Impact of Maternal Weight Gain on the Newborn Metabolome.

Pre-pregnancy obesity and excessive gestational weight gain (GWG) appear to affect birth weight and the offspring's risk of obesity and disease later in life. However, the identification of the mediators of this relationship, could be of clinical interest, taking into account the presence of other confounding factors, such as genetics and other shared influences. The aim of this study was to evaluate the metabolomic profiles of infants at birth (cord blood) and 6 and 12 months after birth to identify offspring metabolites associated with maternal GWG. Nuclear Magnetic Resonance (NMR) metabolic profiles were measured in 154 plasma samples from newborns (82 cord blood samples) and in 46 and 26 of these samples at 6 months and 12 months of age, respectively. The levels of relative abundance of 73 metabolomic parameters were determined in all the samples. We performed univariate and machine-learning analysis of the association between the metabolic levels and maternal weight gain adjusted for mother's age, Body Mass Index (BMI), diabetes, diet adherence and infant sex. Overall, our results showed differences, both at the univariate level and in the machine-learning models, between the offspring, according to the tertiles of maternal weight gain. Some of these differences were resolved at 6 and 12 months of age, whereas some others remained. Lactate and leucine were the metabolites with the strongest and longest association with maternal weight gain during pregnancy. Leucine, as well as other significant metabolites, have been associated in the past with metabolic wellness in both general and obese populations. Our results suggest that the metabolic changes associated to excessive GWG are present in children from early life.

Dynamic balance improvement in children with Autism Spectrum Disorder after an extracurricular Service-Learning Physical Education program.

This study aimed to examine the acute changes in dynamic balance Postural Control experienced by children with Autism Spectrum Disorder (ASD) who undertook a 6-month extracurricular Service-Learning Physical Education (PE) program. The study used a quasi-experimental design with 23 participants divided into an experimental group and a control group. Limits of Stability protocol was used to measure the children's postural control. The results showed that the experimental group achieved statistically significant improvements. To conclude, this study provides substantial input about how extracurricular PE activities aimed at developing the general motor proficiency of ASD children can improve their dynamic balance.

La selección de atletas élite desde edad temprana: reflexión desde la perspectiva de los sistemas complejos / A seleção de atletas de elite desde cedo: reflexão a partir da perspectiva de sistemas complexos / The selection of elite athletes from an early age: reflection from the perspective of complex systems

RESUMEN Introducción: En este estudio, se examinó la idoneidad de seleccionar atletas élite desde una edad temprana. Objetivo: El objetivo de este estudio consistió en verificar si el comportamiento identificado en investigaciones antecedentes también ocurre entre los mejores atletas mundiales de todos los tiempos que compiten en la carrera de 100 metros. Materiales y métodos: Para ello, y con base en un análisis de las clasificaciones oficiales de todos los tiempos masculinos y femeninos de la Asociación Internacional de Federaciones de atletismo en la referida prueba, se utilizó muestras en categoría absoluta (Top-1,000 hasta el 31/12/2018) y categoría sub-20 (Top-100 hasta el 31/12/2003); se aplicaron leyes de potencia y otras estrategias típicas de los sistemas complejos para analizar los datos. Resultados: los resultados muestran cómo, con demasiada frecuencia, los mejores corredores masculinos y femeninos del mundo en la categoría sub-20 no evolucionan positivamente durante el resto de su carrera deportiva mejorando sus registros en las categorías superiores. Solo 51 hombres de los 100 mejores corredores sub-20 de todos los tiempos, lograron mejorar sus registros una vez que alcanzaron la categoría sénior, y las mejoras no presentaron siempre progresiones estadísticamente significativas. En consecuencia, una inversión excesiva en recursos humanos y materiales para identificar individuos con altas capacidades atléticas, no es siempre la mejor estrategia. Conclusiones: el deporte de nivel a edades tempranas puede ser un error metodológico que debe evitarse a la hora de construir las reservas deportivas de un país o federación deportiva.

