80 results on '"Rodrigues TB"'
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2. CRISE BLÁSTICA MIELOIDE COMO MANIFESTAÇÃO CLÍNICA INICIAL DE PACIENTE COM DOENÇA MIELOPROLIFERATIVA CRÔNICA ASSOCIADA À REARRANJO DO PDGFR: RELATO DE CASO
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Lima, GG, primary, Rocha, BF, additional, Barreto, GC, additional, Perruso, LL, additional, Rodrigues, TB, additional, Rocha, VG, additional, Melo, RCB, additional, and Seguro, FS, additional
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- 2021
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3. Analysis of human biological samples using porous graphitic carbon columns and liquid chromatography-mass spectrometry: a review.
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Rodrigues TB, Cunha RL, Barci PEP, Santos-Neto ÁJ, and Lanças FM
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Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical technique for analyzing complex biological samples. Among various chromatographic stationary phases, porous graphitic carbon (PGC) columns have attracted significant attention due to their unique properties-such as the ability to separate both polar and non-polar compounds and their stability through all pH ranges and to high temperatures-besides the compatibility with LC-MS. This review discusses the applicability of PGC for SPE and separation in LC-MS-based analyses of human biological samples, highlighting the diverse applications of PGC-LC-MS in analyzing endogenous metabolites, pharmaceuticals, and biomarkers, such as glycans, proteins, oligosaccharides, sugar phosphates, and nucleotides. Additionally, the fundamental principles underlying PGC column chemistry and its advantages, challenges, and advances in method development are explored. This comprehensive review aims to provide researchers and practitioners with a valuable resource for understanding the capabilities and limitations of PGC columns in LC-MS-based analysis of human biological samples, thereby facilitating advancements in analytical methodologies and biomedical research., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)
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- 2024
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4. When the Stomach Takes a Vacation: The Unseen Battles of Gastroparesis.
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R Sousa B, Rodrigues TB, and Ribeiro J
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Gastroparesis is a syndrome characterised by delayed gastric emptying that is usually idiopathic, diabetic, or iatrogenic. This underdiagnosed disease has a substantial influence on the quality of life of its patients. We present the case of an 86-year-old man with dementia, benign prostatic hyperplasia, and gastroesophageal reflux disease who developed symptoms of gastroparesis during a lengthy hospital stay. Computed tomography (CT) and upper digestive endoscopy demonstrated gastric distention and pyloric stenosis. Despite cautious treatment and eventual pyloric dilation, the patient died from aspiration due to refractory respiratory failure. This example emphasises the need for early detection and thorough examination of gastroparesis to optimise patient outcomes and reduce morbidity and mortality., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, R. Sousa et al.)
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- 2024
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5. Pharmacokinetic Properties of Dapagliflozin in Hemodialysis and Peritoneal Dialysis Patients.
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Barreto J, Borges C, Rodrigues TB, Jesus DC, Campos-Staffico AM, Nadruz W, Luiz da Costa J, Bueno de Oliveira R, and Sposito AC
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- Humans, Hypoglycemic Agents therapeutic use, Prospective Studies, Renal Dialysis, Dialysis Solutions therapeutic use, Diabetes Mellitus, Type 2 complications, Diabetes Mellitus, Type 2 drug therapy, Peritoneal Dialysis, Renal Insufficiency chemically induced
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Background: Sodium-glucose cotransporter 2 (SGLT2) inhibitors attenuate incident cardiovascular outcomes, irrespective of baseline GFR, in conservatively managed CKD. As this condition inexorably progresses to demanding KRT, drug withdrawal is supported by the current lack of evidence of safety of SGLT2 inhibitors in dialysis., Methods: This study was a prospective, single-center, open-label trial ( ClinicalTrials.gov identifier: NCT05343078 ) aimed at assessing the pharmacokinetic properties and safety of dapagliflozin in patients with kidney failure on regular dialysis regimens compared with those with type 2 diabetes and age- and sex-matched controls with normal kidney function. Peripheral blood samples were collected from both groups every 30 minutes for 4 hours and again after 48 hours after ingestion of dapagliflozin 10 mg, which occurred immediately before dialysis session initiation in the kidney failure group. This protocol occurred in drug-naïve patients and again after six daily doses of dapagliflozin to assess whether the drug had accumulated. The plasma and dialysate levels of dapagliflozin at each time point were determined by liquid chromatography and used to calculate pharmacokinetics parameters (peak concentration [C max ] and area under the plasma concentration-versus-time curve) for each participant., Results: Dapagliflozin C max was 117 and 97.6 ng/ml in the kidney failure and control groups, respectively, whereas the corresponding accumulation ratios were 26.7% and 9.5%. No serious adverse events were reported for either group. Dapagliflozin recovered from dialysate corresponded to 0.10% of the administered dose., Conclusions: In patients with kidney failure on dialysis, dapagliflozin was well tolerated, was slightly dialyzable, and had nonaccumulating pharmacokinetic properties., Clinical Trial Registry Name and Registration Number: Pharmacokinetics and Dialyzability of Dapagliflozin in Dialysis Patients (DARE-ESKD 1), NCT05343078., (Copyright © 2023 by the American Society of Nephrology.)
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- 2023
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6. AIDS-related mortality in Pará Province, Brazilian Amazon region: Spatial and temporal analysis.
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Rodrigues TB, Dias BRL, Gomes D, Arcêncio RA, Andrade JAA, Ferreira GRON, Gonçalves LHT, and Botelho EP
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- Humans, Brazil epidemiology, Spatial Analysis, Income, Policy, Acquired Immunodeficiency Syndrome epidemiology
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Despite considerable therapeutic advances in the care of people living with human immunodeficiency virus (HIV) and with the acquired immunodeficiency syndrome (AIDS) and an overall reduction of 47% in the AIDS mortality rate in the last decade, the AIDS-mortality rates remains high. The social determinants of health (SDH) have a direct influence on the dynamics of this phenomenon. However, changes in SDH caused by the implemented policies against HIV have been poorly investigated. Moreover, the Brazilian rainforest has had the highest and continuously increasing AIDS mortality rate in Brazil since the 1980s. In this study, AIDS mortality in a province of the Brazilian rainforest was examined by using temporal and spatial analyses., Methods: In this ecological study, data from 2007 to 2018 were extracted from the Mortality Information System provided by the State Department of Public Health of Pará. For the temporal analysis, the integrated autoregressive model of moving average (ARIMA) and locally weighted polynomial regression (STLF) were used to forecast AIDS mortality from 2019 to 2022. For the spatial analysis, spatial autocorrelation and geographically weighted regression (GWR) analyses were employed., Results: The samples consisted of 6,498 notifications for AIDS-related deaths. From 2007 to 2013, the AIDS mortality rates showed an upward trend, followed by a stabilization until 2018 and an upward forecasted trend from 2019 to 2022. High mortality rates and high-high clusters were found in economic pole municipalities. Furthermore, AIDS mortality risk was directly associated with per capita income and demographic density, except in the southwestern region of Pará, which exhibited an inverse association with population density., Conclusion: Although the policies against HIV may have contributed to the stabilization of AIDS mortality rates from 2013 in Pará, the upward forecasted trend until 2022 raises an alert and concern to health authorities to provide reinforcement of the policies. The geographic variability of AIDS mortality promoted by SDH provides subsidies to health authorities to implement SDH-focused strategies for AIDS mortality reduction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rodrigues 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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7. The Temporal and Spatial Epidemiology Employed in the Elimination of the HIV Epidemic in the Largest Capital of the Brazilian Rainforest.
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Dias BRL, Rodrigues TB, Gomes D, Arcêncio RA, Gir E, Ferreira GRON, Polaro SHI, and Botelho EP
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Background: The main goal of this study was to analyze the human immunodeficiency virus (HIV) epidemic temporally and spatially in Belém from 2007 to 2018., Methods: The incidence rates were analyzed according to time using autoregressive integrated moving-average models, as well as spatially using spatial autocorrelation, Kernel density, scan statistics, and regression techniques., Results: During the study period, 6007 notifications of new cases of HIV/AIDS were reported. The time series analysis revealed a stabilized trend of incidence from 2007 to October 2016, followed by irregular fluctuations until the end of December 2018. Seasonal behavior was observed from 2019 to 2022. The high-high incidence clusters were found in the central and transition areas. An expansion of the number of new reported cases was observed in the central area. Three spatial risk zones were observed. The higher relative risk zone was concentrated in the transition area. The spatial regression showed that the incidence rates were positively correlated with the Family Health Strategy (FHS) coverage., Conclusions: To eliminate HIV in Belém, it will be necessary to decentralize testing and ART and expand the coverage of FHS to ensure universal access to healthcare for citizens.
