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Adults with metabolically healthy overweight or obesity present more brown adipose tissue and higher thermogenesis than their metabolically unhealthy counterparts
Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
- Jurado Fasoli, Lucas
- Sánchez Delgado, Guillermo
- Alcántara Alcántara, Juan Manuel
- Acosta, Francisco M.
- Sánchez Sánchez, Rocío
- Labayen Goñi, Idoia
- Ortega, Francisco B.
- Martínez Téllez, Borja
- Ruiz, Jonatan R.
Background: There is a subset of individuals with overweight/obesity characterized by a lower risk of cardiometabolic complications, the so-called metabolically healthy overweight/obesity (MHOO) phenotype. Despite the relatively higher levels of subcutaneous adipose tissue and lower visceral adipose tissue observed in individuals with MHOO than individuals with metabolically unhealthy overweight/obesity (MUOO), little is known about the differences in brown adipose tissue (BAT). Methods: This study included 53 young adults (28 women) with a body mass index (BMI) ¿25 kg/m2 which were classified as MHOO (n = 34) or MUOO (n = 19). BAT was assessed through a static 18F-FDG positron emission tomography/computed tomography scan after a 2-h personalized cooling protocol. Energy expenditure, skin temperature, and thermal perception were assessed during a standardized mixed meal test (3.5 h) and a 1-h personalized cold exposure. Body composition was assessed by dual-energy x-ray absorptiometry, energy intake was determined during an ad libitum meal test and dietary recalls, and physical activity levels were determined by a wrist-worn accelerometer. Findings: Participants with MHOO presented higher BAT volume (+124%, P = 0.008), SUVmean (+63%, P = 0.001), and SUVpeak (+133%, P = 0.003) than MUOO, despite having similar BAT mean radiodensity (P = 0.354). In addition, individuals with MHOO exhibited marginally higher meal-induced thermogenesis (P = 0.096) and cold-induced thermogenesis (+158%, P = 0.050). Moreover, MHOO participants showed higher supraclavicular skin temperature than MUOO during the first hour of the postprandial period and during the cold exposure, while no statistically significant differences were observed in other skin temperature parameters. We observed no statistically significant differences between MHOO and MUOO in thermal perception, body composition, outdoor ambient temperature exposure, resting metabolic rate, energy intake, or physical activity levels. Interpretation: Adults with MHOO present higher BAT volume and activity than MUOO. The higher meal- and cold-induced thermogenesis and cold-induced supraclavicular skin temperature are compatible with a higher BAT activity. Overall, these results suggest that BAT presence and activity might be linked to a healthier phenotype in young adults with overweight or obesity., The study was supported by the Junta de Andalucía, Consejería de Transformación Económica, Industria, Conocimiento y Universidades Dirección General de Investigación y Transferencia del Conocimiento (ref. P18-RT-4455, ref. SOMM17/6107/UGR, and DOC 01151) and European Regional Development Funds (ERDF), the Spanish Ministry of Economy and Competitiveness via the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI13/01393), and PTA-12264, Retos de la Sociedad (DEP2016-79512-R), the Spanish Ministry of Education (FPU13/04365, FPU15/04059 and FPU19/01609), the Fundación Iberoamericana de Nutrición (FINUT), the Redes Temáticas de Investigación Cooperativa RETIC (Red SAMID RD16/0022), the AstraZeneca HealthCare Foundation, the University of Granada Plan Propio de Investigación 2016 -Excellence actions: Unit of Excellence on Exercise and Health (UCEES). G.SD is supported by a Marie Sklodowska-Curie Actions-Individual Fellowship (101028941; Horizon 2020, European Commission). B.MT is supported by a grant for the requalification of the Spanish university system from the Ministry of Universities of the Government of Spain, funded by the European Union, NextGeneration EU (María Zambrano program, reference RR _C_2021_04). J.M.A.A. is supported by the Juan de la Cierva-Formación Grant FJC2020-044453-I funded by MCIN/AEI/10.13039/501100011033 and 'European Union NextGeneration EU/PRTR'
Reproducibility of the energy metabolism response to an oral glucose tolerance test: influence of a postcalorimetric correction procedure
Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
- Alcántara Alcántara, Juan Manuel
- Sánchez Delgado, Guillermo
- Jurado Fasoli, Lucas
- Galgani, Jose E.
