81 results on '"Carnes J"'
Search Results
2. Sensitization to Vitis vinifera pollen in a wine production area. Identification of the allergens involved
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González Mahave, I, primary, Lobera, T, additional, López-Matas, Mª A, additional, Blasco, A, additional, Vidal, I, additional, Álvarez, F, additional, and Carnes, J, additional
- Published
- 2022
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3. Knee cartilage defects in a sample of older adults: natural history, clinical significance and factors influencing change over 2.9 years
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Carnes, J., Stannus, O., Cicuttini, F., Ding, C., and Jones, G.
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- 2012
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4. Intermittent high-dose vitamin D corrects vitamin D deficiency in adolescents: a pilot study
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Carnes, J, Quinn, S, Nelson, M, Jones, G, and Winzenberg, T
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- 2012
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5. In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases
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Carnes, J, McDermott, S, Anupama, A, Oliver, BG, Sather, DN, Stuart, K, Carnes, J, McDermott, S, Anupama, A, Oliver, BG, Sather, DN, and Stuart, K
- Abstract
© The Author(s) 2017. RNA editing is an essential post-transcriptional process that creates functional mitochondrial mRNAs in Kinetoplastids. Multiprotein editosomes catalyze pre-mRNA cleavage, uridine (U) insertion or deletion, and ligation as specified by guide RNAs. Three functionally and compositionally distinct editosomes differ by the mutually exclusive presence of the KREN1, KREN2 or KREN3 endonuclease and their associated partner proteins. Because endonuclease cleavage is a likely point of regulation for RNA editing, we elucidated endonuclease specificity in vivo. We used a mutant gamma ATP synthase allele (MGA) to circumvent the normal essentiality of the editing endonucleases, and created cell lines in which both alleles of one, two or all three of the endonucleases were deleted. Cells lackingmultiple endonucleases had altered editosome sedimentation on glycerol gradients and substantial defects in overall editing. Deep sequencing analysis of RNAs from such cells revealed clear discrimination by editosomes between sites of deletion versus insertion editing and preferential but overlapping specificity for sites of insertion editing. Thus, endonuclease specificities in vivo are distinct but with some functional overlap. The overlapping specificities likely accommodate the more numerous sites of insertion versus deletion editing as editosomes collaborate to accurately edit thousands of distinct editing sites in vivo.
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- 2017
6. In Vivo Diagnosis with Purified Tropomyosin Comparison of Tropomyosin Sensitization in Shellfish and Mite Allergic Patients
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Carnes J, Matas M, Moya R, Larramendi C, Huertas J, Ferrer A, Navaro L, Garcia-Abujeta J, Vicario S, Flores I, Andreu C, Pena M, and Sanchez-Guerrero I
- Published
- 2016
7. In vivo diagnosis with purified tropomyosin in mite and shellfish allergic patients
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Lopez-Matas M, de Larramendi C, Moya R, Sanchez-Guerrero I, Ferrer A, Huertas A, Flores I, Navarro L, Garcia-Abujeta J, Vicario S, Andreu C, Pena M, and Carnes J
- Abstract
Background: Tropomyosin is the most studied shellfish allergen and has been involved in cross-reactivity among different invertebrates (crustacean, mollusks, mites, insects, and nematodes). Objective: To determine the relevance of tropomyosin in mite-and shellfish-sensitized patients using tropomyosin skin testing. Methods: Patients were divided into 3 groups: group M included mite allergic patients (ie, individuals with respiratory symptoms and a positive result on skin prick testing [SPT] to house dust mites), group S included shellfish allergic patients (ie, individuals who reported symptoms with shellfish), and group MS included mite-and shellfish allergic patients (ie, individuals who simultaneously fulfilled the inclusion criteria for groups M and S). Tropomyosin was purified from shrimp, characterized, and used in SPT for diagnosis in the patient population. Results: Eight hundred fifty patients were included in the study: 790 (92.9%) in group M, 21 (2.5%) in group S, and 39 (4.6%) in group MS. Tropomyosin was purified from shrimp with a purity higher than 95%. Forty-two individuals tested positive to tropomyosin: the prevalence was 2.7% in group M, 28.6% in group S, and 38.5% in patients of group MS. Twenty-one (50%) of the tropomyosin-positive individuals had symptoms with shellfish, and 3 (14.3%) reported anaphylaxis. Conclusion: The prevalence of tropomyosin was low in mite-sensitized patients (2.7 %) and high in shellfish allergic patients (28.6%). The higher prevalence of tropomyosin was found in patients sensitized to both mite and shellfish (38.5%). The selection of tropomyosin-sensitized patients by SPT might help in the choice of appropriate treatments or management for these patients. (C) 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
- Published
- 2016
8. Modulation of the humoral response to dermatophagoides pteronyssinus allergens in BALB/c mice by extract modification and adjuvant use
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Pico de Coaña, Yago, Carnes, J., Gallego, M. Teresa, Alonso, Carlos, and Parody, Nuria
- Abstract
Background: Currently, several strategies are being used in order to improve the safety and efficacy of allergen-specific immunotherapy; these strategies include the use of modified hypoallergenic extracts as well as different adjuvants with immunomodulatory properties in combination with native or modified extracts. The objectives of this study were to investigate the humoral response generated in mice to modified Dermatophagoides pteronyssinus extracts in the presence or absence of two different adjuvants. Methods: BALB/c mice were inoculated either with native, depigmented or depigmented-polymerised D. pteronyssinus without adjuvants or combined with aluminium hydroxide or oligodeoxinucleotides containing CpG motifs. IgE concentration, specific total IgG, IgG1 and IgG2a titres were measured in mice sera and cross-reactivity inhibition experiments were performed. IgG antigenic profiles were obtained by immunoblotting for all formulations. Results: Inoculation of depigmented-polymerised extract induced statistically significant lower IgE levels than the native extract even when adsorbed onto aluminium hydroxide. When this extract was inoculated in the presence of oligodeoxinucleotides containing CpG motifs, it elicited high IgG levels, a high IgG2a/lgG1 ratio and low IgE production. Furthermore, the antigenic profiles observed after extract inoculation showed punctual differences between the depigmented-polymerised extract and the native or depigmented extracts. Conclusions: Our results suggest that the depigmentation and polymerisation process modifies the native extract's antigenic and immunogenic properties and converts the depigmented-polymerised extract into a better choice for allergen-specific immunotherapy. Copyright © 2011 S. Karger AG, Basel.
- Published
- 2012
9. PyCogent: a toolkit for making sense from sequence
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Knight, R, Maxwell, P, Birmingham, A, Carnes, J, Caporaso, JG, Easton, BC, Eaton, M, Hamady, M, Lindsay, H, Liu, Z, Lozupone, C, McDonald, D, Robeson, M, Sammut, R, Smit, S, Wakefield, MJ, Widmann, J, Wikman, S, Wilson, S, Ying, H, Huttley, GA, Knight, R, Maxwell, P, Birmingham, A, Carnes, J, Caporaso, JG, Easton, BC, Eaton, M, Hamady, M, Lindsay, H, Liu, Z, Lozupone, C, McDonald, D, Robeson, M, Sammut, R, Smit, S, Wakefield, MJ, Widmann, J, Wikman, S, Wilson, S, Ying, H, and Huttley, GA
- Abstract
We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.