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- 2022
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8. Toxicity of a novel dsRNA-based insecticide to the Colorado potato beetle in laboratory and field trials.
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Pallis S, Alyokhin A, Manley B, Rodrigues TB, Buzza A, Barnes E, and Narva K
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- Animals, Pest Control, RNA, Double-Stranded pharmacology, Coleoptera, Insecticides pharmacology, Solanum tuberosum genetics
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Background: The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) is one of the most notorious pests of the potato, Solanum tuberosum. Potato beetles are capable of developing resistance to various insecticides in relatively few generations. Novel and effective means of controlling Colorado potato beetle populations are constantly required to protect potato crops and prevent loss of yield. The knockdown of gene function through ribonucleic acid interference has been demonstrated in Colorado potato beetles, suggesting the use of this technology as a means of beetle management. A novel double-stranded RNA-based insecticide with the active ingredient, ledprona, has been tested in variable dose laboratory bioassays, followed by field studies., Results: Exposure to ledprona resulted in both increased beetle mortality and decreased foliage consumption in all four instars and adult beetles. Effects decreased from earlier to later life stages. No ovicidal activity was detected. Onset of mortality was slower compared with existing chemical insecticides. Nevertheless, field applications of formulated ledprona to potato plots resulted in their protection comparable with that provided by spinosad and chlorantraniliprole., Conclusion: Based on the results of this study, formulated ledprona has attributes to become a useful tool in controlling Colorado potato beetle populations that is likely to be a good fit in integrated pest management protocols. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)
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- 2022
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9. HIV epidemic among Brazilian women who have sex with women: An ecological study.
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Bezerra ALL, Sorensen W, Rodrigues TB, Sousa SML, Carneiro MS, Polaro SHI, Ramos AMPC, Ferreira GRON, Gir E, Reis RK, and Botelho EP
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- Brazil epidemiology, Female, Humans, Male, Sexual Behavior, HIV Infections epidemiology, Homosexuality, Female, Sexual and Gender Minorities
- Abstract
The influences of public policies fighting HIV among women who have sex with women is scarcely studied. This study aimed to analyse the time series of human immunodeficiency virus (HIV) epidemic, between 2007 and 2020, among Brazilian women who have sex with women, in order to evaluate the effect of Brazilian policies for fighting HIV in this subpopulation compared to women who have sex with men (WSM). This ecological study employed HIV and acquired immunodeficiency syndrome (AIDS) new cases among women who have sex strictly with women (WSW), women who have sex with men and women (WSMW), and WSM reported to the Sistema de Informação de Agravos de Notificação from 2007 to 2020. Crude Brazilian and regional annual age-adjusted HIV/AIDS population-level incidence rates were calculated for WSW, WSMW and WSM. The rates were then analyzed using the Joinpoint regression model. A total of 102,890, 757, and 1,699 notifications of WSW, WSMW, and WSM living with HIV/AIDS were reported during the study period, respectively. South Brazilian region had the greatest HIV/AIDS incidence rates among WSM and bisexual women while the North region had the greatest incidence among WSW. In the WSM population, the temporal trends showed at least one stable or an increasing trend period from 2007 to 2013 or 2014, followed by one decreasing trend in all Brazilian regions. While among the WSMW most of the regions had a stable trend period from 2007 to 2020, in WSW group most of the trends had only one decreasing period. The decreasing trends were faster in WSM than in WSW. These results suggest a low efficiency of Brazilian policies for fighting HIV among WSW and WSMW and show the necessity of implementing new policies specific to this population., 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 Bezerra, Sorensen, Rodrigues, Sousa, Carneiro, Polaro, Ramos, Ferreira, Gir, Reis and Botelho.)
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- 2022
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10. Temporal trend and spatial analysis of the HIV epidemic in young men who have sex with men in the second largest Brazilian Amazonian province.
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Seabra IL, Pedroso AO, Rodrigues TB, Ferreira GRON, da Silva Ferreira AL, Arcêncio RA, Gomes D, da Silva RAR, and Botelho EP
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- Adolescent, Brazil epidemiology, Homosexuality, Male, Humans, Male, Spatial Analysis, Acquired Immunodeficiency Syndrome epidemiology, HIV Infections epidemiology, Sexual and Gender Minorities
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Background: After 40 years of its starting, the HIV epidemic in Brazilian Amazon region remains on an increasing trend. The young men who have sex with men (MSM) have been the most impacted by the HIV in the last decade. However, much more than attributing the risk behavior to HIV uniquely to the individual, behaviors are shaped by social determinants of health (SDH). Despite the problem, there is a scarcity of studies evaluating the impact of SDH on HIV among young MSM and none of them were done in the Northern of Brazil. Therefore, the main goal of this study was to analyse the HIV epidemic among Brazilian Amazonian young MSM using temporal trends and spatial analysis., Methods: We conducted an ecological study using reported cases of HIV/AIDS in young MSM living in Pará, the second larger Brazilian Amazonian province, between 2007 and 2018. Data were obtained from the Information System for Notifiable Diseases. For the temporal analysis, we employed a Seasonal and Trend decomposition using Loess Forecasting model (STLF), which is a hybrid time-series forecast model, that combines the Autoregressive-Integrated Moving Average (ARIMA) forecasting model with the Seasonal-Trend by Loess (STL) decomposition method. For the spatial analysis, Moran's spatial autocorrelation, spatial scan, and spatial regression techniques were used., Results: A total of 2192 notifications were included in the study. Greater variabilities in HIV/AIDS population-level diagnosis rates were found in the festive months. The HIV/AIDS population-level diagnosis rates exhibited an upward trend from 2013 and this trend is forecasted to continue until 2022. Belém, the capital of Pará, presented the highest spatial risk for HIV/AIDS and was the only city to present spatiotemporal risk from 2014 to 2018. The geographic variation of the HIV epidemic was associated with the number of men with formal jobs, the average salary of men, and the percentage of people over 18 years old with elementary education., Conclusion: The upward trend of HIV/AIDS population-level diagnosis rate forecasted until 2022 and the variability of the epidemic promoted by the SDH brings an alert and subsidies to health authorities to implement more efficient and focalized public policies against HIV among young MSM in Pará., (© 2022. The Author(s).)
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- 2022
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11. Synthetic cannabinoid receptor agonists profile in infused papers seized in Brazilian prisons.
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Rodrigues TB, Souza MP, de Melo Barbosa L, de Carvalho Ponce J, Júnior LFN, Yonamine M, and Costa JL
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- Humans, Prisons, Brazil epidemiology, Cannabinoid Receptor Agonists, Prisoners
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Purpose: Synthetic cannabinoid receptor agonists (SCRAs) are a class of varied compounds that mimic the effects of natural cannabinoids found in cannabis. Because they have a wide range of diverse structures, they vary widely in their potency. The abuse of new psychoactive substances (NPS) in prisons was reported in many European countries and in the USA. In the present study, we have described the identification of SCRAs in 56 infused paper sheet samples, seized mainly in Brazilian prisons between 2016 and 2020., Methods: The materials were seized by local or federal law enforcement and analyzed by São Paulo State Police or Brazilian Federal Police using gas chromatography-mass spectrometry, attenuated total reflection-Fourier transform infrared spectroscopy, liquid chromatography-high-resolution mass spectrometry or nuclear magnetic resonance spectrometry., Results: Most of these samples (87.5%) were seized in 2019-2020; seven different SCRAs were identified in samples, and the most frequently identified substances were MDMB-4en-PINACA (23.6%) and 5F-MDMB-PICA (36.4%), the newest SCRAs emerging recently., Conclusions: As observed in Europe and the USA, Brazil also shows the prevalence of indazole-3-carboxamides and indole-3-carboxamides among SCRAs seizures in the prison system. This phenomenon is spreading all over the world at this moment. These data on the prevalence could help to alert judicial authorities to shutting down the introduction of NPS, including SCRAs, into prisons to ensure safety and security for avoiding health risks of prisoners and staff, leading to positive effects in this population. To our knowledge, this is the first demonstration of SCRAs smuggling into prisons in Latin America., (© 2021. Japanese Association of Forensic Toxicology.)