- Labayen Goñi, Idoia
- Ruiz, Jonatan R.
Purpose Metabolic fexibility (MetF), which is a surrogate of metabolic health, can be assessed by the change in the respiratory exchange ratio (RER) in response to an oral glucose tolerance test (OGTT). We aimed to determine the day-to-day
reproducibility of the energy expenditure (EE) and RER response to an OGTT, and whether a simulation-based postcalorimetric correction of metabolic cart readouts improves day-to-day reproducibility.
Methods The EE was assessed (12 young adults, 6 women, 27±2 years old) using an Omnical metabolic cart (Maastricht
Instruments, Maastricht, The Netherlands) after an overnight fast (12 h) and after a 75-g oral glucose dose on 2 separate
days (48 h). On both days, we assessed EE in 7 periods (one 30-min baseline and six 15-min postprandial). The ICcE was
performed immediately after each recording period, and capillary glucose concentration (using a digital glucometer) was
determined.
Results We observed a high day-to-day reproducibility for the assessed RER (coefcients of variation [CV]<4%) and EE
(CVs<9%) in the 7 diferent periods. In contrast, the RER and EE areas under the curve showed a low day-to-day reproducibility (CV=22% and 56%, respectively). Contrary to our expectations, the postcalorimetric correction procedure did
not infuence the day-to-day reproducibility of the energy metabolism response, possibly because the Omnical’s accuracy
was~100%.
Conclusion Our study demonstrates that the energy metabolism response to an OGTT is poorly reproducible (CVs>20%)
even using a very accurate metabolic cart. Furthermore, the postcalorimetric correction procedure did not infuence the
day-to-day reproducibility.
Trial registration NCT04320433; March 25, 2020., Funding for open access charge: Universidad de Granada / CBUA. Supported by the Spanish Ministry of Economy and Competitiveness via Retos de la Sociedad grant DEP2016-79512-R (to JRR), and European Regional Development Funds (ERDF); Spanish Ministry of Education grant (FPU15/04059 to JMAA; and FPU19/01609 to LJ-F); the University of Granada Plan Propio de Investigación 2016-Excellence actions: Unit of Excellence on Exercise and Health (to JRR)—Plan Propio de Investigación 2018 and 2020 Programa Contratos-Puente and Programa Perfeccionamiento de Doctores (to GS-D, and to JMAA respectively); Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades grant SOMM17/6107/UGR (to JRR) via the ERDF; and the Fundación Alfonso Martín Escudero (to GS-D).
reproducibility of the energy expenditure (EE) and RER response to an OGTT, and whether a simulation-based postcalorimetric correction of metabolic cart readouts improves day-to-day reproducibility.
Methods The EE was assessed (12 young adults, 6 women, 27±2 years old) using an Omnical metabolic cart (Maastricht
Instruments, Maastricht, The Netherlands) after an overnight fast (12 h) and after a 75-g oral glucose dose on 2 separate
days (48 h). On both days, we assessed EE in 7 periods (one 30-min baseline and six 15-min postprandial). The ICcE was
performed immediately after each recording period, and capillary glucose concentration (using a digital glucometer) was
determined.
Results We observed a high day-to-day reproducibility for the assessed RER (coefcients of variation [CV]<4%) and EE
(CVs<9%) in the 7 diferent periods. In contrast, the RER and EE areas under the curve showed a low day-to-day reproducibility (CV=22% and 56%, respectively). Contrary to our expectations, the postcalorimetric correction procedure did
not infuence the day-to-day reproducibility of the energy metabolism response, possibly because the Omnical’s accuracy
was~100%.
Conclusion Our study demonstrates that the energy metabolism response to an OGTT is poorly reproducible (CVs>20%)
even using a very accurate metabolic cart. Furthermore, the postcalorimetric correction procedure did not infuence the
day-to-day reproducibility.
