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1. A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

Author

Lane, Dan, Allsopp, Rebecca, Holmes, Christopher W., Slingsby, Oliver C., Jukes-Jones, Rebekah, Bird, Paul, Anderson, N. Leigh, Razavi, Morteza, Yip, Richard, Pearson, Terry W., Pope, Matt, Khunti, Kamlesh, Doykov, Ivan, Hällqvist, Jenny, Mills, Kevin, Skipp, Paul, Carling, Rachel, Ng, Leong, Shaw, Jacqui, and Gupta, Pankaj

Subjects
IMMUNOAFFINITY chromatography, DENSITOMETRY, ANTIGEN analysis, GENE amplification, REVERSE transcriptase polymerase chain reaction, MASS spectrometry, LIQUID chromatography-mass spectrometry, SARS-CoV-2
Abstract

Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput. [ABSTRACT FROM AUTHOR]

Published
2024
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2. Infections with immunogenic trypanosomes reduce tsetse reproductive fitness: potential impact of different parasite strains on vector population structure.

Author

Hu, Changyun, Rio, Rita VM, Medlock, Jan, Haines, Lee R, Nayduch, Dana, Savage, Amy F, Guz, Nurper, Attardo, Geoffrey M, Pearson, Terry W, Galvani, Alison P, and Aksoy, Serap

Subjects
Animals, Tsetse Flies, Trypanosoma brucei rhodesiense, Protozoan Proteins, Blotting, Western, Blotting, Northern, Reverse Transcriptase Polymerase Chain Reaction, Reproduction, Fertility, Female, Male, Host-Parasite Interactions, Blotting, Northern, Western, Biological Sciences, Medical and Health Sciences, Tropical Medicine
Abstract

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6-8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite-host coevolution, and disease prevalence are discussed.

Published
2008

4. Theileria

Author

Bishop, Richard P., Odongo, David O., Mann, David J., Pearson, Terry W., Sugimoto, Chihiro, Haines, Lee R., Glass, Elizabeth, Jensen, Kirsty, Seitzer, Ulrike, Ahmed, Jabbar S., Graham, Simon P., de Villiers, Etienne P., Nene, Vishvanath, editor, and Kole, Chittaranjan, editor

Published
2009
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5. Written Discourse in Scientific Communities: A Conversation with Two Scientists About their Views of Science, Use of Language, Role of Writing in Doing Science, and Compatibility Between their Epistemic Views and Language

Author

Yore, Larry D., Florence, Marilyn K., and Pearson, Terry W.

Abstract

This autobiographical case study of two scientists involved in earlier studies documents a profile of each scientist. These profiles were used to develop semi-structured interview protocols and email surveys for each scientist. The central issues of these data collections were whether these modern, evaluativist scientists believe that the review-react-revise process of publishing a peer-reviewed research report simply improves the quality of the language or actually changes the science, and how their metacognitive awareness and executive control were demonstrated in their science inquiry and science writing. The scientists served both as informants and co-authors. Both scientists believed that writing and revising research reports improved the science as well as the clarity of the text; that their use of absolutist language related to their beliefs about inquiry and not about science knowledge; that addressing comments about their writing forced them to assess, monitor, and regulate their science inquiries and research reports; and that traditional forms of knowledge about nature and natural events were valuable information sources that stress description rather than physical causality

Published
2006

6. Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis

Author

Hober, Andreas, primary, Tran-Minh, Khue Hua, additional, Foley, Dominic, additional, McDonald, Thomas, additional, Vissers, Johannes PC, additional, Pattison, Rebecca, additional, Ferries, Samantha, additional, Hermansson, Sigurd, additional, Betner, Ingvar, additional, Uhlén, Mathias, additional, Razavi, Morteza, additional, Yip, Richard, additional, Pope, Matthew E, additional, Pearson, Terry W, additional, Andersson, Leigh N, additional, Bartlett, Amy, additional, Calton, Lisa, additional, Alm, Jessica J, additional, Engstrand, Lars, additional, and Edfors, Fredrik, additional