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- 2022
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12. Stability of tropical forest tree carbon-water relations in a rainfall exclusion treatment through shifts in effective water uptake depth.
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Pivovaroff AL, McDowell NG, Rodrigues TB, Brodribb T, Cernusak LA, Choat B, Grossiord C, Ishida Y, Jardine KJ, Laurance S, Leff R, Li W, Liddell M, Mackay DS, Pacheco H, Peters J, de J Sampaio Filho I, Souza DC, Wang W, Zhang P, and Chambers J
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- Carbon, Droughts, Forests, Plant Leaves, Rainforest, Trees, Water
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Increasing severity and frequency of drought is predicted for large portions of the terrestrial biosphere, with major impacts already documented in wet tropical forests. Using a 4-year rainfall exclusion experiment in the Daintree Rainforest in northeast Australia, we examined canopy tree responses to reduced precipitation and soil water availability by quantifying seasonal changes in plant hydraulic and carbon traits for 11 tree species between control and drought treatments. Even with reduced soil volumetric water content in the upper 1 m of soil in the drought treatment, we found no significant difference between treatments for predawn and midday leaf water potential, photosynthesis, stomatal conductance, foliar stable carbon isotope composition, leaf mass per area, turgor loss point, xylem vessel anatomy, or leaf and stem nonstructural carbohydrates. While empirical measurements of aboveground traits revealed homeostatic maintenance of plant water status and traits in response to reduced soil moisture, modeled belowground dynamics revealed that trees in the drought treatment shifted the depth from which water was acquired to deeper soil layers. These findings reveal that belowground acclimation of tree water uptake depth may buffer tropical rainforests from more severe droughts that may arise in future with climate change., (© 2021 John Wiley & Sons Ltd.)
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- 2021
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13. First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle ( Leptinotarsa decemlineata ).
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Rodrigues TB, Mishra SK, Sridharan K, Barnes ER, Alyokhin A, Tuttle R, Kokulapalan W, Garby D, Skizim NJ, Tang YW, Manley B, Aulisa L, Flannagan RD, Cobb C, and Narva KE
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Colorado potato beetle (CPB, Leptinotarsa decemlineata ) is a major pest of potato and other solanaceous vegetables in the Northern Hemisphere. The insect feeds on leaves and can completely defoliate crops. Because of the repeated use of single insecticide classes without rotating active ingredients, many chemicals are no longer effective in controlling CPB. Ledprona is a sprayable double-stranded RNA biopesticide with a new mode of action that triggers the RNA interference pathway. Laboratory assays with second instar larvae fed Ledprona showed a dose-response where 25×10
-6 g/L of dsPSMB5 caused 90% mortality after 6days of initial exposure. We also showed that exposure to Ledprona for 6h caused larval mortality and decreased target messenger RNA (mRNA) expression. Decrease in PSMB5 protein levels was observed after 48h of larval exposure to Ledprona. Both PSMB5 mRNA and protein levels did not recover over time. Ledprona efficacy was demonstrated in a whole plant greenhouse trial and performed similarly to spinosad. Ledprona, currently pending registration at EPA, represents a new biopesticide class integrated pest management and insecticide resistance management programs directed against CPB., Competing Interests: Authors TR, SM, KS, EB, RT, BM, BF and KN were employed by the company GreenLight Biosciences, Inc. and conduct research in developing products based on RNAi. The remaining 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 © 2021 Rodrigues, Mishra, Sridharan, Barnes, Alyokhin, Tuttle, Kokulapalan, Garby, Skizim, Tang, Manley, Aulisa, Flannagan, Cobb and Narva.)- Published
- 2021
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14. Development and Validation of a Method for Quantification of 28 Psychotropic Drugs in Postmortem Blood Samples by Modified Micro-QuEChERS and LC-MS-MS.
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Rodrigues TB, Morais DR, Gianvecchio VAP, Aquino EM, Cunha RL, Huestis MA, and Costa JL
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- Chromatography, Liquid, Forensic Toxicology, Humans, Limit of Detection, Psychotropic Drugs, Tandem Mass Spectrometry
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The development of new sample preparation alternatives in analytical toxicology leading to quick, effective, automated and environmentally friendly procedures is growing in importance. One of these alternatives is the QuEChERS, originally developed for the analysis of pesticide residues, producing cleaner extracts than liquid-liquid extraction, and easier separation of aqueous and organic phases. However, there are few published studies on the miniaturization of this technique for forensic toxicology, especially in postmortem analysis. We developed and validated a modified micro-QuEChERS and LC-MS-MS assay to quantify 16 antidepressants, 7 antipsychotics and 3 metabolites and semi-quantify norfluoxetine and norsertraline in postmortem blood. The calibration curve was linear from 1 to 500 ng/mL, achieved an r > 0.99, with all standards quantifying within ±15% of target except ±20% at the limit of quantification of 1 ng/mL for 26 substances. The F test was applied to evaluate if the variance between replicates remained constant for all calibrators. Six weighting factors were analyzed (1/x, 1/x2, 1/x0,5, 1/y, 1/y2 and 1/y0,5), with the weighting factor with the lowest sum of residual regression errors (1/x2) selected. No endogenous or exogenous interferences were observed. Method imprecision and bias were <19.0% and 19.7%, respectively. Advantages of this method include a low sample volume of 100 µL, simple but effective sample preparation and a rapid 8.5-min run time. The validated analytical method was successfully applied to the analysis of 100 authentic postmortem samples., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2021
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15. Integrative review on the incidence of HIV infection and its socio-spatial determinants.
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Dias BRL, Rodrigues TB, Botelho EP, Oliveira MFV, Feijão AR, and Polaro SHI
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- Humans, Incidence, Poverty, Risk-Taking, HIV Infections epidemiology
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Objectives: to identify the socio-spatial determinants associated with the incidence of infection by the human immunodeficiency virus (HIV) described in national and international literature., Methods: integrative literature review that included original articles indexed in the LILACS, PUBMED, CINAHL, and Scopus databases, from 2015 to 2019. The synthesis of the articles was done in a descriptive manner in two categories according to the type of approach., Results: 13 articles were selected, with a predominance of ecological studies. Regions with deficient access to health services, high population density, higher rates of poverty, intense migratory flow, border areas, as well as precarious socioeconomic conditions, and risk behaviors are associated with higher rates of infection., Final Considerations: the influence of regional inequalities on the incidence of HIV infection is revealed, confirming the relevance of spatial analysis techniques for mapping the distribution of diseases and identifying risk areas.
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- 2021
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16. Optimization of QuEChERS extraction for detection and quantification of 20 antidepressants in postmortem blood samples by LC-MS/MS.
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Campêlo JM, Rodrigues TB, Costa JL, and Santos JM
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- Chromatography, Liquid, Humans, Limit of Detection, Solid Phase Extraction, Tandem Mass Spectrometry, Antidepressive Agents blood, Forensic Toxicology methods
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In this study, a comprehensively optimization of QuEChERS (quick, easy, cheap, effective, rugged and safe) method using design of experiments (DOE) was conducted to evaluate the best conditions to obtain the most effective extraction. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was performed to identify and quantify the antidepressants, with electrospray ionization acquired in positive mode. The method was validated for all analytes; the calibration curves were linear from 10-1000ng/mL, with R
2 >0.98, and with LOD and LOQ defined as 10ng/mL. Method imprecision and bias were less than 14.3% and 18.9%, respectively. Neither carryover nor interferences were observed. Overall, the optimized method was applied in postmortem real sample analysis to quantify the antidepressants. This study showed a viable method that can be applied for routine forensic analysis, with a quick and easy sample preparation and a rapid total run time of 8min for each analysis., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2021
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17. Stimulation of isoprene emissions and electron transport rates as key mechanisms of thermal tolerance in the tropical species Vismia guianensis.