Trial registration NCT04320433; March 25, 2020., Funding for open access charge: Universidad de Granada / CBUA. Supported by the Spanish Ministry of Economy and Competitiveness via Retos de la Sociedad grant DEP2016-79512-R (to JRR), and European Regional Development Funds (ERDF); Spanish Ministry of Education grant (FPU15/04059 to JMAA; and FPU19/01609 to LJ-F); the University of Granada Plan Propio de Investigación 2016-Excellence actions: Unit of Excellence on Exercise and Health (to JRR)—Plan Propio de Investigación 2018 and 2020 Programa Contratos-Puente and Programa Perfeccionamiento de Doctores (to GS-D, and to JMAA respectively); Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades grant SOMM17/6107/UGR (to JRR) via the ERDF; and the Fundación Alfonso Martín Escudero (to GS-D).
Eating behavior, physical activity and exercise training: a randomized controlled trial in young healthy adults
Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
- Martínez Ávila, Wendy D.
- Sánchez Delgado, Guillermo
- Acosta, Francisco M.
- Jurado Fasoli, Lucas
- Oustric, Pauline
- Labayen Goñi, Idoia
- Blundell, John E.
- Ruiz, Jonatan R.
Incluye material complementario, Regular physical activity (PA) is an important part of the treatment of several medical conditions, including overweight and obesity, in which there may be a weakened appetite control. Eating behaviour traits influence weight control and may be different in active and sedentary subjects. This paper reports the relationships between the time spent in sedentary behaviour and physical activity (PA) of different intensity, and eating behaviour traits in young, healthy adults. Additionally, it reports the results of a six-month-long, randomized, controlled trial to examine the effect of an exercise intervention on eating behaviour traits. A total of 139 young (22.06 ± 2.26 years) healthy adults (68.35% women) with a Body Mass Index (BMI) of 24.95 ± 4.57 kg/m2 were enrolled. Baseline assessments of habitual PA were made using wrist-worn triaxial accelerometers; eating behaviour traits were examined via the self-reported questionnaires: Binge Eating, Three-Factor Eating Questionnaire-R18 and Control of Eating Questionnaire. The subjects were then randomly assigned to one of three groups: control (usual lifestyle), moderate-intensity exercise (aerobic and resistance training 3¨C4 days/week at a heart rate equivalent to 60% of the heart rate reserve (HRres) for the aerobic component, and at 50% of the 1 repetition maximum (RM) for the resistance component), or vigorous-intensity exercise (the same training but at 80% HRres for half of the aerobic training, and 70% RM for the resistance training). At baseline, sedentary behaviour was inversely associated with binge eating (r = −0.181, p < 0.05) and with uncontrolled eating (r = −0.286, p = 0.001). Moderate PA (MPA) was inversely associated with craving control (r = −0.188, p <0.05). Moderate-to-vigorous PA (MVPA) was directly associated with binge eating (r = 0.302, p < 0.001) and uncontrolled eating (r = 0.346, p < 0.001), and inversely associated with craving control (r = −0.170, p < 0.015). Overall, PA was directly associated with binge eating (r = 0.275, p = 0.001), uncontrolled eating (r = 0.321, p < 0.001) and emotional eating (r = 0.204, p < 0.05). Additionally, only emotional eating was modified by the intervention, increasing in the vigorous-intensity exercise group (p < 0.05). In summary, we observed that time spent in sedentary behaviour/PA of different intensity is associated with eating behaviour traits, especially binge eating in young adults. In contrast, the six-month exercise intervention did not lead to appreciable changes in eating behaviour traits., Funding for this work came from the Spanish Ministry of Economy and Competitiveness via the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI13/01393) and PTA 12264-I, Retos de la Sociedad (DEP2016-79512-R) and European Regional Development Fund (ERDF), the Spanish Ministry of Education (FPU 13/04365 and FPU19/01609), the Fundación Iberoamericana de Nutrición (FINUT), the Redes Temáticas de Investigación Cooperativa (RETIC) (Red SAMID RD16/0022), the AstraZeneca HealthCare Foundation, the University of Granada Research and Knowledge Transfer Fund (PPIT) 2016 Excellence Actions Programme: Scientific Unit of Excellence on Exercise and Health (UCEES) and the Research and Knowledge Transfer Fund (PPIT) 2018 Bridging Contracts Programme and the Andalusian Regional Government, Regional Ministry of Economy, Knowledge, Enterprises and University (ERDF, SOMM17/6107/UGR). Also, international doctoral studies scholarship no. 440575 from the Mexican National Council of Science and Technology (CONACYT).