- Published
- 2007
10. Intermittent high-dose vitamin D corrects vitamin D deficiency in adolescents: a pilot study
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Carnes, J, primary, Quinn, S, additional, Nelson, M, additional, Jones, G, additional, and Winzenberg, T, additional
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- 2011
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11. Spectral characterization of biophysical characteristics in a boreal forest - Relationship between Thematic Mapper band reflectance and leaf area index for Aspen
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Badhwar, G. D, Macdonald, R. B, Hall, F. G, and Carnes, J. G
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Earth Resources And Remote Sensing - Abstract
Results from analysis of a data set of simultaneous measurements of Thematic Mapper band reflectance and leaf area index are presented. The measurements were made over pure stands of Aspen in the Superior National Forest of northern Minnesota. The analysis indicates that the reflectance may be sensitive to the leaf area index of the Aspen early in the season. The sensitivity disappears as the season progresses. Based on the results of model calculations, an explanation for the observed relationship is developed. The model calculations indicate that the sensitivity of the reflectance to the Aspen overstory depends on the amount of understory present.
- Published
- 1986
12. Separability of boreal forest species in the Lake Jennette area, Minnesota
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Shen, S. S, Carnes, J. G, and Badhwar, G. D
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Earth Resources And Remote Sensing - Abstract
In order to exploit the use of thematic mapper (TM) data to obtain vegetation and net primary productivity maps in the boreal forest, three aircraft flights were undertaken over the area near Ely, MN, with the NS 001 Thematic Mapper Simulator. Attention is presently given to an analysis of these 1983 data, which attempted to separate coniferous trees from deciduous ones. Canopy reflectance models and measured optical properties of the scattering elements have been used to deepen understanding of this separability, and to relate the ratio of nadir view reflectances in TM bands 4 and 3 to the overstory leaf area index. A map that is proportional to the leaf area index for deciduous species is presented.
- Published
- 1985
13. Evaluation of corn/soybeans separability using Thematic Mapper and Thematic Mapper Simulator data
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Pitts, D. E, Badhwar, G. D, Thompson, D. R, Henderson, K. E, Shen, S. S, Sorensen, C. T, and Carnes, J. G
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Earth Resources And Remote Sensing - Abstract
Multitemporal Thematic Mapper, Thematic Mapper Simulator, and detailed ground truth data were collected for a 9- by 11-km sample segment in Webster County, IA, in the summer of 1982. Three dates were acquired each with Thematic Mapper Simulator (June 7, June 23, and July 31) and Thematic Mapper (August 2, September 3, and October 21). The Thematic Mapper Simulator data were converted to equivalent TM count values using TM and TMS calibration data and model based estimates of atmospheric effects. The July 31, TMS image was compared to the August 2, TM image to verify the conversion process. A quantitative measure of proportion estimation variance (Fisher information) was used to evaluate the corn/soybeans separability for each TM band as a function of time during the growing season. The additional bands in the middle infrared allowed corn and soybeans to be separated much earlier than was possible with the visible and near-infrared bands alone. Using the TM and TMS data, temporal profiles of the TM principal components were developed. The greenness and brightness exhibited behavior similar to MSS greenness and brightness for corn and soybeans.
- Published
- 1984
14. The effect of gas loading on the RUS spectra of spheres
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Sorbello, R. S., primary, Feller, J., additional, Levy, Moisés, additional, Isaak, D. G., additional, Carnes, J. D., additional, and Anderson, O. L., additional
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- 2000
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15. Radiation impedance of resonant ultrasound spectroscopy modes in fused silica
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Zhang, Hong, primary, Sorbello, R. S., additional, Hucho, Carsten, additional, Herro, Joseph, additional, Feller, Jeffrey R., additional, Beck, D. E., additional, Levy, Moises, additional, Isaak, D., additional, Carnes, J. D., additional, and Anderson, O., additional
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- 1998
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16. Tributes To The Late Dr. Walter F. Kosonocky
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Carnes, J., primary, Savoye, E.D., additional, Dyck, R., additional, Chamberlain, S., additional, Shepherd, F., additional, Bredthauer, D., additional, Elabd, H., additional, Hynecek, J., additional, Fossum, E.R., additional, Theuwissen, A.J.P., additional, McGrath, R.D., additional, and Yang, G., additional
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- 1997
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17. Radiation impedance of RUS modes in fused silica and KCl.
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Herro, J., primary, Zhang, H., additional, Hucho, C., additional, Beck, D., additional, Levy, M., additional, Isaak, D., additional, Carnes, J. D., additional, and Anderson, O., additional
- Published
- 1996
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18. New pressure measurements of elasticity using the rectangular parallelepiped resonance method.
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Isaak, Donald G., primary, Anderson, O. L., additional, Carnes, J. D., additional, and Cynn, H., additional
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- 1996
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19. Address, on the duty of the slave states in the present crisis,: delivered in Galveston, Dec. 12th, 1860, by Rev. J.E. Carnes, by special invitation of the Committee of Safety and Correspondence, and many of the oldest citizens.
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Carnes, J. E., Carnes, J. E., Carnes, J. E., and Carnes, J. E.
- Abstract
16 p ; 21 cm., Electronic text and image data. Ann Arbor, Mich. : University of Michigan, Digital Library Initiatives, 1996. Includes both TIFF files and keyword searchable text. [Making of America] This volume is made possible by a grant from the Andrew W. Mellon Foundation., Making of America (MOA), (dlps) ABJ4987.0001.001, (lccallno) See URL for access, http://quod.lib.umich.edu/t/text/accesspolicy.html
20. Address, on the duty of the slave states in the present crisis,: delivered in Galveston, Dec. 12th, 1860, by Rev. J.E. Carnes, by special invitation of the Committee of Safety and Correspondence, and many of the oldest citizens.
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Carnes, J. E., Carnes, J. E., Carnes, J. E., and Carnes, J. E.
- Abstract
16 p ; 21 cm., Electronic text and image data. Ann Arbor, Mich. : University of Michigan, Digital Library Initiatives, 1996. Includes both TIFF files and keyword searchable text. [Making of America] This volume is made possible by a grant from the Andrew W. Mellon Foundation., Making of America (MOA), (dlps) ABJ4987.0001.001, (lccallno) See URL for access, http://quod.lib.umich.edu/t/text/accesspolicy.html
21. A Micro-Pulse Differential Absorption Lidar Test Network
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Spuler Scott, Bernatsky Todd, Bunn Catharine, Carnes Joshua, Hayman Matthew, Repasky Kevin, Stillwell Robert, and Weckwerth Tammy
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Physics ,QC1-999 - Abstract
The National Center for Atmospheric Research (NCAR) and Montana State University (MSU) have developed a test network of five micro-pulse Differential Absorption Lidar (DIAL) instruments to continuously measure high-vertical-resolution water vapor in the lower atmosphere. The instruments are accurate, low-cost, operate unattended, and eye-safe – all key features to enable larger ‘national-scale’ networks needed to characterize atmo-spheric moisture variability which influences important processes related to weather and climate.
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- 2020
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22. CCD Page Reader for Mail-Scanning Applications.