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Rodrigues TB, Baker CR, Walker AP, McDowell N, Rogers A, Higuchi N, Chambers JQ, and Jardine KJ
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- Carbon Dioxide, Electron Transport, Photosynthesis, Plant Leaves, Butadienes, Hemiterpenes
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Tropical forests absorb large amounts of atmospheric CO
2 through photosynthesis, but high surface temperatures suppress this absorption while promoting isoprene emissions. While mechanistic isoprene emission models predict a tight coupling to photosynthetic electron transport (ETR) as a function of temperature, direct field observations of this phenomenon are lacking in the tropics and are necessary to assess the impact of a warming climate on global isoprene emissions. Here we demonstrate that in the early successional species Vismia guianensis in the central Amazon, ETR rates increased with temperature in concert with isoprene emissions, even as stomatal conductance (gs ) and net photosynthetic carbon fixation (Pn ) declined. We observed the highest temperatures of continually increasing isoprene emissions yet reported (50°C). While Pn showed an optimum value of 32.6 ± 0.4°C, isoprene emissions, ETR, and the oxidation state of PSII reaction centers (qL ) increased with leaf temperature with strong linear correlations for ETR (ƿ = 0.98) and qL (ƿ = 0.99) with leaf isoprene emissions. In contrast, other photoprotective mechanisms, such as non-photochemical quenching, were not activated at elevated temperatures. Inhibition of isoprenoid biosynthesis repressed Pn at high temperatures through a mechanism that was independent of stomatal closure. While extreme warming will decrease gs and Pn in tropical species, our observations support a thermal tolerance mechanism where the maintenance of high photosynthetic capacity under extreme warming is assisted by the simultaneous stimulation of ETR and metabolic pathways that consume the direct products of ETR including photorespiration and the biosynthesis of thermoprotective isoprenoids. Our results confirm that models which link isoprene emissions to the rate of ETR hold true in tropical species and provide necessary "ground-truthing" for simulations of the large predicted increases in tropical isoprene emissions with climate warming., (© 2020 John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)- Published
- 2020
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18. Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops.
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Roberts A, Boeckman CJ, Mühl M, Romeis J, Teem JL, Valicente FH, Brown JK, Edwards MG, Levine SL, Melnick RL, Rodrigues TB, Vélez AM, Zhou X, and Hellmich RL
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Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis ( Bt ) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non- Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms., (Copyright © 2020 Roberts, Boeckman, Mühl, Romeis, Teem, Valicente, Brown, Edwards, Levine, Melnick, Rodrigues, Vélez, Zhou and Hellmich.)
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- 2020
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19. Safety Considerations for Humans and Other Vertebrates Regarding Agricultural Uses of Externally Applied RNA Molecules.
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Rodrigues TB and Petrick JS
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The potential of double-stranded RNAs (dsRNAs) for use as topical biopesticides in agriculture was recently discussed during an OECD (Organisation for Economic Co-operation and Development) Conference on RNA interference (RNAi)-based pesticides. Several topics were presented and these covered different aspects of RNAi technology, its application, and its potential effects on target and non-target organisms (including both mammals and non-mammals). This review presents information relating to RNAi mechanisms in vertebrates, the history of safe RNA consumption, the biological barriers that contribute to the safety of its consumption, and effects related to humans and other vertebrates as discussed during the conference. We also review literature related to vertebrates exposed to RNA molecules and further consider human health safety assessments of RNAi-based biopesticides. This includes possible routes of exposure other than the ingestion of potential residual material in food and water (such as dermal and inhalation exposures during application in the field), the implications of different types of formulations and RNA structures, and the possibility of non-specific effects such as the activation of the innate immune system or saturation of the RNAi machinery., (Copyright © 2020 Rodrigues and Petrick.)
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- 2020
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20. Traffic accidents among drivers: incidence and differences between motorcyclists and car drivers in population-based study.
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Rios PAA, Mota ELA, Ferreira LN, Cardoso JP, Santos GJ, and Rodrigues TB
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- Adult, Age Factors, Brazil epidemiology, Educational Status, Female, Humans, Incidence, Male, Middle Aged, Qualitative Research, Sex Factors, Young Adult, Accidents, Traffic statistics & numerical data, Automobile Driving statistics & numerical data, Automobiles statistics & numerical data, Motorcycles statistics & numerical data
- Abstract
Objectives: To estimate the incidence of traffic accidents among land vehicle drivers and to verify differences between motorcyclist and car driver accident victims., Methods: A household survey was conducted in Jequié city, Bahia, Brazil, in 2013, with 1,406 drivers who were selected by cluster sampling strategy. Estimates of the cumulative incidence of traffic accidents were weighted by the sample design and, to compare groups of drivers, incidence rates (IR) and 95% confidence intervals were calculated with Poisson Regression. The Chi-Square test were used with Rao-Scott correction (p ≤ 0.05) to evaluate differentials of the event between motorcyclists and car driver victims., Results: Involvement in traffic accidents in the last 12 months before the interview was reported by 10.6% of the drivers. The cumulative incidence was 4.3% for accidents that caused injuries. Motorcyclists had double the risk of being involved in accidents (IR = 2.03; 95%CI 1.40 - 2.94) and higher proportions of injuries (p < 0.001), interruption in daily life activities (p = 0.003) and use of emergency services (p = 0.008). Factors related to time and place of accident were not different between groups., Conclusion: Higher incidence of traffic accidents and higher proportions of injuries and others repercussions of these events were seen in motorcyclists. These findings reaffirm the vulnerability of this group and explains the different impact that a preventable cause of morbidity and mortality has on each driver group.
- Published
- 2019
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21. RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis.
- Author
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Kyre BR, Rodrigues TB, and Rieske LK
- Subjects
- Animals, Coleoptera genetics, Forests, Gene Silencing physiology, Genes, Essential genetics, Insecta genetics, Pinus, RNA Interference physiology, RNA, Double-Stranded pharmacology, Real-Time Polymerase Chain Reaction methods, Real-Time Polymerase Chain Reaction standards, Pest Control methods, Weevils genetics
- Abstract
RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.
- Published
- 2019
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22. [Mammographic over-screening: evaluation based on probabilistic linkage of records databases from the Breast Cancer Information System (SISMAMA)].
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Rodrigues TB, Stavola B, Bustamante-Teixeira MT, Guerra MR, Nogueira MC, Fayer VA, Corrêa CSL, and Dos-Santos-Silva I
- Subjects
- Aged, Brazil epidemiology, Databases, Factual, Early Detection of Cancer, Female, Humans, Mammography statistics & numerical data, Mass Screening statistics & numerical data, Middle Aged, Pilot Projects, Risk Factors, Breast Neoplasms diagnostic imaging, Breast Neoplasms epidemiology, Health Information Systems statistics & numerical data, Mammography methods, Mass Screening methods
- Abstract
The Brazilian Ministry of Health recommends biennial mammographic screening for women aged between 50 and 69 years. Since screening is opportunistic in the country, the actual periodicity varies. This study sought to test a methodology for estimating over-screening due to excessive periodicity, defined as a smaller than recommended interval between exams, and its association with socio-demographic characteristics. A cohort of women who underwent mammography in 2010, and whose result was normal, was assembled through probabilistic linkage SISMAMA records based on a set of personal identifiers. We used data from women living in the micro health region of Juiz de Fora/Lima Duarte/Bom Jardim, Minas Gerais State, Brazil, who were followed in the System until the end of 2012. The rate of over-screening was 150/1,000 women/year (95%CI: 144.9-155.9), affecting 21% of women. Over-screening increased by 24% during Pink October campaigns (adjusted HR = 1.24; 95%CI: 1.15-1.35). The shorter the time passed since the last mammogram, the greater the odds of over-screening. Compared with women who had never had a mammogram prior to 2010, women who had had one in the previous 2 years were two times more likely to be over-screened (adjusted HR = 2.01; 95%CI: 1.74-2.31) whilst those who had had a mammogram ≤ 1 year previously were three times more likely to be over-screened (adjusted HR = 3.27; 95%CI: 2.87-3.73). Over-screening was substantial in this population, excessively exposing women to the risks of screening with no additional benefits and overestimating mammogram coverage. The methodology proved to be successful and should be applied to representative populations in order to guide breast cancer control policies.
- Published
- 2019
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23. Analysis of 13 C and 14 C labeling in pyruvate and lactate in tumor and blood of lymphoma-bearing mice injected with 13 C- and 14 C-labeled pyruvate.