Impact of methods for data selection on the day-to-day reproducibility of resting metabolic rate assessed with four different metabolic carts
Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
- Alcántara Alcántara, Juan Manuel
- Jurado Fasoli, Lucas
- Dote-Montero, Manuel
- Merchán Ramírez, Elisa
- Amaro Gahete, Francisco J.
- Labayen Goñi, Idoia
- Ruiz, Jonatan R.
- Sánchez Delgado, Guillermo
Background and aims: Accomplishing a high day-to-day reproducibility is important to detect changes in resting metabolic rate (RMR) and respiratory exchange ratio (RER) that may be produced after an intervention or for monitoring patients’ metabolism over time. We aimed to analyze: (i) the influence of different methods for selecting indirect calorimetry data on RMR and RER assessments; and, (ii) whether these methods influence RMR and RER day-to-day reproducibility. Methods and results: Twenty-eight young adults accomplished 4 consecutive RMR assessments (30-min each), using the Q-NRG (Cosmed, Rome, Italy), the Vyntus CPX (Jaeger-CareFusion, Höchberg, Germany), the Omnical (Maastricht Instruments, Maastricht, The Netherlands), and the Ultima CardiO2 (Medgraphics Corporation, St. Paul, Minnesota, USA) carts, on 2 consecutive mornings. Three types of methods were used: (i) short (periods of 5 consecutive minutes; 6e10, 11e15, 16e20, 21e25, and 26e30 min) and long time intervals (TI) methods (6e25 and 6 e30 min); (ii) steady state (SSt methods); and, (iii) methods filtering the data by thresholding from the mean RMR (filtering methods). RMR and RER were similar when using different methods (except RMR for the Vyntus and RER for the Q-NRG). Conversely, using different methods impacted RMR (all P 0.037) and/or RER (P 0.009) day-to-day reproducibility in all carts. The 6e25 min and the 6e30 min long TI methods yielded more reproducible measurements for all metabolic carts.
Conclusion: The 6e25 min and 6e30 min should be the preferred methods for selecting data, as they result in the highest day-to-day reproducibility of RMR and RER assessments., This work was supported by the Spanish Ministry of Economy and Competitiveness via Retos de la Sociedad grant DEP2016-79512-R (to JRR), and European Regional Development Funds (ERDF); Spanish Ministry of Education grant (FPU15/04059 to JMAA; FPU19/01609 to LJ-F; and FPU18/03357 to MD-M); the University of Granada Plan Propio de Investigación 2016-Excellence actions: Unit of Excellence on Exercise and Health (to JRR); the University of Granada Plan Propio 2020 and 2018 Programa Contratos-Puente (to JMA and GS-D, respectively), and Programa Perfeccionamiento de Doctores (to GS-D); Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades grant SOMM17/6107/UGR (to JRR) via the ERDF; Grant FJC2020-044453-I funded by MCIN/AEI/10.13039/501100011033 and by “European Union NextGenerationEU/PRTR” (to JMA); the Fundación Alfonso Martín Escudero (to GS-D); and a Marie Skłodowska-Curie Actions-Individual Fellowship grant (Horizon2020, 101028941, to GS-D).
Conclusion: The 6e25 min and 6e30 min should be the preferred methods for selecting data, as they result in the highest day-to-day reproducibility of RMR and RER assessments., This work was supported by the Spanish Ministry of Economy and Competitiveness via Retos de la Sociedad grant DEP2016-79512-R (to JRR), and European Regional Development Funds (ERDF); Spanish Ministry of Education grant (FPU15/04059 to JMAA; FPU19/01609 to LJ-F; and FPU18/03357 to MD-M); the University of Granada Plan Propio de Investigación 2016-Excellence actions: Unit of Excellence on Exercise and Health (to JRR); the University of Granada Plan Propio 2020 and 2018 Programa Contratos-Puente (to JMA and GS-D, respectively), and Programa Perfeccionamiento de Doctores (to GS-D); Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades grant SOMM17/6107/UGR (to JRR) via the ERDF; Grant FJC2020-044453-I funded by MCIN/AEI/10.13039/501100011033 and by “European Union NextGenerationEU/PRTR” (to JMA); the Fundación Alfonso Martín Escudero (to GS-D); and a Marie Skłodowska-Curie Actions-Individual Fellowship grant (Horizon2020, 101028941, to GS-D).