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RCA LABS PRINCETON N J, Kosonocky,W F, Angle,R L, Carnes,J E, Criado,A R, Sauer,D J, RCA LABS PRINCETON N J, Kosonocky,W F, Angle,R L, Carnes,J E, Criado,A R, and Sauer,D J
- Abstract
The following new design concepts for construction of a high-speed 2200x96-element TDI-CCD line sensor were developed and demonstrated with a 748x96-element page-reader test chip. (1) The four-phase electrode-per-bit clock to improve the vertical resolution of the TDI array by a factor of 1.5 was proposed and designed in the 748x96-element page-reader test chip. (2) To improve horizontal resolution and achieve the required effective output scanning rate of 8.4 x 10 to the 7th power picture elements (PELs) per second. The 748x96-element TDI array with 0.6 mil x 0.6 mil PELs and built with two-level polysilicon buried-channel CCDs can be read out by two types of 4:1 multiplexed outputs. One side of the array can be scanned-out by four parallel output registers with 2.4-mil stages. This mode of operation was experimentally demonstrated. The other side of the array can be scanned-out by two pairs of 2:1 multiplexed output registers with 1.2-mil stages.
- Published
- 1977
23. Correlation between Alt a 1 levels and clinical symptoms in Alternaria alternata-monosensitized patients
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Feo Brito, F., Alonso, A. M., Carnes, J., Martin-Martin, R., Fernandez-Caldas, E., Galindo, P. A., Alfaya, T., and Mariano Amo-Salas
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Adult ,Male ,Spores ,Adolescent ,Air Microbiology ,Colony Count, Microbial ,Dose-Response Relationship, Immunologic ,Alternaria ,Antibodies, Monoclonal ,Allergens ,Asthma ,Fungal Proteins ,Young Adult ,Humans ,Female ,Rhinitis - Abstract
Alternaria alternata is a risk factor for developing asthma.Alt a 1, which has been described as the major allergen in A alternata, shows a good correlation with A alternata spores only when they have germinated.The objective of this study was to determine the correlation between spore counts and clinical symptoms in patients with allergic asthma and/or rhinitis monosensitized to A alternata.Two types of samplers were used to determine exposure: a Burkard spore trap to collect A alternata spores and a high-volume air sampler to collect airborne particles. A total of 366 air filters were collected. Alt a 1 levels were measured by monoclonal antibody-based enzyme-linked immunosorbent assay. Eighteen monosensitized patients were asked to record their daily symptoms throughout the year.A alternata spores were detected throughout the year, whereas Alt a 1 was detected only between March and December. Symptoms showed positive and significant correlations with spore counts (r=0.459, P.001), and Alt a 1 levels (r=0.294, P.001). The correlation between spores and Alt a 1 was low. The negative binomial model proved that an increase of 10 pg/m3 in Alt a 1 levels increased the number of symptoms at a 3-day lag by 5%.In patients who are allergic to A alternata, Alt a 1 levels can be considered an important marker for predicting the risk of respiratory symptoms.
24. Usefulness of manufactured tomato extracts in the diagnosis of tomato sensitization: Comparison with the prick-prick method
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López-Matas María A, Pagán Juan A, Andreu Carmen, Lavín Jose R, Bartra Joan, García-Abujeta Jose L, Larramendi Carlos H, Huertas Ángel J, Ferrer Ángel, Fernández-Caldas Enrique, and Carnés Jerónimo
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Immunologic diseases. Allergy ,RC581-607 - Abstract
Abstract Background Commercial available skin prick test with fruits can be negative in sensitized or allergic patients due to a reduction in biological activity during the manufacturing process. Prick-prick tests with fresh foods are often preferred, but they are a non-standardized procedure. The usefulness of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method in the diagnosis of tomato sensitization has been analyzed. The objective of the study was to assess the potential diagnostic of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method. Methods Two groups of patients were analyzed: Group I: 26 individuals reporting clinical symptoms induced by tomato contact or ingestion. Group II: 71 control individuals with no symptoms induced by tomato: 12 of them were previously skin prick test positive to a tomato extract, 39 were atopic and 20 were non-atopic. All individuals underwent prick-prick with fresh ripe peel Canary tomatoes and skin prick tested with freeze-dried peel and pulp extracts obtained from peel and pulp of Canary tomatoes at 10 mg/ml. Wheal sizes and prick test positivity (≥ 7 mm2) were compared between groups. Results In group I, 21 (81%) out of 26 patients were prick-prick positive. Twenty patients (77%) had positive skin prick test to peel extracts and 12 (46%) to pulp extracts. Prick-prick induced a mean wheal size of 43.81 ± 40.19 mm2 compared with 44.25 ± 36.68 mm2 induced by the peel extract (Not significant), and 17.79 ± 9.39 mm2 induced by the pulp extract (p < 0.01). In group II, 13 (18%) out of 71 control patients were prick-prick positive. Twelve patients (all of them previously positive to peel extract) had positive skin prick test to peel and 3 to pulp. Prick-prick induced a mean wheal size of 28.88 ± 13.12 mm2 compared with 33.17 ± 17.55 mm2 induced by peel extract (Not significant), and 13.33 ± 4.80 mm2 induced by pulp extract (p < 0.05 with peel extract and prick-prick). Conclusion Canary peel tomato extract seems to be as efficient as prick-prick tests with ripe tomatoes to diagnose patients sensitized to tomato. The wheal sizes induced by prick-prick and peel extracts were very similar and showed a high correlation coefficient.
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- 2008
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25. Separability of boreal forest species in the Lake Jennette Area, Minnesota
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Badhwar, G. D., Carnes, J. G., and Shen, S. S.
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FORESTS & forestry ,REMOTE sensing - Published
- 1985
26. 2D signal estimation for sparse distributed target photon counting data.
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Hayman M, Stillwell RA, Carnes J, Kirchhoff GJ, Spuler SM, and Thayer JP
- Abstract
In this study, we explore the utilization of penalized likelihood estimation for the analysis of sparse photon counting data obtained from distributed target lidar systems. Specifically, we adapt the Poisson Total Variation processing technique to cater to this application. By assuming a Poisson noise model for the photon count observations, our approach yields denoised estimates of backscatter photon flux and related parameters. This facilitates the processing of raw photon counting signals with exceptionally high temporal and range resolutions (demonstrated here to 50 Hz and 75 cm resolutions), including data acquired through time-correlated single photon counting, without significant sacrifice of resolution. Through examination involving both simulated and real-world 2D atmospheric data, our method consistently demonstrates superior accuracy in signal recovery compared to the conventional histogram-based approach commonly employed in distributed target lidar applications., (© 2024. The Author(s).)
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- 2024
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27. Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4.
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McDermott SM, Pham V, Oliver B, Carnes J, Sather DN, and Stuart KD
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- Amino Acid Substitution, DNA Mutational Analysis, High-Throughput Nucleotide Sequencing, Mutation, Protein Domains genetics, Protozoan Proteins genetics, Protozoan Proteins metabolism, Ribonuclease III genetics, Ribonuclease III metabolism, RNA Editing, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei metabolism, Trypanosoma brucei brucei growth & development
- Abstract
Kinetoplastid pathogens including Trypanosoma brucei , T. cruzi , and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei . We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei . These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 McDermott, Pham, Oliver, Carnes, Sather and Stuart.)