- Author
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Serrao EM, Kettunen MI, Rodrigues TB, Lewis DY, Gallagher FA, Hu DE, and Brindle KM
- Subjects
- Animals, Injections, Isotope Labeling, Mice, Inbred C57BL, Tissue Distribution, Carbon Isotopes metabolism, Carbon Radioisotopes metabolism, Lactic Acid blood, Lymphoma blood, Pyruvic Acid blood
- Abstract
Measurements of hyperpolarized
13 C label exchange between injected [1-13 C]pyruvate and the endogenous tumor lactate pool can give an apparent first-order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1-14 C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first-order rate constant for hyperpolarized13 C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non-polarized [3-13 C]pyruvate, rapid excision of the tumor after 30 s and measurement of13 C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1-13 C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1-13 C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized13 C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined., (© 2018 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)- Published
- 2018
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24. Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.
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Rodrigues TB, Duan JJ, Palli SR, and Rieske LK
- Subjects
- Administration, Oral, Animals, Female, Humans, Insect Proteins genetics, Introduced Species, Larva genetics, Male, Pest Control, Biological methods, Species Specificity, Coleoptera genetics, Fraxinus parasitology, Insect Control methods, RNA Interference, RNA, Double-Stranded administration & dosage
- Abstract
Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.
- Published
- 2018
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25. Sub-minute kinetics of human red cell fumarase: 1 H spin-echo NMR spectroscopy and 13 C rapid-dissolution dynamic nuclear polarization.
- Author
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Shishmarev D, Wright AJ, Rodrigues TB, Pileio G, Stevanato G, Brindle KM, and Kuchel PW
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- Fumarates chemistry, Fumarates metabolism, Humans, Kinetics, Malates chemistry, Malates metabolism, Markov Chains, Models, Biological, Monte Carlo Method, Time Factors, Carbon-13 Magnetic Resonance Spectroscopy, Erythrocytes enzymology, Fumarate Hydratase metabolism, Proton Magnetic Resonance Spectroscopy
- Abstract
Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of
1 H spin-echo and13 C-NMR spectra.1 H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized13 C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13 C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis-Menten equations. In RD-DNP experiments, the decline in the13 C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when13 C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy., (Copyright © 2018 John Wiley & Sons, Ltd.)- Published
- 2018
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26. RNA interference in the Asian Longhorned Beetle:Identification of Key RNAi Genes and Reference Genes for RT-qPCR.
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Rodrigues TB, Dhandapani RK, Duan JJ, and Palli SR
- Subjects
- Animals, Coleoptera classification, Computational Biology methods, Gene Expression Profiling, Gene Expression Regulation, Phylogeny, Reproducibility of Results, Coleoptera genetics, Genes, Insect, RNA Interference
- Abstract
Asian Longhorned Beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi) technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes including those coding for Dicer, Argonaute, and double-stranded RNA-binding proteins (dsRBP) as well as for proteins involved in dsRNA transport and the systemic RNAi. We also compared expression of six potential reference genes that could be used to normalize gene expression and selected gapdh and rpl32 as the most reliable genes among different tissues and stages of ALB. Injection of double-stranded RNA (dsRNA) targeting gene coding for inhibitor of apoptosis (IAP) into larvae and adults resulted in a significant knockdown of this gene and caused the death of 90% of the larvae and 100% of adults. No mortality of both larvae and adults injected with dsRNA targeting gene coding for green fluorescence protein (GFP, as a negative control) was observed. These data suggest that functional RNAi machinery exists in ALB and a potential RNAi-based method could be developed for controlling this insect.
- Published
- 2017
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27. Development of RNAi method for screening candidate genes to control emerald ash borer, Agrilus planipennis.
- Author
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Rodrigues TB, Rieske LK, J Duan J, Mogilicherla K, and Palli SR
- Subjects
- Animals, Gene Expression, Genetic Association Studies, Genetic Testing, Larva, RNA, Double-Stranded, Coleoptera genetics, Genes, Insect, RNA Interference
- Abstract
The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed to siRNAs by EAB larvae within 72 h after ingestion. Also, feeding neonate larvae with IAP (inhibitor of apoptosis) or COP (COPI coatomer, β subunit) dsRNA silenced their target genes and caused mortality. Both an increase in the concentration of dsRNA fed and sequential feeding of two different dsRNAs increased mortality. Here we provide evidence for successful RNAi in EAB, and demonstrate the development of a rapid and effective bioassay for oral delivery of dsRNA to screen additional genes.
- Published
- 2017
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28. ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.
- Author
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Juaristi I, García-Martín ML, Rodrigues TB, Satrústegui J, Llorente-Folch I, and Pardo B
- Subjects
- Amino Acid Transport Systems, Acidic genetics, Animals, Antiporters genetics, Astrocytes metabolism, Brain Chemistry genetics, Glucose metabolism, Glucosephosphate Dehydrogenase genetics, Glucosephosphate Dehydrogenase metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Oxygen Consumption genetics, Aggrecans genetics, Aggrecans metabolism, Amino Acid Transport Systems, Acidic deficiency, Antiporters deficiency, Hereditary Central Nervous System Demyelinating Diseases genetics, Lactic Acid metabolism, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Diseases genetics, Nervous System Diseases genetics, Nervous System Diseases metabolism, Neurons metabolism, Psychomotor Disorders genetics
- Abstract
ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells., (© 2017 International Society for Neurochemistry.)
- Published
- 2017
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29. Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I.
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Neves AA, Xie B, Fawcett S, Alam IS, Witney TH, de Backer MM, Summers J, Hughes W, McGuire S, Soloviev D, Miller J, Howat WJ, Hu DE, Rodrigues TB, Lewis DY, and Brindle KM
- Subjects
- Animals, Biomarkers metabolism, Cell Line, Tumor, Female, Humans, Mice, Mice, Inbred C57BL, Mice, Nude, Neoplasms, Experimental diagnostic imaging, Protein Domains, Radiopharmaceuticals pharmacokinetics, Synaptotagmin I chemistry, Tissue Distribution, Apoptosis, Molecular Imaging methods, Neoplasms, Experimental metabolism, Neoplasms, Experimental pathology, Positron-Emission Tomography methods, Synaptotagmin I pharmacokinetics
- Abstract
Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. Methods: The capability of near-infrared fluorophore-labeled and
99m Tc- and111 In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft. Results: The fluorophore-labeled C2Am derivative showed predominantly renal clearance and high specificity and sensitivity for detecting low levels of tumor cell death (2%-5%). There was a significant correlation ( R > 0.9, P < 0.05) between fluorescently labeled C2Am binding and histologic markers of cell death, including cleaved caspase-3, whereas there was no such correlation with a site-directed mutant of C2Am (iC2Am) that does not bind phosphatidylserine.99m Tc-C2Am and111 In-C2Am also showed favorable biodistribution profiles, with predominantly renal clearance and low nonspecific retention in the liver and spleen at 24 h after probe administration.99m Tc-C2Am and111 In-C2Am generated tumor-to-muscle ratios in drug-treated tumors of 4.3× and 2.2×, respectively, at 2 h and 7.3× and 4.1×, respectively, at 24 h after administration. Conclusion: Given the favorable biodistribution profile of99m Tc- and111 In-labeled C2Am, and their ability to produce rapid and cell death-specific image contrast, these agents have potential for clinical translation., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)- Published
- 2017
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30. (13) C magnetic resonance spectroscopy measurements with hyperpolarized [1-(13) C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo.
- Author
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Dzien P, Tee SS, Kettunen MI, Lyons SK, Larkin TJ, Timm KN, Hu DE, Wright A, Rodrigues TB, Serrao EM, Marco-Rius I, Mannion E, D'Santos P, Kennedy BW, and Brindle KM
- Subjects
- Animals, Enzyme Activation, Female, Genes, Reporter physiology, HEK293 Cells, Humans, Mice, Mice, SCID, Recombinant Proteins genetics, Reproducibility of Results, Sensitivity and Specificity, Tissue Distribution, Zymomonas genetics, Carbon-13 Magnetic Resonance Spectroscopy methods, Magnetic Resonance Imaging methods, Molecular Imaging methods, Pyruvate Decarboxylase metabolism, Pyruvic Acid pharmacokinetics, Recombinant Proteins metabolism, Zymomonas enzymology
- Abstract
Purpose: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide., Methods: Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using (13) C MRS measurements of the conversion of hyperpolarized [1-(13) C] pyruvate to H(13) CO3-., Results: Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two-fold increase in the H(13) CO3-/[1-(13) C] pyruvate signal ratio following intravenous injection of hyperpolarized [1-(13) C] pyruvate., Conclusion: We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized (13) C MRS. Magn Reson Med 76:391-401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., (© 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)
- Published
- 2016
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31. Effects of fasting on serial measurements of hyperpolarized [1-(13) C]pyruvate metabolism in tumors.