RESUMO Introdução: Neste estudo, examinou-se a adequação da seleção de atletas de elite desde tenra idade. Objetivo: O objetivo deste estudo foi verificar se o comportamento identificado em pesquisas anteriores também ocorre entre os melhores atletas do mundo de todos os tempos que competem na prova de 100 metros. Materiais e métodos: Para isso, e com base na análise das classificações oficiais de todos os tempos para homens e mulheres da Associação Internacional de Federações de Atletismo na prova supracitada, foram utilizadas amostras em categoria absoluta (Top-1.000 até 31/12 /2018) e sub-20 (Top-100 até 31/12/2003); Leis de potência e outras estratégias típicas de sistemas complexos foram aplicadas para analisar os dados. Resultados: Os resultados mostram como, muitas vezes, os melhores corredores masculinos e femininos do mundo na categoria sub-20 não evoluem positivamente durante o resto de sua carreira esportiva, melhorando seus registros nas categorias superiores. Apenas 51 homens dos 100 melhores corredores sub-20 de todos os tempos conseguiram melhorar seus tempos quando chegaram à categoria sênior, e as melhorias nem sempre mostraram progressões estatisticamente significativas. Consequentemente, um investimento excessivo em recursos humanos e materiais para identificar indivíduos com altas habilidades atléticas nem sempre é a melhor estratégia. Conclusões: o esporte de nível em idade precoce pode ser um erro metodológico que deve ser evitado na construção das reservas esportivas de um país ou federação esportiva.

ABSTRACT Introduction: In this study, the suitability of selecting elite athletes from an early age was examined. Objective: The objective of this study was to verify if the behavior identified in previous research also occurs among the world's best athletes of all time who compete in the 100-meter race. Materials and methods: For this, and based on an analysis of the official classifications of all times for men and women of the International Association of Athletics Federations in the referred test, samples were used in absolute category (Top-1,000 up to the 12/31/2018) and sub -20 category (Top - 100 until 12/31/2003); power laws and other strategies typical of complex systems will be applied to analyze the data. Results: The results show how, all too often, the best male and female runners in the world in the under-20 category do not evolve positively during the rest of their sports career. improving their records in the higher categories. Only 51 men out of the top 100 U-20 runners of all time managed to improve their times once they reached the senior category, and the improvements did not always show statistically significant progressions. Consequently, an excessive investment in human and material resources to identify individuals with high athletic abilities is not always the best strategy. Conclusions: level sport at an early age can be a methodological error that should be avoided when building the sports reserves of a country or sports federation.

How Can Service-Learning Shape the Political Perspectives of Pre-Service Teachers? A Program in the Field of Physical Education.

Active and democratic citizenship promotion has become a critical challenge for higher education, and civic engagement is a fundamental axis not only in education but also in fostering democratic systems. Consequently, teacher educators held a prominent role through their own teaching practices to generate contexts promoting critical thinking, skills and attitudes. The aim of this study was to analyze the learning related to the political dimension developed by pre-service teachers (n = 123) after participating in a Service-Learning program through physical education with children at risk or/and student with educational needs. This research followed a qualitative research approach and we based the analysis of reflective diaries on Gorham's (2005) categories regarding political learning: Critical political thinking, Interest in politics, Deliberation and Political judgment. The findings show a development in learning such as civic attitudes, critical political thinking, awareness of social justice problems, increased civic compromise and responsibility. The Service-Learning program, therefore, may have been an adequate option to develop pre-service teachers' learning related to a political perspective. Therefore, these findings let us understand how Service-Learning may foster equity and social justice among future teachers.

A Longitudinal Study of the Effects of Service-Learning on Physical Education Teacher Education Students.

Research examining Service-Learning (SL) in Physical Education Teacher Education (PETE) is ample. However, long-term investigations are still scarce and literature demands the application of this type of design to uncover the effects of SL on the long run. This study followed a longitudinal quantitative approach; thus, the participants completed the Civic Attitudes and Skills Questionnaire (CASQ) in three occasions (pretest-postest1-postest2). Results show that there exist significant differences between mean values of the global outcomes of the CASQ; concretely, there was an improvement in the first interval followed by a decrease in the second period. Regarding the different dimensions of the CASQ, leadership skills, attitudes towards social justice and attitudes towards diversity showed significant differences too. This research leads towards better understanding of methodological strategies promoting quality education, positing SL as an adequate possibility in this respect, also in the long term.

Behaviour of salivary testosterone and cortisol in men during an Ironman Triathlon.