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- 2024
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28. Multiple domains of the integral KREPA3 protein are critical for the structure and precise functions of RNA editing catalytic complexes in Trypanosoma brucei .
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Davidge B, McDermott SM, Carnes J, Lewis I, Tracy M, and Stuart KD
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- RNA Editing, Protozoan Proteins genetics, Protozoan Proteins metabolism, Mutation, RNA, Protozoan genetics, RNA, Protozoan metabolism, RNA genetics, Trypanosoma brucei brucei metabolism
- Abstract
The gRNA directed U-insertion and deletion editing of mitochondrial mRNAs that is essential in different life-cycle stages for the protozoan parasite Trypanosoma brucei is performed by three similar multiprotein catalytic complexes (CCs) that contain the requisite enzymes. These CCs also contain a common set of eight proteins that have no apparent direct catalytic function, including six that have an OB-fold domain. We show here that one of these OB-fold proteins, KREPA3 (A3), has structural homology to other editing proteins, is essential for editing, and is multifunctional. We investigated A3 function by analyzing the effects of single amino acid loss of function mutations, most of which were identified by screening bloodstream form (BF) parasites for loss of growth following random mutagenesis. Mutations in the zinc fingers (ZFs), an intrinsically disordered region (IDR), and several within or near the carboxy-terminal OB-fold domain variably impacted CC structural integrity and editing. Some mutations resulted in almost complete loss of CCs and its proteins and editing, whereas others retained CCs but had aberrant editing. All but a mutation which is near the OB-fold affected growth and editing in BF but not procyclic form (PF) parasites. These data indicate that multiple positions within A3 have essential functions that contribute to the structural integrity of CCs, the precision of editing and the developmental differences in editing between BF and PF stages., (© 2023 Davidge et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2023
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29. Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge.
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Oyong DA, Duffy FJ, Neal ML, Du Y, Carnes J, Schwedhelm KV, Hertoghs N, Jun SH, Miller H, Aitchison JD, De Rosa SC, Newell EW, McElrath MJ, McDermott SM, and Stuart KD
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- Humans, Animals, Plasmodium falciparum genetics, Vaccination, Interferons, Immunity, Sporozoites, Malaria, Falciparum prevention & control, Malaria, Malaria Vaccines
- Abstract
Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration: ClinicalTrials.gov NCT01994525., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Oyong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2023
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30. KRGG1 function in RNA editing in Trypanosoma brucei .
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Carnes J, Gendrin C, McDermott SM, and Stuart K
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- RNA genetics, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Guide, Kinetoplastida genetics, RNA, Protozoan genetics, RNA, Protozoan metabolism, Protozoan Proteins genetics, Protozoan Proteins metabolism, RNA Editing, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei metabolism
- Abstract
Mitochondrial gene expression in trypanosomes requires numerous multiprotein complexes that are unique to kinetoplastids. Among these, the most well characterized are RNA editing catalytic complexes (RECCs) that catalyze the guide RNA (gRNA)-specified insertion and deletion of uridines during mitochondrial mRNA maturation. This post-transcriptional resequencing of mitochondrial mRNAs can be extensive, involving dozens of different gRNAs and hundreds of editing sites with most of the mature mRNA sequences resulting from the editing process. Proper coordination of the editing with the cognate gRNAs is attributed to RNA editing substrate-binding complexes (RESCs), which are also required for RNA editing. Although the precise mechanism of RESC function is less well understood, their affinity for binding both editing substrates and products suggests that these complexes may provide a scaffold for RECC catalytic processing. KRGG1 has been shown to bind RNAs, and although affinity purification co-isolates RESC complexes, its role in RNA editing remains uncertain. We show here that KRGG1 is essential in BF parasites and required for normal editing. KRGG1 repression results in reduced amounts of edited A6 mRNA and increased amounts of edited ND8 mRNA. Sequence and structure analysis of KRGG1 identified a region of homology with RESC6, and both proteins have predicted tandem helical repeats that resemble ARM/HEAT motifs. The ARM/HEAT-like region is critical for function as exclusive expression of mutated KRGG1 results in growth inhibition and disruption of KRGG1 association with RESCs. These results indicate that KRGG1 is critical for RNA editing and its specific function is associated with RESC activity., (© 2023 Carnes et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2023
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31. Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.
- Author
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Duffy FJ, Hertoghs N, Du Y, Neal ML, Oyong D, McDermott S, Minkah N, Carnes J, Schwedhelm KV, McElrath MJ, De Rosa SC, Newell E, Aitchison JD, and Stuart K
- Subjects
- Animals, Humans, CD8-Positive T-Lymphocytes, Immunity, Interferons, Leukocytes, Mononuclear, Vaccination methods, Clinical Trials as Topic, Malaria prevention & control, Sporozoites
- Abstract
Background: Identifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge., Methods: To identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each., Results: RAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1
st immunization, but not the 2nd -5th . Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations., Conclusions: This study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Duffy, Hertoghs, Du, Neal, Oyong, McDermott, Minkah, Carnes, Schwedhelm, McElrath, De Rosa, Newell, Aitchison and Stuart.)- Published
- 2022
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32. Domain function and predicted structure of three heterodimeric endonuclease subunits of RNA editing catalytic complexes in Trypanosoma brucei.
- Author
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Carnes J, McDermott SM, Lewis I, Tracy M, and Stuart K
- Subjects
- Endonucleases genetics, Endonucleases metabolism, RNA metabolism, RNA Editing, RNA, Guide, Kinetoplastida genetics, RNA, Guide, Kinetoplastida metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Protozoan genetics, RNA, Protozoan metabolism, Uridine metabolism, Protozoan Proteins metabolism, Ribonuclease III metabolism, Trypanosoma brucei brucei metabolism
- Abstract
Each of the three similar RNA Editing Catalytic Complexes (RECCs) that perform gRNA-directed uridine insertion and deletion during Trypanosoma brucei mitochondrial (mt) mRNA editing has a distinct endonuclease activity that requires two related RNase III proteins, with only one competent for catalysis. We identified multiple loss-of-function mutations in the RNase III and other motifs of the non-catalytic KREPB6, KREPB7, and KREPB8 components by random mutagenesis and screening. These mutations had various effects on growth, editing, and both the abundances and RECC associations of these RNase III protein pairs in bloodstream form (BF) and procyclic form (PF) cells. Protein structure modelling predicted that the Zinc Finger (ZnF) of each paired RNase III protein contacts RNA positioned at the heterodimeric active site which is flanked by helices of a novel RNase III-Associated Motif (RAM). The results indicate that the protein domains of the non-catalytic subunits function together in RECC integrity, substrate binding, and editing site recognition during the multistep RNA editing process. Additionally, several mutants display distinct functional consequences in different life cycle stages. These results highlight the complementary roles of protein pairs and three RECCs within the complicated T. brucei mRNA editing machinery that matures mt mRNAs differentially between developmental stages., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
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33. Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity.