- Author
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Serrao EM, Rodrigues TB, Gallagher FA, Kettunen MI, Kennedy BW, Vowler SL, Burling KA, and Brindle KM
- Subjects
- Animals, Female, Mice, Mice, Inbred C57BL, Neoplasms, Experimental pathology, Reproducibility of Results, Sensitivity and Specificity, Algorithms, Biomarkers, Tumor metabolism, Carbon-13 Magnetic Resonance Spectroscopy methods, Fasting metabolism, Neoplasms, Experimental metabolism, Pyruvic Acid metabolism, Signal Processing, Computer-Assisted
- Abstract
Imaging of the metabolism of hyperpolarized [1-(13) C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of (13) C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non-fasted animals. The fasted state showed lower intra-individual variability, although the [1-(13) C]lactate/[1-(13) C]pyruvate signal ratio was significantly greater in fasted than in non-fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of (13) C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd., (© 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)
- Published
- 2016
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32. Development of Timd2 as a reporter gene for MRI.
- Author
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Patrick PS, Rodrigues TB, Kettunen MI, Lyons SK, Neves AA, and Brindle KM
- Subjects
- Animals, Female, Ferritins administration & dosage, Ferritins chemistry, Ferritins metabolism, Fluorescent Dyes administration & dosage, Fluorescent Dyes chemistry, Fluorescent Dyes metabolism, HEK293 Cells, Humans, Membrane Proteins chemistry, Mice, Mice, SCID, Genes, Reporter genetics, Magnetic Resonance Imaging methods, Membrane Proteins genetics, Membrane Proteins metabolism
- Abstract
Purpose: To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), using T1- and T2-weighted imaging., Methods: Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron-loaded or manganese-loaded ferritin, were imaged using T1- and T2-weighted MRI. Mice were also implanted subcutaneously with Timd2-expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2-weighted imaging., Results: Timd2-expressing cells, but not control cells, showed a large increase in both R2 and R1 in vitro following incubation with iron-loaded and manganese-loaded ferritin, respectively. Expression of Timd2 had no effect on cell viability or proliferation; however, manganese-loaded ferritin, but not iron-loaded ferritin, was toxic to Timd2-expressing cells. Timd2-expressing xenografts in vivo showed much smaller changes in R2 following injection of iron-loaded ferritin than the same cells incubated in vitro with iron-loaded ferritin., Conclusion: Timd2 has demonstrated potential as an MRI reporter gene, producing large increases in R2 and R1 with ferritin and manganese-loaded ferritin respectively in vitro, although more modest changes in R2 in vivo. Manganese-loaded apoferritin was not used in vivo due to the toxicity observed in vitro. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance., (© 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)
- Published
- 2016
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33. MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model.
- Author
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Serrao EM, Kettunen MI, Rodrigues TB, Dzien P, Wright AJ, Gopinathan A, Gallagher FA, Lewis DY, Frese KK, Almeida J, Howat WJ, Tuveson DA, and Brindle KM
- Subjects
- Animals, Biomarkers metabolism, Carcinoma, Pancreatic Ductal metabolism, Disease Progression, Mice, Mice, Transgenic, Pancreas pathology, Pancreatic Neoplasms metabolism, Pancreatitis diagnosis, Pancreatitis metabolism, Precancerous Conditions metabolism, Pyruvic Acid, Carbon-13 Magnetic Resonance Spectroscopy methods, Carcinoma, Pancreatic Ductal diagnosis, Pancreas metabolism, Pancreatic Neoplasms diagnosis, Precancerous Conditions diagnosis
- Abstract
Objectives: Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging., Design: The concentrations of alanine and lactate and the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were measured in extracts prepared from the pancreas of animals at different stages of disease progression; from pancreatitis, through tissue with predominantly low-grade and then high-grade pancreatic intraepithelial neoplasia and then tumour. (13)C magnetic resonance spectroscopic imaging ((13)C-MRSI) was used to measure non-invasively changes in (13)C labelling of alanine and lactate with disease progression, following injection of hyperpolarised [1-(13)C]pyruvate., Results: Progressive decreases in the alanine/lactate concentration ratio and ALT/LDH activity ratio with disease progression were accompanied by a corresponding decrease in the [1-(13)C]alanine/[1-(13)C]lactate signal ratio observed in (13)C-MRSI images of the pancreas., Conclusions: Metabolic imaging with hyperpolarised [1-(13)C]pyruvate enables detection and monitoring of the progression of PCa precursor lesions. Translation of this MRI technique to the clinic has the potential to improve the management of patients at high risk of developing PCa., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)
- Published
- 2016
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34. Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.
- Author
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Neves AA, Wainman YA, Wright A, Kettunen MI, Rodrigues TB, McGuire S, Hu DE, Bulat F, Geninatti Crich S, Stöckmann H, Leeper FJ, and Brindle KM
- Subjects
- Animals, Gadolinium pharmacokinetics, Glycosylation, Mice, Molecular Probes, Tissue Distribution, Carbohydrates chemistry, Magnetic Resonance Imaging methods
- Abstract
Glycosylation is a ubiquitous post-translational modification, present in over 50% of the proteins in the human genome, with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, including cancer. We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe. Significant N-azidoacetylgalactosamine dependent T1 contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10-fold). This approach has the potential to enable the rapid and non-invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
- Published
- 2016
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35. Carbon dioxide receptor genes and their expression profile in Diabrotica virgifera virgifera.
- Author
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Rodrigues TB, Moriyama EN, Wang H, Khajuria C, and Siegfried BD
- Subjects
- Animals, Gene Expression Regulation, Developmental, Likelihood Functions, Phylogeny, Receptors, Cell Surface metabolism, Coleoptera genetics, Genes, Insect, Insect Proteins genetics, Receptors, Cell Surface genetics, Transcriptome
- Abstract
Background: Diabrotica virgifera virgifera, western corn rootworm, is one of the most devastating species in North America. D. v. virgifera neonates crawl through the soil to locate the roots on which they feed. Carbon dioxide (CO2) is one of the important volatile cues that attract D. v. virgifera larvae to roots., Results: In this study, we identified three putative D. v. virgifera gustatory receptor genes (Dvv_Gr1, Dvv_Gr2, and Dvv_Gr3). Phylogenetic analyses confirmed their orthologous relationships with known insect CO2 receptor genes from Drosophila, mosquitoes, and Tribolium. The phylogenetic reconstruction of insect CO2 receptor proteins and the gene expression profiles were analyzed. Quantitative analysis of gene expression indicated that the patterns of expression of these three candidate genes vary among larval tissues (i.e., head, integument, fat body, and midgut) and different development stages (i.e., egg, three larval stages, adult male and female)., Conclusion: The Dvv_Gr2 gene exhibited highest expression in heads and neonates, suggesting its importance in allowing neonate larvae to orient to its host plant. Similar expression patterns across tissues and developmental stages for Dvv_Gr1 and Dvv_Gr3 suggest a potentially different role. Findings from this study will allow further exploration of the functional role of specific CO2 receptor proteins in D. v. virgifera.
- Published
- 2016
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36. Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.
- Author
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Timm KN, Hartl J, Keller MA, Hu DE, Kettunen MI, Rodrigues TB, Ralser M, and Brindle KM
- Subjects
- Animals, Cell Line, Tumor, Female, Magnetic Resonance Spectroscopy methods, Metabolic Clearance Rate, Mice, Mice, Inbred C57BL, Radiopharmaceuticals pharmacokinetics, Reproducibility of Results, Saccharomyces cerevisiae metabolism, Sensitivity and Specificity, Glucose pharmacokinetics, Glycolysis, Neoplasms, Experimental metabolism, Pentose Phosphate Pathway, Uranium pharmacokinetics
- Abstract
Purpose: A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. (13) C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-(2) H, U-(13) C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG CONCLUSION: The resonance at ∼181 ppm in (13) C MR spectra following injection of hyperpolarized [U-(2) H, U-(13) C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells., (© 2014 Wiley Periodicals, Inc.)