No evidence of brown adipose tissue activation after 24 weeks of supervised exercise training in young sedentary adults in the ACTIBATE randomized controlled trial
Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
- Martínez Téllez, Borja
- Sánchez Delgado, Guillermo
- Acosta, Francisco M.
- Alcántara Alcántara, Juan Manuel
- Amaro Gahete, Francisco J.
- Martínez Ávila, Wendy D.
- Merchán Ramírez, Elisa
- Muñoz-Hernández, Victoria
- Osuna Prieto, Francisco J.
- Jurado Fasoli, Lucas
- Xu, Huiwen
- Ortiz Álvarez, Lourdes
- Arias Téllez, María J.
- Méndez Gutiérrez, Andrea
- Labayen Goñi, Idoia
- Ortega, Francisco B.
- Schönke, Milena
- Rensen, Patrick C. N
- Aguilera, Concepción María
- Llamas Elvira, José M.
- Gil, Ángel
- Ruiz, Jonatan R.
Exercise modulates both brown adipose tissue (BAT) metabolism and white
adipose tissue (WAT) browning in murine models. Whether this is true in
humans, however, has remained unknown. An unblinded randomized controlled trial (ClinicalTrials.gov ID: NCT02365129) was therefore conducted to
study the effects of a 24-week supervised exercise intervention, combining
endurance and resistance training, on BAT volume and activity (primary outcome). The study was carried out in the Sport and Health University Research
Institute and the Virgen de las Nieves University Hospital of the University of
Granada (Spain). One hundred and forty-five young sedentary adults were
assigned to either (i) a control group (no exercise, n = 54), (ii) a moderate
intensity exercise group (MOD-EX, n = 48), or (iii) a vigorous intensity exercise
group (VIG-EX n = 43) by unrestricted randomization. No relevant adverse
events were recorded. 97 participants (34 men, 63 women) were included in
the final analysis (Control; n = 35, MOD-EX; n = 31, and VIG-EX; n = 31). We
observed no changes in BAT volume (Δ Control: −22.2 ± 52.6 ml; Δ MOD-EX:
−15.5 ± 62.1 ml, Δ VIG-EX: −6.8 ± 66.4 ml; P = 0.771) or 18F-fluorodeoxyglucose
uptake (SUVpeak Δ Control: −2.6 ± 3.1 ml; Δ MOD-EX: −1.2 ± 4.8, Δ VIG-EX:
−2.2 ± 5.1; p = 0.476) in either the control or the exercise groups. Thus, we did
not find any evidence of an exercise-induced change on BAT volume or activity
in young sedentary adults., This study was funded by the Spanish Ministry of Economy and Competitiveness via the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI13/01393; J.R.R.) and PTA-12264I, Retos de la Sociedad (DEP2016-79512-R; J.R.R.) and European Regional Development Funds (ERDF; J.R.R.), the Spanish Ministry of Education (FPU13/04365 (G.S.D.), FPU14/04172 (F.A.G.), FPU15/04059 (J.M.A.), FPU16/03653 (A.M.G.), FPU16/02828 (F.J.O.P.), FPU16/05159 (H.X.), FPU17/01523 (L.O.A.), FPU19/01609 (L.J.F.)), International Doctoral Studies Scholarship no. 440575 from the Mexican National Council of Science and Technology (CONACYT; WDMA), the Fundación Iberoamericana de Nutrición (FINUT; JRR), the Redes Temáticas de Investigación Cooperativa RETIC (Red SAMID RD16/0022; J.R.R.), the AstraZeneca HealthCare Foundation (J.R.R.), the University of Granada Plan Propio de Investigación 2016 -Excellence actions: Unit of Excellence on Exercise and Health (UCEES) (J.R.R.)- and Plan Propio de Investigación 2018 - Programa Contratos-Puente and Programa Perfecionamiento de Doctores (G.S.D.), the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades (ERDF; ref. SOMM17/6107/UGR; JRR), the Junta de Andalucía, Consejería de Economía, Conocimiento, Empresas y Universidad (ref. P18-RT-4455; J.R.R.), the Fundación Alfonso Martín Escudero (B.M.T. and G.S.D.), the Maria Zambrano fellowship by the Ministerio de Universidades y la Unión Europea NextGenerationEU (RR_C_2021_04; B.M.T.), and the Novo Nordisk Foundation (NNF18OC0032394; M.S.).