- Author
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Du Y, Hertoghs N, Duffy FJ, Carnes J, McDermott SM, Neal ML, Schwedhelm KV, McElrath MJ, De Rosa SC, Aitchison JD, and Stuart KD
- Subjects
- Adult, Animals, Anopheles parasitology, Female, Humans, Immunization methods, Insect Bites and Stings immunology, Malaria, Falciparum parasitology, Male, Mosquito Vectors parasitology, T-Lymphocytes immunology, Vaccination methods, Vaccines, Attenuated immunology, Immunity, Inflammation, Malaria Vaccines immunology, Malaria, Falciparum immunology, Plasmodium falciparum immunology, Sporozoites immunology
- Abstract
Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites (IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development. Trial registration: ClinicalTrials.gov NCT01994525., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2022
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34. Transcriptional correlates of malaria in RTS,S/AS01-vaccinated African children: a matched case-control study.
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Moncunill G, Carnes J, Chad Young W, Carpp L, De Rosa S, Campo JJ, Nhabomba A, Mpina M, Jairoce C, Finak G, Haas P, Muriel C, Van P, Sanz H, Dutta S, Mordmüller B, Agnandji ST, Díez-Padrisa N, Williams NA, Aponte JJ, Valim C, Neafsey DE, Daubenberger C, McElrath MJ, Dobaño C, Stuart K, and Gottardo R
- Subjects
- Antibodies, Protozoan immunology, Antigens, Protozoan immunology, B-Lymphocytes immunology, B-Lymphocytes metabolism, Case-Control Studies, Child, Preschool, Clinical Trials, Phase III as Topic, Humans, Infant, Mozambique, T-Lymphocytes immunology, T-Lymphocytes metabolism, Tanzania, Leukocytes, Mononuclear immunology, Leukocytes, Mononuclear metabolism, Malaria Vaccines immunology, Malaria, Falciparum immunology, Malaria, Falciparum prevention & control, Transcriptome genetics, Transcriptome immunology, Vaccines, Synthetic immunology
- Abstract
Background: In a phase 3 trial in African infants and children, the RTS,S/AS01 vaccine (GSK) showed moderate efficacy against clinical malaria. We sought to further understand RTS,S/AS01-induced immune responses associated with vaccine protection., Methods: Applying the blood transcriptional module (BTM) framework, we characterized the transcriptomic response to RTS,S/AS01 vaccination in antigen-stimulated (and vehicle control) peripheral blood mononuclear cells sampled from a subset of trial participants at baseline and month 3 (1-month post-third dose). Using a matched case-control study design, we evaluated which of these 'RTS,S/AS01 signature BTMs' associated with malaria case status in RTS,S/AS01 vaccinees. Antigen-specific T-cell responses were analyzed by flow cytometry. We also performed a cross-study correlates analysis where we assessed the generalizability of our findings across three controlled human malaria infection studies of healthy, malaria-naive adult RTS,S/AS01 recipients., Results: RTS,S/AS01 vaccination was associated with downregulation of B-cell and monocyte-related BTMs and upregulation of T-cell-related BTMs, as well as higher month 3 (vs. baseline) circumsporozoite protein-specific CD4
+ T-cell responses. There were few RTS,S/AS01-associated BTMs whose month 3 levels correlated with malaria risk. In contrast, baseline levels of BTMs associated with dendritic cells and with monocytes (among others) correlated with malaria risk. The baseline dendritic cell- and monocyte-related BTM correlations with malaria risk appeared to generalize to healthy, malaria-naive adults., Conclusions: A prevaccination transcriptomic signature associates with malaria in RTS,S/AS01-vaccinated African children, and elements of this signature may be broadly generalizable. The consistent presence of monocyte-related modules suggests that certain monocyte subsets may inhibit protective RTS,S/AS01-induced responses., Funding: Funding was obtained from the NIH-NIAID (R01AI095789), NIH-NIAID (U19AI128914), PATH Malaria Vaccine Initiative (MVI), and Ministerio de Economía y Competitividad (Instituto de Salud Carlos III, PI11/00423 and PI14/01422). The RNA-seq project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute. This study was also supported by the Vaccine Statistical Support (Bill and Melinda Gates Foundation award INV-008576/OPP1154739 to R.G.). C.D. was the recipient of a Ramon y Cajal Contract from the Ministerio de Economía y Competitividad (RYC-2008-02631). G.M. was the recipient of a Sara Borrell-ISCIII fellowship (CD010/00156) and work was performed with the support of Department of Health, Catalan Government grant (SLT006/17/00109). This research is part of the ISGlobal's Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces and we acknowledge support from the Spanish Ministry of Science and Innovation through the 'Centro de Excelencia Severo Ochoa 2019-2023' Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program., Competing Interests: GM, JC, WC, LC, SD, AN, MM, CJ, GF, PH, CM, PV, HS, SD, BM, SA, ND, NW, JA, CV, DN, CD, MM, CD, KS, RG No competing interests declared, JC is an employee of Antigen Discovery Inc. The author declare that no other competing interests exist- Published
- 2022
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35. Early whole blood transcriptional responses to radiation-attenuated Plasmodium falciparum sporozoite vaccination in malaria naïve and malaria pre-exposed adult volunteers.
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Duffy FJ, Du Y, Carnes J, Epstein JE, Hoffman SL, Abdulla S, Jongo S, Mpina M, Daubenberger C, Aitchison JD, and Stuart K
- Subjects
- Antibodies, Protozoan blood, Clinical Trials as Topic, Humans, Malaria Vaccines administration & dosage, Plasmodium falciparum genetics, Plasmodium falciparum immunology, Protozoan Proteins genetics, Sporozoites genetics, Sporozoites immunology, Transcription, Genetic, Vaccines, Attenuated administration & dosage, Vaccines, Attenuated therapeutic use, Malaria Vaccines therapeutic use, Malaria, Falciparum prevention & control
- Abstract
Background: Vaccination with radiation-attenuated Plasmodium falciparum sporozoites is known to induce protective immunity. However, the mechanisms underlying this protection remain unclear. In this work, two recent radiation-attenuated sporozoite vaccination studies were used to identify potential transcriptional correlates of vaccination-induced protection., Methods: Longitudinal whole blood RNAseq transcriptome responses to immunization with radiation-attenuated P. falciparum sporozoites were analysed and compared across malaria-naïve adult participants (IMRAS) and malaria-experienced adult participants (BSPZV1). Parasite dose and method of delivery differed between trials, and immunization regimens were designed to achieve incomplete protective efficacy. Observed protective efficacy was 55% in IMRAS and 20% in BSPZV1. Study vaccine dosings were chosen to elicit both protected and non-protected subjects, so that protection-associated responses could be identified., Results: Analysis of comparable time points up to 1 week after the first vaccination revealed a shared cross-study transcriptional response programme, despite large differences in number and magnitude of differentially expressed genes between trials. A time-dependent regulatory programme of coherent blood transcriptional modular responses was observed, involving induction of inflammatory responses 1-3 days post-vaccination, with cell cycle responses apparent by day 7 in protected individuals from both trials. Additionally, strongly increased induction of inflammation and interferon-associated responses was seen in non-protected IMRAS participants. All individuals, except for non-protected BSPZV1 participants, showed robust upregulation of cell-cycle associated transcriptional responses post vaccination., Conclusions: In summary, despite stark differences between the two studies, including route of vaccination and status of malaria exposure, responses were identified that were associated with protection after PfRAS vaccination. These comprised a moderate early interferon response peaking 2 days post vaccination, followed by a later proliferative cell cycle response steadily increasing over the first 7 days post vaccination. Non-protection is associated with deviations from this model, observed in this study with over-induction of early interferon responses in IMRAS and failure to mount a cell cycle response in BSPZV1., (© 2021. The Author(s).)