Published
2021
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7. Author response: Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis

Author

Hober, Andreas, primary, Tran-Minh, Khue Hua, additional, Foley, Dominic, additional, McDonald, Thomas, additional, Vissers, Johannes PC, additional, Pattison, Rebecca, additional, Ferries, Samantha, additional, Hermansson, Sigurd, additional, Betner, Ingvar, additional, Uhlén, Mathias, additional, Razavi, Morteza, additional, Yip, Richard, additional, Pope, Matthew E, additional, Pearson, Terry W, additional, Andersson, Leigh N, additional, Bartlett, Amy, additional, Calton, Lisa, additional, Alm, Jessica J, additional, Engstrand, Lars, additional, and Edfors, Fredrik, additional

Published
2021
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10. Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis

Author

Hober, Andreas, Hua, Tranh-Min Khue, Foley, Dominic, McDonald, Thomas, Vissers, Johannes Pc, Pattison, Rebecca, Ferries, Samantha, Hermansson, Sigurd, Betner, Ingvar, Uhlén, Mathias, Razavi, Morteza, Yip, Richard, Pope, Matthew E., Pearson, Terry W., Andersson, Leigh N., Bartlett, Amy, Calton, Lisa, Alm, Jessica J., Engstrand, Lars, Edfors, Fredrik, Hober, Andreas, Hua, Tranh-Min Khue, Foley, Dominic, McDonald, Thomas, Vissers, Johannes Pc, Pattison, Rebecca, Ferries, Samantha, Hermansson, Sigurd, Betner, Ingvar, Uhlén, Mathias, Razavi, Morteza, Yip, Richard, Pope, Matthew E., Pearson, Terry W., Andersson, Leigh N., Bartlett, Amy, Calton, Lisa, Alm, Jessica J., Engstrand, Lars, and Edfors, Fredrik

Abstract

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct <= 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies., See also peer review documents at DOI 10.7554/eLife.70843.sa0 10.7554/eLife.70843.sa1 10.7554/eLife.70843.sa2QC 20211217

Published
2021
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11. Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive

Author

Srakaew, Nopparat, Young, Charlene D., Sae-wu, Arpornrad, Xu, Hongbin, Quesnel, Krista L., di Brisco, Riccardo, Kongmanas, Kessiri, Fongmoon, Duriya, Hommalai, Greanggrai, Weerachatyanukul, Wattana, Hall, Susan H., Zhang, Yong-Lian, Panza, Luigi, Franchini, Laura, Compostella, Federica, Pearson, Terry W., Hancock, Robert E., Oko, Richard J., Hermo, Louis S., and Tanphaichitr, Nongnuj

Published
2014
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24. Structural characterization reveals a novel bilobed architecture for the ectodomains of insect stage expressed Trypanosoma brucei PSSA‐2 and Trypanosoma congolense ISA

Author

Ramaswamy, Raghavendran, Goomeshi Nobary, Sarah, Eyford, Brett A., Pearson, Terry W., and Boulanger, Martin J.

Subjects
Protein Domains, Tsetse Flies, Trypanosoma congolense, parasitic diseases, Trypanosoma brucei brucei, Protozoan Proteins, Animals, Antigens, Protozoan, Protein Structure Reports
Abstract

African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector-borne diseases of humans and livestock in sub-Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic-Specific Surface Antigen-2 (TbPSSA-2). To investigate the underlying molecular mechanism of TbPSSA-2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen (TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter-lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector.

Published
2016

29. Structural Characterization and Epitope Mapping of the Glutamic Acid/Alanine-rich Protein from Trypanosoma congolense: DEFINING ASSEMBLY ON THE PARASITE CELL SURFACE*♦

Author

Loveless, Bianca C., Mason, Jeremy W., Sakurai, Tatsuya, Inoue, Noboru, Razavi, Morteza, Pearson, Terry W., and Boulanger, Martin J.