adipose tissue (WAT) browning in murine models. Whether this is true in
humans, however, has remained unknown. An unblinded randomized controlled trial (ClinicalTrials.gov ID: NCT02365129) was therefore conducted to
study the effects of a 24-week supervised exercise intervention, combining
endurance and resistance training, on BAT volume and activity (primary outcome). The study was carried out in the Sport and Health University Research
Institute and the Virgen de las Nieves University Hospital of the University of
Granada (Spain). One hundred and forty-five young sedentary adults were
assigned to either (i) a control group (no exercise, n = 54), (ii) a moderate
intensity exercise group (MOD-EX, n = 48), or (iii) a vigorous intensity exercise
group (VIG-EX n = 43) by unrestricted randomization. No relevant adverse
events were recorded. 97 participants (34 men, 63 women) were included in
the final analysis (Control; n = 35, MOD-EX; n = 31, and VIG-EX; n = 31). We
observed no changes in BAT volume (Δ Control: −22.2 ± 52.6 ml; Δ MOD-EX:
−15.5 ± 62.1 ml, Δ VIG-EX: −6.8 ± 66.4 ml; P = 0.771) or 18F-fluorodeoxyglucose
uptake (SUVpeak Δ Control: −2.6 ± 3.1 ml; Δ MOD-EX: −1.2 ± 4.8, Δ VIG-EX:
−2.2 ± 5.1; p = 0.476) in either the control or the exercise groups. Thus, we did
not find any evidence of an exercise-induced change on BAT volume or activity
in young sedentary adults., This study was funded by the Spanish Ministry of Economy and Competitiveness via the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI13/01393; J.R.R.) and PTA-12264I, Retos de la Sociedad (DEP2016-79512-R; J.R.R.) and European Regional Development Funds (ERDF; J.R.R.), the Spanish Ministry of Education (FPU13/04365 (G.S.D.), FPU14/04172 (F.A.G.), FPU15/04059 (J.M.A.), FPU16/03653 (A.M.G.), FPU16/02828 (F.J.O.P.), FPU16/05159 (H.X.), FPU17/01523 (L.O.A.), FPU19/01609 (L.J.F.)), International Doctoral Studies Scholarship no. 440575 from the Mexican National Council of Science and Technology (CONACYT; WDMA), the Fundación Iberoamericana de Nutrición (FINUT; JRR), the Redes Temáticas de Investigación Cooperativa RETIC (Red SAMID RD16/0022; J.R.R.), the AstraZeneca HealthCare Foundation (J.R.R.), the University of Granada Plan Propio de Investigación 2016 -Excellence actions: Unit of Excellence on Exercise and Health (UCEES) (J.R.R.)- and Plan Propio de Investigación 2018 - Programa Contratos-Puente and Programa Perfecionamiento de Doctores (G.S.D.), the Junta de Andalucía, Consejería de Conocimiento, Investigación y Universidades (ERDF; ref. SOMM17/6107/UGR; JRR), the Junta de Andalucía, Consejería de Economía, Conocimiento, Empresas y Universidad (ref. P18-RT-4455; J.R.R.), the Fundación Alfonso Martín Escudero (B.M.T. and G.S.D.), the Maria Zambrano fellowship by the Ministerio de Universidades y la Unión Europea NextGenerationEU (RR_C_2021_04; B.M.T.), and the Novo Nordisk Foundation (NNF18OC0032394; M.S.).