- Published
- 2021
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36. Editosome RNase III domain interactions are essential for editing and differ between life cycle stages in Trypanosoma brucei .
- Author
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McDermott SM, Carnes J, and Stuart K
- Subjects
- Animals, Cell Line, Endonucleases genetics, Mutation genetics, RNA, Guide, Kinetoplastida genetics, RNA, Messenger genetics, RNA, Mitochondrial genetics, RNA, Protozoan genetics, Uridine genetics, Protozoan Proteins genetics, RNA Editing genetics, Ribonuclease III genetics, Trypanosoma brucei brucei genetics
- Abstract
Multiprotein editosomes catalyze gRNA-specified insertion and deletion of uridines to create functional mitochondrial mRNAs in Trypanosoma brucei Three functionally distinct editosomes are distinguished by their single KREN1, KREN2, or KREN3 RNase III endonuclease and, respectively, KREPB8, KREPB7, and KREPB6 partner proteins. These endonucleases perform the first catalytic step of editing, cleaving mRNA in diverse mRNA/gRNA heteroduplex substrates. We identified divergent and likely noncatalytic RNase III domains in KREPB4, KREPB5, KREPB6, KREPB7, KREPB8, KREPB9, and KREPB10 editosome proteins. Because known RNase III endonuclease functional domains are dimeric, the editing endonucleases may form heterodimers with one or more of these divergent RNase III proteins. We show here using conditional null cell lines that KREPB6, KREPB7, and KREPB8 are essential in both procyclic form (PF) and bloodstream (BF) cells. Loss of these proteins results in growth defects and loss of editing in vivo, as does mutation of their RNase III domain that is predicted to prevent dimerization. Loss of KREPB6, KREPB7, or KREPB8 also dramatically reduces cognate endonuclease abundance, as does the RNase III mutation, indicating that RNase III interactions with their partner proteins stabilize the endonucleases. The phenotypic consequences of repression are more severe in BF than in PF, indicating differences in endonuclease function between developmental stages that could impact regulation of editing. These results suggest that KREPB6, KREPB7, and KREPB8 form heterodimers with their respective endonucleases to perform mRNA cleavage. We also present a model wherein editosome proteins with divergent RNase III domains function in substrate selection via enzyme-pseudoenzyme interactions., (© 2019 McDermott et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2019
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37. Whole blood transcriptome changes following controlled human malaria infection in malaria pre-exposed volunteers correlate with parasite prepatent period.
- Author
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Rothen J, Murie C, Carnes J, Anupama A, Abdulla S, Chemba M, Mpina M, Tanner M, Lee Sim BK, Hoffman SL, Gottardo R, Daubenberger C, and Stuart K
- Subjects
- Blood Proteins genetics, Gene Expression Profiling, Gene Expression Regulation genetics, Humans, Malaria, Falciparum blood, Malaria, Falciparum parasitology, Parasitemia genetics, Plasmodium falciparum pathogenicity, Volunteers, Malaria, Falciparum genetics, Parasitemia blood, Plasmodium falciparum isolation & purification, Transcriptome genetics
- Abstract
Malaria continues to be one of mankind's most devastating diseases despite the many and varied efforts to combat it. Indispensable for malaria elimination and eventual eradication is the development of effective vaccines. Controlled human malaria infection (CHMI) is an invaluable tool for vaccine efficacy assessment and investigation of early immunological and molecular responses against Plasmodium falciparum infection. Here, we investigated gene expression changes following CHMI using RNA-Seq. Peripheral blood samples were collected in Bagamoyo, Tanzania, from ten adults who were injected intradermally (ID) with 2.5x104 aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria® PfSPZ Challenge). A total of 2,758 genes were identified as differentially expressed following CHMI. Transcriptional changes were most pronounced on day 5 after inoculation, during the clinically silent liver phase. A secondary analysis, grouping the volunteers according to their prepatent period duration, identified 265 genes whose expression levels were linked to time of blood stage parasitemia detection. Gene modules associated with these 265 genes were linked to regulation of transcription, cell cycle, phosphatidylinositol signaling and erythrocyte development. Our study showed that in malaria pre-exposed volunteers, parasite prepatent period in each individual is linked to magnitude and timing of early gene expression changes after ID CHMI., Competing Interests: Authors KLS and SH are affiliated with the commercial company Sanaria Inc., the manufacturer of PfSPZ Challenge. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
- Published
- 2018
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38. RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei .
- Author
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Carnes J, McDermott SM, and Stuart K
- Abstract
Editosomes are the multiprotein complexes that catalyze the insertion and deletion of uridines to create translatable mRNAs in the mitochondria of kinetoplastids. Recognition and cleavage of a broad diversity of RNA substrates in vivo require three functionally distinct RNase III-type endonucleases, as well as five additional editosome proteins that contain noncatalytic RNase III domains. RNase III domains have recently been identified in the editosome accessory proteins KREPB9 and KREPB10, suggesting a role related to editing endonuclease function. In this report, we definitively show that KREPB9 and KREPB10 are not essential in either bloodstream-form parasites (BF) or procyclic-form parasites (PF) by creating null or conditional null cell lines. While preedited and edited transcripts are largely unaffected by the loss of KREPB9 in both PF and BF, loss of KREPB10 produces distinct responses in BF and PF. BF cells lacking KREPB10 also lack edited CYb, while PF cells have increased edited A6, RPS12, ND3, and COII after loss of KREPB10. We also demonstrate that mutation of the RNase III domain of either KREPB9 or KREPB10 results in decreased association with ~20S editosomes. Editosome interactions with KREPB9 and KREPB10 are therefore mediated by the noncatalytic RNase III domain, consistent with a role in endonuclease specialization in Trypanosoma brucei . IMPORTANCE Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in T. brucei generates mature mitochondrial mRNAs. Editing is essential for survival in mammalian hosts and tsetse fly vectors and is differentially regulated during the parasite life cycle. Three multiprotein "editosomes," typified by exclusive RNase III endonucleases that act at distinct sites, catalyze editing. Here, we show that editosome accessory proteins KREPB9 and KREPB10 are not essential for mammalian blood- or insect-form parasite survival but have specific and differential effects on edited RNA abundance in different stages. We also characterize KREPB9 and KREPB10 noncatalytic RNase III domains and show they are essential for editosome association, potentially via dimerization with RNase III domains in other editosome proteins. This work enhances the understanding of distinct editosome and accessory protein functions, and thus differential editing, during the parasite life cycle and highlights the importance of RNase III domain interactions to editosome architecture.
- Published
- 2018
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39. In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases.