Subjects
Models, Molecular, Structure-Activity Relationship, Tsetse Flies, Trypanosoma congolense, parasitic diseases, Protozoan Proteins, Animals, Molecular Bases of Disease, Cattle, Crystallography, X-Ray, Epitope Mapping, Protein Structure, Secondary
Abstract

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.

Published
2011

30. Quantification of serum apolipoproteins A-I and B-100 in clinical samples using an automated SISCAPA–MALDI-TOF-MS workflow

Author

van den Broek, Irene, primary, Nouta, Jan, additional, Razavi, Morteza, additional, Yip, Richard, additional, Bladergroen, Marco R., additional, Romijn, Fred P.H.T.M., additional, Smit, Nico P.M., additional, Drews, Oliver, additional, Paape, Rainer, additional, Suckau, Detlev, additional, Deelder, André M., additional, van der Burgt, Yuri E.M., additional, Pearson, Terry W., additional, Anderson, N. Leigh, additional, and Cobbaert, Christa M., additional

Published
2015
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31. Tsetse EP protein protects the fly midgut from trypanosome establishment

Author

Haines, Lee, Lehane, Stella, Pearson, Terry W, and Lehane, Mike

Subjects
wb_710, wc_680, qx_650, wc_705, parasitic diseases, fungi, qx_70, wc_755, qx_505, wc_695
Abstract

African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.

Published
2010

32. Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Author

Koch Institute for Integrative Cancer Research at MIT, Carr, Steven A., Zhao, Lei, Whiteaker, Jeffrey R., Pope, Matthew E., Kuhn, Eric, Jackson, Angela, Anderson, N. Leigh, Pearson, Terry W., Paulovich, Amanda G., Carr, Steven A, Koch Institute for Integrative Cancer Research at MIT, Carr, Steven A., Zhao, Lei, Whiteaker, Jeffrey R., Pope, Matthew E., Kuhn, Eric, Jackson, Angela, Anderson, N. Leigh, Pearson, Terry W., Paulovich, Amanda G., and Carr, Steven A

Abstract

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of in, National Cancer Institute (U.S.) (NCI Clinical Proteomic Technology Assessment Center (CPTAC) grant (#U24 CA126476)), Entertainment Industry Foundation (grant), Entertainment Industry Foundation (EFI Women's Cancer Research Fund to the Breast Cancer Biomarker Discovery Consortium), W. M. Keck Foundation, Canary Foundation, Paul G. Allen Family Foundation

Published
2014

34. Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Author

Koch Institute for Integrative Cancer Research at MIT, Carr, Steven A., Eyford, Brett A., Ahmad, Rushdy, Enyaru, John C., Pearson, Terry W., Carr, Steven A, Koch Institute for Integrative Cancer Research at MIT, Carr, Steven A., Eyford, Brett A., Ahmad, Rushdy, Enyaru, John C., Pearson, Terry W., and Carr, Steven A

Abstract

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.

Published
2013

41. Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

Author

Kuhn, Eric, primary, Whiteaker, Jeffrey R., additional, Mani, D.R., additional, Jackson, Angela M., additional, Zhao, Lei, additional, Pope, Matthew E., additional, Smith, Derek, additional, Rivera, Keith D., additional, Anderson, N. Leigh, additional, Skates, Steven J., additional, Pearson, Terry W., additional, Paulovich, Amanda G., additional, and Carr, Steven A., additional

Published
2012
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46. Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry

Author

Whiteaker, Jeffrey R., primary, Zhao, Lei, additional, Abbatiello, Susan E., additional, Burgess, Michael, additional, Kuhn, Eric, additional, Lin, ChenWei, additional, Pope, Matthew E., additional, Razavi, Morteza, additional, Anderson, N. Leigh, additional, Pearson, Terry W., additional, Carr, Steven A., additional, and Paulovich, Amanda G., additional

Published
2011
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