic mutants of Colletotrichum magna and Fusarium oxysporum, and the cucurbit host defense system. We had shown previously that a nonpathogenic endophytic mutant path- 1 of C. magna, caused no visible disease symptoms but protected watermelon seedlings from disease caused by the wildtype isolate and F. o. niveum. Objectives were: 1) Determine the microscopic, biochemical and molecular genetic interaction between "protected" (path- 1 colonized) cucurbit hosts and wildtype isolates of C. magna; 2) Isolate non-pathogenic mutants of F.o. melonis and test feasibility for protecting plants against fungal diseases. We found that path-1 caused no visible disease symptoms in cucurbit seedlings but conferred disease resistance against pathogenic isolates of C. magna, C. orbiculare, and F. oxysporum. Disease resistance conferred by path-1 correlated to a decrease in the time of activation of host defense systems after exposure of path-1 colonized plants to virulent pathogens. This was determined by monitoring the biochemical activity of PAL and peroxidase, and the deposition of lignin. It appears that path-1-conferred disease resistance is a multigenic phenomenon which should be more difficult for pathogen to overcome than single gene conferred resistance. Based on the benefits conferred by path-1, we have defined this mutant as expressing a mutualistic lifestyle. REMI (restriction enzyme-mediated integration) nonpathogenic mutants were also isolated using pHA1.3 plasmid linearized with Hind III and transformed into wildtype C. magna. The integrated vector and flanking genomic DNA sequences in REMI mutant R1 was re-isolated and cloned resulting in a product of approximately 11 kb designated pGMR1. Transformations of wildtype C. magna with pGMR1 resulted in the same non-pathogenic phenotype. A nonpathogenic mutant of F.o. melonis (pathogenic to melon) was isolated that colonized melon plants but elicited no disease symptoms in seedlings and conferred 25 - 50% disease protection against the virulent wildtype isolate. Subsequently, nonpathogenic mutant isolates of F.o. niveum (pathogenic to watermelon) were also isolated. Their protection capacity against the respective wildtype parent is currently under investigation. This research has provided information toward a better understanding of host-parasite interactions; specifically, endophytes, pathogens and their hosts. It will also allow us to assess the potential for utilizing nonpathogenic mutants as biological control agents against fungal pathogens and isolating molecular genetic factors of pathogenicity in Fusarium.