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Carnes J, McDermott S, Anupama A, Oliver BG, Sather DN, and Stuart K
- Subjects
- Base Sequence, Endonucleases metabolism, Gene Deletion, Glycerol pharmacology, Isoenzymes genetics, Isoenzymes metabolism, Mitochondrial Proton-Translocating ATPases genetics, Mitochondrial Proton-Translocating ATPases metabolism, Protozoan Proteins metabolism, RNA Cleavage, RNA Precursors genetics, RNA Precursors metabolism, RNA, Guide, Kinetoplastida genetics, RNA, Guide, Kinetoplastida metabolism, RNA, Messenger metabolism, RNA, Mitochondrial, RNA, Protozoan metabolism, Sequence Alignment, Substrate Specificity, Transfection, Trypanosoma brucei brucei drug effects, Trypanosoma brucei brucei enzymology, Endonucleases genetics, Protozoan Proteins genetics, RNA Editing, RNA, Messenger genetics, RNA, Protozoan genetics, Trypanosoma brucei brucei genetics
- Abstract
RNA editing is an essential post-transcriptional process that creates functional mitochondrial mRNAs in Kinetoplastids. Multiprotein editosomes catalyze pre-mRNA cleavage, uridine (U) insertion or deletion, and ligation as specified by guide RNAs. Three functionally and compositionally distinct editosomes differ by the mutually exclusive presence of the KREN1, KREN2 or KREN3 endonuclease and their associated partner proteins. Because endonuclease cleavage is a likely point of regulation for RNA editing, we elucidated endonuclease specificity in vivo. We used a mutant gamma ATP synthase allele (MGA) to circumvent the normal essentiality of the editing endonucleases, and created cell lines in which both alleles of one, two or all three of the endonucleases were deleted. Cells lacking multiple endonucleases had altered editosome sedimentation on glycerol gradients and substantial defects in overall editing. Deep sequencing analysis of RNAs from such cells revealed clear discrimination by editosomes between sites of deletion versus insertion editing and preferential but overlapping specificity for sites of insertion editing. Thus, endonuclease specificities in vivo are distinct but with some functional overlap. The overlapping specificities likely accommodate the more numerous sites of insertion versus deletion editing as editosomes collaborate to accurately edit thousands of distinct editing sites in vivo., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2017
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40. The Architecture of Trypanosoma brucei editosomes.
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McDermott SM, Luo J, Carnes J, Ranish JA, and Stuart K
- Subjects
- Computational Biology methods, Endonucleases metabolism, Models, Molecular, Multiprotein Complexes metabolism, Protein Binding, Protein Conformation, Protein Interaction Domains and Motifs, Protein Interaction Maps, Proteome, Proteomics methods, Protozoan Proteins chemistry, RNA-Binding Proteins metabolism, Reproducibility of Results, Protozoan Proteins metabolism, RNA Editing, RNA, Protozoan genetics, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei metabolism
- Abstract
Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra- and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture., Competing Interests: The authors declare no conflict of interest.
- Published
- 2016
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41. Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei.
- Author
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McDermott SM, Carnes J, and Stuart K
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Crystallography, X-Ray, Humans, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, Protozoan Proteins chemistry, Protozoan Proteins genetics, RNA, Protozoan genetics, RNA, Protozoan metabolism, Ribonuclease III chemistry, Ribonuclease III genetics, Sequence Alignment, Trypanosoma brucei brucei metabolism, Protozoan Proteins metabolism, RNA Editing, Ribonuclease III metabolism, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei growth & development, Trypanosomiasis, African parasitology
- Abstract
KREPB5 is an essential component of ∼ 20S editosomes in Trypanosoma brucei which contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional null T. brucei bloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼ 20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages of T. brucei that differentially edit mRNAs., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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42. Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei.
- Author
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McDermott SM, Guo X, Carnes J, and Stuart K
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Blood parasitology, Cell Line, Drug Resistance genetics, Models, Molecular, Molecular Sequence Data, Mutation, Protein Structure, Tertiary, Protozoan Proteins chemistry, Protozoan Proteins genetics, Ribonuclease III chemistry, Ribonuclease III metabolism, Tetracycline pharmacology, Trypanosoma brucei brucei drug effects, Trypanosoma brucei brucei metabolism, Life Cycle Stages drug effects, Protozoan Proteins metabolism, RNA Editing, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei growth & development
- Abstract
Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei. The mRNAs are differentially edited in bloodstream form (BF) and procyclic form (PF) life cycle stages, and this correlates with the differential utilization of glycolysis and oxidative phosphorylation between the stages. The mechanism that controls this differential editing is unknown. Editing is catalyzed by multiprotein ∼20S editosomes that contain endonuclease, 3'-terminal uridylyltransferase, exonuclease, and ligase activities. These editosomes also contain KREPB5 and KREPA3 proteins, which have no functional catalytic motifs, but they are essential for parasite viability, editing, and editosome integrity in BF cells. We show here that repression of KREPB5 or KREPA3 is also lethal in PF, but the effects on editosome structure differ from those in BF. In addition, we found that point mutations in KREPB5 or KREPA3 differentially affect cell growth, editosome integrity, and RNA editing between BF and PF stages. These results indicate that the functions of KREPB5 and KREPA3 editosome proteins are adjusted between the life cycle stages. This implies that these proteins are involved in the processes that control differential editing and that the 20S editosomes differ between the life cycle stages., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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43. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.
- Author
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Carnes J, Anupama A, Balmer O, Jackson A, Lewis M, Brown R, Cestari I, Desquesnes M, Gendrin C, Hertz-Fowler C, Imamura H, Ivens A, Kořený L, Lai DH, MacLeod A, McDermott SM, Merritt C, Monnerat S, Moon W, Myler P, Phan I, Ramasamy G, Sivam D, Lun ZR, Lukeš J, Stuart K, and Schnaufer A
- Subjects
- Gene Expression Regulation, Microsatellite Repeats, Polymorphism, Single Nucleotide, Principal Component Analysis, Protozoan Proteins genetics, Variant Surface Glycoproteins, Trypanosoma genetics, Variant Surface Glycoproteins, Trypanosoma metabolism, Genome, Protozoan, Phylogeny, Protozoan Proteins metabolism, Trypanosoma classification, Trypanosoma genetics
- Abstract
Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.
- Published
- 2015
- Full Text
- View/download PDF
44. Oral antiplatelet agent hypersensitivity and cross-reactivity managed by successful desensitisation.
- Author
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Chin N, Rangamuwa K, Mariasoosai R, Carnes J, and Thien F
- Abstract
Oral platelet aggregation inhibitors are widely used for the treatment and prevention of cardiovascular diseases, including coronary stent thrombosis. Premature discontinuation following percutaneous coronary intervention would pose a grave risk of in-stent thrombosis, acute myocardial infarction and eventual death. Although they share the same mechanism of adenosine diphosphate P2Y12 platelet receptor inhibition, they belong to either the chemical class of thienopyridines (clopidogrel, prasugrel, and ticlopidine) or cyclopentyl-triazolo-pyrimidines (ticagrelor and cangrelor). This case describes the first documented cross-reactive hypersensitivity of clopidogrel towards both its fellow thienopyridine, prasugrel, as well as the structurally dissimilar ticagrelor, and its subsequent successful desensitisation.