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- 2015
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37. Carbonic Anhydrase Activity Monitored In Vivo by Hyperpolarized 13C-Magnetic Resonance Spectroscopy Demonstrates Its Importance for pH Regulation in Tumors.
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Gallagher FA, Sladen H, Kettunen MI, Serrao EM, Rodrigues TB, Wright A, Gill AB, McGuire S, Booth TC, Boren J, McIntyre A, Miller JL, Lee SH, Honess D, Day SE, Hu DE, Howat WJ, Harris AL, and Brindle KM
- Subjects
- Animals, Antigens, Neoplasm analysis, Antigens, Neoplasm genetics, Bicarbonates metabolism, Carbon Dioxide metabolism, Carbon Isotopes analysis, Carbonic Anhydrase IX, Carbonic Anhydrases analysis, Carbonic Anhydrases genetics, Cell Line, Tumor, Colorectal Neoplasms pathology, Heterografts, Humans, Male, Mice, Mice, Inbred NOD, Mice, SCID, Neoplasm Proteins analysis, Recombinant Fusion Proteins analysis, Tumor Microenvironment, Antigens, Neoplasm physiology, Carbonic Anhydrases physiology, Colorectal Neoplasms metabolism, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Neoplasm Proteins physiology
- Abstract
Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH., (©2015 American Association for Cancer Research.)
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- 2015
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38. Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains.
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Machado TS, Macabelli CH, Sangalli JR, Rodrigues TB, Smith LC, Meirelles FV, and Chiaratti MR
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- Animals, Female, Male, Mice, Mice, Inbred C57BL, Mice, Inbred NZB, DNA, Mitochondrial genetics, Real-Time Polymerase Chain Reaction
- Abstract
Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.
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- 2015
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39. (13) C magnetic resonance spectroscopic imaging of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation can be used to assess aldehyde dehydrogenase activity in vivo.
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Dzien P, Kettunen MI, Marco-Rius I, Serrao EM, Rodrigues TB, Larkin TJ, Timm KN, and Brindle KM
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- Animals, Blood Alcohol Content, Disulfiram pharmacology, Dose-Response Relationship, Drug, Female, Liver drug effects, Liver enzymology, Mice, Oxidation-Reduction drug effects, Predictive Value of Tests, Alcohol Dehydrogenase metabolism, Carbon-13 Magnetic Resonance Spectroscopy methods, Ethanol metabolism
- Abstract
Purpose: Aldehyde dehydrogenase (ALDH2) is an emerging drug target for the treatment of heart disease, cocaine and alcohol dependence, and conditions caused by genetic polymorphisms in ALDH2. Noninvasive measurement of ALDH2 activity in vivo could inform the development of these drugs and accelerate their translation to the clinic., Methods: [1-(13) C, U-(2) H5 ] ethanol was hyperpolarized using dynamic nuclear polarization, injected into mice and its oxidation in the liver monitored using (13) C MR spectroscopy and spectroscopic imaging., Results: Oxidation of [1-(13) C, U-(2) H5 ] ethanol to [1-(13) C] acetate was observed. Saturation of the acetaldehyde resonance, which was below the level of detection in vivo, demonstrated that acetate was produced via acetaldehyde. Irreversible inhibition of ALDH2 activity with disulfiram resulted in a proportional decrease in the amplitude of the acetate resonance., Conclusion: (13) C magnetic resonance spectroscopy measurements of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation allow real-time assessment of ALDH2 activity in liver in vivo., (© 2014 The Authors. Magnetic Resonance in Medicine Published by Wiley Periodicals, Inc. on behalf of International Society of Medicine in Resonance.)
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- 2015
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40. Detection of transgene expression using hyperpolarized 13C urea and diffusion-weighted magnetic resonance spectroscopy.
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Patrick PS, Kettunen MI, Tee SS, Rodrigues TB, Serrao E, Timm KN, McGuire S, and Brindle KM
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- Animals, Carbon Isotopes pharmacokinetics, HEK293 Cells, Humans, Membrane Transport Proteins genetics, Mice, Mice, SCID, Reproducibility of Results, Sensitivity and Specificity, Tissue Distribution, Transgenes genetics, Urea Transporters, Magnetic Resonance Spectroscopy methods, Membrane Transport Proteins metabolism, Urea pharmacokinetics
- Abstract
Purpose: To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [(13) C]urea., Methods: Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [(13) C]urea, a spin echo sequence was used to measure urea concentration, T1 , and diffusion in control and UTB-expressing tissue., Results: The apparent diffusion coefficient of hyperpolarized urea was 21% lower in tissue expressing UTB, in comparison with control tissue (P < 0.05, 1-tailed t-test, n = 6 in each group). No difference in water apparent diffusion coefficient or cellularity between these tissues was found, indicating that they were otherwise similar in composition., Conclusion: Expression of UTB, by mediating cell uptake of urea, lowers the apparent diffusion coefficient of hyperpolarized (13) C urea in tissue and thus the transporter has the potential to be used as a magnetic resonance-based gene reporter in vivo. Magn Reson Med 73:1401-1406, 2015. © 2014 Wiley Periodicals, Inc., (© 2014 Wiley Periodicals, Inc.)
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- 2015
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41. Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: linker effects on the relaxivity.
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Ferreira MF, Gonçalves J, Mousavi B, Prata MI, Rodrigues SP, Calle D, López-Larrubia P, Cerdan S, Rodrigues TB, Ferreira PM, Helm L, Martins JA, and Geraldes CF
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- Animals, Chelating Agents pharmacokinetics, Contrast Media pharmacokinetics, Coordination Complexes pharmacokinetics, Gadolinium pharmacokinetics, Gold pharmacokinetics, Male, Mice, Rats, Wistar, Tissue Distribution, Water chemistry, Chelating Agents chemistry, Contrast Media chemistry, Coordination Complexes chemistry, Gadolinium chemistry, Gold chemistry, Metal Nanoparticles chemistry
- Abstract
The relaxivity displayed by Gd(3+) chelates immobilized onto gold nanoparticles is the result of the complex interplay between the nanoparticle size, the water exchange rate and the chelate structure. In this work we study the effect of the length of ω-thioalkyl linkers, anchoring fast water exchanging Gd(3+) chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd(3+) chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(α-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM(-1) s(-1) (30 MHz, 25 °C), were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles was determined mainly by size. Small nanoparticles (HD = 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD = 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggest that functionalized gold nanoparticles hold great potential for further investigation as MRI contrast agents. This study contributes to a better understanding of the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd(3+) complexes. It is a relevant contribution towards "design rules" for nanostructures functionalized with Gd(3+) chelates as Contrast Agents for MRI and multimodal imaging.
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- 2015
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42. Validation of reference housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera).
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Rodrigues TB, Khajuria C, Wang H, Matz N, Cunha Cardoso D, Valicente FH, Zhou X, and Siegfried B
- Subjects
- Animals, Coleoptera genetics, Gene Expression Profiling, Insect Proteins genetics, Coleoptera metabolism, Gene Expression Regulation physiology, Genes, Essential physiology, Genes, Insect physiology, Insect Proteins biosynthesis
- Abstract
Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.
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- 2014
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43. Oatp1 enhances bioluminescence by acting as a plasma membrane transporter for D-luciferin.
- Author
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Patrick PS, Lyons SK, Rodrigues TB, and Brindle KM
- Subjects
- Animals, Cell Line, Humans, Kinetics, Luciferases metabolism, Mice, SCID, Photons, Benzothiazoles metabolism, Cell Membrane metabolism, Luminescent Measurements, Organic Anion Transporters, Sodium-Independent metabolism
- Abstract
Purpose: Bioluminescence imaging is a powerful tool for studying gene expression and cell migration in intact living organisms. However, production of bioluminescence by cells transfected to express luciferase can be limited by the rate of plasma membrane transport of its substrate D-luciferin. We sought to identify a plasma membrane transporter for D-luciferin that could be expressed alongside luciferase to increase transmembrane flux of its substrate and thereby increase light output., Procedures: Luciferase-expressing cells were transfected with a lentivirus encoding the rat reno-hepatic organic anion transporter protein, Oatp1, which was identified as a potential transporter for D-luciferin. Light output was compared between cells expressing luciferase and those also expressing Oatp1., Results: In two cell lines and in mouse xenografts, co-expression of Oatp1 with luciferase increased light output by several fold, following addition of luciferin., Conclusions: The increase in light output thus obtained will allow more sensitive detection of luciferase-expressing cells in vivo.