- Published
- 2015
- Full Text
- View/download PDF
45. Bloodstream form Trypanosoma brucei do not require mRPN1 for gRNA processing.
- Author
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Carnes J, Lerch M, Kurtz I, and Stuart K
- Subjects
- Cell Line, Gene Knockout Techniques, Protozoan Proteins metabolism, RNA Processing, Post-Transcriptional, Ribonuclease III metabolism, Protozoan Proteins genetics, RNA, Guide, Kinetoplastida metabolism, Ribonuclease III genetics, Trypanosoma brucei brucei enzymology
- Abstract
Mitochondrial RNA processing in the kinetoplastid parasite Trypanosoma brucei involves numerous specialized catalytic activities that are incompletely understood. The mitochondrial genome consists of maxicircles that primarily encode rRNAs and mRNAs, and minicircles that encode a diverse array of guide RNAs (gRNAs). RNA editing uses these gRNAs as templates to recode mRNAs by insertion and deletion of uridine (U) residues. While the multiprotein complex that catalyzes RNA editing has been extensively studied, other players involved in mitochondrial RNA processing have remained enigmatic. The proteins required for processing mitochondrial polycistronic transcripts into mature species was essentially unknown until an RNase III endonuclease, called mRPN1, was reported to be involved in gRNA processing in procyclic form parasites. In this work, we examine the role of mRPN1 in gRNA processing in bloodstream form parasites, and show that complete elimination of mRPN1 by gene knockout does not alter gRNA maturation. These results indicate that another enzyme must be involved in gRNA processing., (© 2014 Carnes et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2015
- Full Text
- View/download PDF
46. Anaphylaxis due to Eruca sativa.
- Author
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Ruiz Garcia M, Carnes J, Cedena JR, and Nieto MF
- Subjects
- Adult, Anaphylaxis diagnosis, Female, Humans, Anaphylaxis etiology, Brassicaceae immunology
- Published
- 2014
47. Trypanosoma brucei Tb927.2.6100 is an essential protein associated with kinetoplast DNA.
- Author
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Beck K, Acestor N, Schulfer A, Anupama A, Carnes J, Panigrahi AK, and Stuart K
- Subjects
- Chromatography, Affinity, DNA, Mitochondrial metabolism, Polymerase Chain Reaction, Protein Transport, RNA Interference, RNA, Protozoan metabolism, Subcellular Fractions metabolism, Trypanosoma brucei brucei cytology, Trypanosoma brucei brucei drug effects, Trypanosoma brucei brucei growth & development, DNA, Kinetoplast metabolism, Protozoan Proteins metabolism, Trypanosoma brucei brucei metabolism
- Abstract
The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.
- Published
- 2013
- Full Text
- View/download PDF
48. Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function.
- Author
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Carnes J, Schnaufer A, McDermott SM, Domingo G, Proff R, Steinberg AG, Kurtz I, and Stuart K
- Subjects
- Amino Acid Sequence, Amino Acid Substitution physiology, Catalytic Domain genetics, DNA Mutational Analysis, Genome, Protozoan, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Structure, Tertiary genetics, Protein Structure, Tertiary physiology, Protozoan Proteins chemistry, Protozoan Proteins genetics, Protozoan Proteins metabolism, RNA, Messenger analysis, RNA, Messenger genetics, Ribonucleoproteins chemistry, Ribonucleoproteins metabolism, Sequence Homology, Trypanosoma brucei brucei metabolism, RNA Editing genetics, RNA, Protozoan genetics, Ribonucleoproteins genetics, Ribonucleoproteins physiology, Trypanosoma brucei brucei genetics
- Abstract
The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. We show here that various point mutations to KREPB4 and KREPB5 identify essential domains, and suggest that these proteins do not themselves perform RNase III catalysis. The zinc finger of KREPB4 but not KREPB5 is essential for editosome integrity and parasite viability, and mutation of the RNase III signature motif in KREPB5 prevents integration into editosomes, which is lethal. Isolated TAP-tagged KREPB4 and KREPB5 complexes preferentially associate with components of the deletion subcomplex, providing additional insights into editosome architecture. A new alignment of editosome RNase III sequences from several kinetoplastid species implies that KREPB4 and KREPB5 lack catalytic activity and reveals that the PUF motif is present in the editing endonucleases KREN1, KREN2, and KREN3. The data presented here are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.
- Published
- 2012
- Full Text
- View/download PDF
49. Editosome accessory factors KREPB9 and KREPB10 in Trypanosoma brucei.
- Author
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Lerch M, Carnes J, Acestor N, Guo X, Schnaufer A, and Stuart K
- Subjects
- Mutagenesis, Insertional, Protozoan Proteins chemistry, Protozoan Proteins genetics, RNA, Protozoan chemistry, RNA, Protozoan genetics, RNA, Protozoan metabolism, Trypanosoma brucei brucei chemistry, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei metabolism, Zinc Fingers, Protozoan Proteins metabolism, RNA Editing, Trypanosoma brucei brucei enzymology
- Abstract
Multiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ~20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5' OH on the 3' product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei.
- Published
- 2012
- Full Text
- View/download PDF
50. KREPB6, KREPB7, and KREPB8 are important for editing endonuclease function in Trypanosoma brucei.
- Author
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Guo X, Carnes J, Ernst NL, Winkler M, and Stuart K
- Subjects
- Gene Knockdown Techniques methods, Mutagenesis, Insertional, Promoter Regions, Genetic, RNA, Messenger genetics, RNA, Mitochondrial, RNA, Protozoan genetics, Trypanosoma brucei brucei enzymology, Trypanosoma brucei brucei growth & development, Uridine metabolism, Endonucleases genetics, Endonucleases metabolism, Protozoan Proteins genetics, Protozoan Proteins metabolism, RNA Editing, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei metabolism
- Abstract
Three distinct editosomes are required for the uridine insertion/deletion editing that creates translatable mitochondrial mRNAs in Trypanosoma brucei. They contain KREPB6, KREPB7, or KREPB8 proteins and their respective endonucleases KREN3, KREN2, or KREN1. RNAi knockdowns of KREPB6, KREPB7, and KREPB8 variably affect growth and RNA editing. KREPB6 and KREPB7 knockdowns substantially reduced in vitro insertion site cleavage activity of their respective editosomes, while KREPB8 knockdown did not affect its editosome deletion site cleavage activity despite inhibition of growth and editing. KREPB6, KREPB7, and KREPB8 knockdowns disrupted tagged KREN3, KREN2, or KREN1 editosomes, respectively, to varying degrees, and in the case of KREN1 editosomes, the deletion editing site cleavage activity shifted to a smaller S value. The varying effects correlate with a combination of the relative abundances of the KREPB6-8 proteins and of the different insertion and deletion sites. Tagged KREPB6-8 were physically associated with deletion subcomplexes upon knockdown of the centrally interactive KREPA3 protein, while KREN1-3 endonucleases were associated with insertion subcomplexes. The results indicate that KREPB6-8 occupy similar positions in editosomes and are important for the activity and specificity of their respective endonucleases. This suggests that they contribute to the accurate recognition of the numerous similar but diverse editing site substrates.
- Published
- 2012
- Full Text
- View/download PDF
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