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- 2014
- Full Text
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44. Astrocytic-Neuronal-Astrocytic Pathway Selection for Formation and Degradation of Glutamate/GABA.
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Hertz L and Rodrigues TB
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- 2014
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45. Cerebral oedema is not responsible for motor or cognitive deficits in rats with hepatic encephalopathy.
- Author
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Cauli O, Llansola M, Agustí A, Rodrigo R, Hernández-Rabaza V, Rodrigues TB, López-Larrubia P, Cerdán S, and Felipo V
- Subjects
- Animals, Cognition, Disease Models, Animal, Magnetic Resonance Imaging, Male, Maze Learning, Memory, Motor Activity, Portacaval Shunt, Surgical, Radionuclide Imaging, Rats, Rats, Wistar, Brain Edema diagnosis, Cerebellum diagnostic imaging, Hepatic Encephalopathy complications
- Abstract
Background & Aims: Low-grade cytotoxic oedema is considered a main contributor to the neurological (motor and cognitive) alterations in patients with hepatic encephalopathy (HE). This assumption is mainly based on studies with cultured astrocytes treated with very large ammonia concentrations or with animal models of acute liver failure with strong HE. However, the possible contribution of cerebral oedema (vasogenic or cytotoxic) to cognitive or motor alterations in chronic mild HE has not been demonstrated. The aim of this work was to assess whether cerebral oedema contributes to cognitive and/or motor alterations in rats with chronic mild HE., Methods: Motor activity and coordination and different types of learning and memory were assessed in rats with porta-caval shunts (PCS). Brain oedema was assessed by gravimetry in cerebellum and cortex and apparent diffusion coefficient (ADC) by magnetic resonance in 16 areas., Results: Four weeks after surgery, PCS rats show reduced motor activity and coordination, impaired ability to learn a conditional discrimination task in the Y maze and reduced spatial memory in the Morris water maze. PCS rats did not show increased brain water content at 4 or 10 weeks or changes in ADC at 4 weeks. At 10 weeks, increased ADC in some areas is compatible with vasogenic but not cytotoxic oedema., Conclusion: Cerebral oedema is not involved in motor and cognitive alterations in rats (and likely in humans) with mild HE. Proper understanding of the mechanisms responsible for the neurological alterations in HE is necessary to design efficient treatments., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)
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- 2014
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46. Dual-modality gene reporter for in vivo imaging.
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Patrick PS, Hammersley J, Loizou L, Kettunen MI, Rodrigues TB, Hu DE, Tee SS, Hesketh R, Lyons SK, Soloviev D, Lewis DY, Aime S, Fulton SM, and Brindle KM
- Subjects
- Animals, Cell Line, Tumor, Contrast Media chemistry, Female, Gadolinium chemistry, Gadolinium DTPA chemistry, HCT116 Cells, HEK293 Cells, Humans, Image Enhancement methods, Ions, MCF-7 Cells, Mice, Mice, SCID, Microscopy, Fluorescence methods, Neoplasm Transplantation, Organic Anion Transporters metabolism, Tomography, Emission-Computed, Single-Photon methods, Genes, Reporter, Magnetic Resonance Imaging methods
- Abstract
The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.
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- 2014
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47. Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose.
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Rodrigues TB, Serrao EM, Kennedy BW, Hu DE, Kettunen MI, and Brindle KM
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- Animals, Carbon Isotopes metabolism, Glucose metabolism, Lactic Acid metabolism, Lung Neoplasms diagnosis, Lymphoma diagnosis, Mice, Pentose Phosphate Pathway physiology, Glycolysis physiology, Lung Neoplasms physiopathology, Lymphoma physiopathology, Magnetic Resonance Spectroscopy methods
- Abstract
In this study, we monitored glycolysis in mouse lymphoma and lung tumors by measuring the conversion of hyperpolarized [U-2H, U-13C]glucose to lactate using 13C magnetic resonance spectroscopy and spectroscopic imaging. We observed labeled lactate only in tumors and not in surrounding normal tissue or other tissues in the body and found that it was markedly decreased at 24 h after treatment with a chemotherapeutic drug. We also detected an increase in a resonance assigned to 6-phosphogluconate in the pentose phosphate pathway. This technique could provide a new way of detecting early evidence of tumor treatment response in the clinic and of monitoring tumor pentose phosphate pathway activity.
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- 2014
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48. (13)C NMR spectroscopy applications to brain energy metabolism.
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Rodrigues TB, Valette J, and Bouzier-Sore AK
- Abstract
(13)C nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying brain metabolism. Indeed, the most convincing data obtained to decipher metabolic exchanges between neurons and astrocytes have been obtained using this technique, thus illustrating its power. It may be difficult for non-specialists, however, to grasp thefull implication of data presented in articles written by spectroscopists. The aim of the review is, therefore, to provide a fundamental understanding of this topic to facilitate the non-specialists in their reading of this literature. In the first part of this review, we present the metabolic fate of (13)C-labeled substrates in the brain in a detailed way, including an overview of some general neurochemical principles. We also address and compare the various spectroscopic strategies that can be used to study brain metabolism. Then, we provide an overview of the (13)C NMR experiments performed to analyze both intracellular and intercellular metabolic fluxes. More particularly, the role of lactate as a potential energy substrate for neurons is discussed in the light of (13)C NMR data. Finally, new perspectives and applications offered by (13)C hyperpolarization are described.
- Published
- 2013
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49. Hyperpolarized singlet lifetimes of pyruvate in human blood and in the mouse.
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Marco-Rius I, Tayler MC, Kettunen MI, Larkin TJ, Timm KN, Serrao EM, Rodrigues TB, Pileio G, Ardenkjaer-Larsen JH, Levitt MH, and Brindle KM
- Subjects
- Animals, Cattle, Cell Line, Tumor, Female, Humans, Magnetic Fields, Mice, Mice, Inbred C57BL, Serum Albumin, Bovine metabolism, Solutions, Time Factors, Magnetic Resonance Spectroscopy, Pyruvic Acid blood
- Abstract
Hyperpolarized NMR is a promising technique for non-invasive imaging of tissue metabolism in vivo. However, the pathways that can be studied are limited by the fast T1 decay of the nuclear spin order. In metabolites containing pairs of coupled nuclear spins-1/2, the spin order may be maintained by exploiting the non-magnetic singlet (spin-0) state of the pair. This may allow preservation of the hyperpolarization in vivo during transport to tissues of interest, such as tumors, or to detect slower metabolic reactions. We show here that in human blood and in a mouse in vivo at millitesla fields the (13)C singlet lifetime of [1,2-(13)C2]pyruvate was significantly longer than the (13)C T1, although it was shorter than the T1 at field strengths of several tesla. We also examine the singlet-derived NMR spectrum observed for hyperpolarized [1,2-(13)C2]lactate, originating from the metabolism of [1,2-(13)C2]pyruvate., (© 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.)
- Published
- 2013
- Full Text
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50. Increased oxidative metabolism and neurotransmitter cycling in the brain of mice lacking the thyroid hormone transporter SLC16A2 (MCT8).
- Author
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Rodrigues TB, Ceballos A, Grijota-Martínez C, Nuñez B, Refetoff S, Cerdán S, Morte B, and Bernal J
- Subjects
- Animals, Enzyme Activation, Gene Knockout Techniques, Hyperthyroidism metabolism, Iodide Peroxidase metabolism, Male, Mice, Monocarboxylic Acid Transporters, Oxidation-Reduction, Symporters, Thyroid Hormones metabolism, Iodothyronine Deiodinase Type II, Brain metabolism, Membrane Transport Proteins deficiency, Membrane Transport Proteins genetics, Neurotransmitter Agents metabolism
- Abstract
Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T4 and T3) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T4 and T3. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-(13)C) glucose and brain extracts prepared and analyzed by (13)C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T3 by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.
- Published
- 2013
- Full Text
- View/download PDF
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