Endurance exercise induces notable acute hormonal responses on the gonadal and adrenal hormones. The purpose of this study was to assess the changes in salivary testosterone (Ts), salivary cortisol (Cs) and T/C ratio during long-distance triathlon. Ten well-trained male triathletes participated in the study and were assessed for hormonal changes at four time-points (pre-competition, post-swimming, post-cycling, and post-running phases). Ts decreased from pre-competition to post-swimming (from 93.37 pg/mL to 57.63 pg/mL; p < .01) and increased during two other parts of the competition to almost pre-competition values (cycling: 79.20 pg/mL, p = .02; running: 89,66 pg/mL, p = .04, respectively). Cs showed a similar behaviour; decreasing in the post-swimming phase (1.74 pg/mL) and increasing in the other transitions (post-cycling: 7.30 pg/mL; post-running: 13.31 pg/mL), with significant differences between pre-competition and post- competition values (p = .01). Conversely, T/C increased significantly from pre-competition to post-swimming phase (p = .04) to later decrease until the end of the competition. Overall, T/C significantly decreased (p < .05). In conclusion, during an Ironman triathlon, hormone values fluctuate in response to the demands of the competition. Ts and Cs decrease after-swimming, increase after-cycling and reach the maximum values after-running. T/C reflects overall catabolic status.

Age as a determining variable in the practice or abandonment of physical exercise among young people.

BACKGROUND: Numerous studies have reported a gradual decline in the practice of physical exercise with age, confirming high dropout rates in the adolescent period. The aim of this study was to investigate the motivation or barriers that lead to greater or lesser adherence to physical exercise among children and adolescents. METHODS: In this study, 1,081 individuals volunteered to participate [239 children (age: 10.89 ± .66 years) and 842 adolescents (age: 14.9 ± 1.75 years)]. For this purpose, we used the Self-Report on Motivation for Exercising (AMPEF) and Self-Report on Barriers to Exercising (ABPEF) questionnaires. RESULTS: The most significant motivational factors in the practice of physical exercise (in both children and adolescents) are competition, social recognition and challenge (t = -16.02, p < .001), prevention and positive health (t = 5.24, p < .001) and affiliation, fun and well-being (t = -19.54, p < .001), while the barriers focus on fatigue and laziness (t = -13.20, p < .001) and body image and physical-social anxiety (t = -5.71, p < .001). It is also observed that adolescents showed significantly higher motivation than children and greater presence of barriers in the practice of physical exercise. CONCLUSIONS: The age is a determining variable to include in preventive and intervention programs for the practice of physical exercise.

Sustainable Service-Learning in Physical Education Teacher Education: Examining Postural Control to Promote ASD Children's Well-Being.

As classrooms become more and more diverse, it is imperative to provide physical education teacher education (PETE) students with opportunities to develop competencies that promote quality education for all students. In this study, PETE students applied a physical education service-learning (SL) program aimed at enhancing Autism Spectrum Disorder (ASD) children's motor domain and general well-being-objectives that are connected to the third focus of the United Nations' Sustainable Development Goals (SDGs). Traditionally, research on SL has focused on students' outcomes, and there is a call to examine SL's effects on service receivers, which is the gap this paper aspires to fill. The aim of this study was to measure the postural control of children with ASD who were involved in a 6-month SL program in comparison to ASD peers in a control group. A quasi-experimental design was used in which a total of 29 children with ASD participated. The results of the experimental group showed a significant improvement in the vestibular pathways, an improvement trend in the somatosensorial and visual pathways and improvements in the dynamic tests. This study provides valuable feedback about how SL programs can benefit ASD children to improve their postural control, thus contributing to the third SDG concerned with well-being promotion.

Tactical behavior of high-level male marathon runners.

This study analyzes the strategy used by the best male runners who participated in one of the major city marathons (Frankfurt Marathon, 2008-2018), the all-time performances <2:04:00, the male world records achieved during the 21st century and the Nike Breaking2 Project and INEOS 1:59 Challenge (total = 235 races). The races of the best runners in the Frankfurt Marathon (top 10) were analyzed (n = 110 runners, range: 2:03:42-2:14:05 hours); the runners were divided into two groups according to the tactical used. The pace of Group A (stable pace) remained steady throughout the race, while in Group B (decrease in running speed toward the end of the race) a moderate, but significant drop in speed was detected (P ≤ .001), starting from halfway through the race and getting sharper from the 30th kilometer (30-35 km = 1.6%, P ≤ .001 - 35-40 km = 4.3%, P ≤ .001 - 40-42.195 km: 3.9%, P ≤ .001, total = ≈10%). In the races in which the world record is achieved, the running speed tends to be steady and relatively conservative during the first stretch of the race, increasing smoothly in the second half and achieving a significant increase in the last 2195 m of the race (P = .016, ES = 1.14). Among all the possible strategies, running at a steady pace throughout the race seems the most effective option, especially when priority is given to time rather than position (ie, world records and best all-time races).

Gender Differences in Motivation and Barriers for The Practice of Physical Exercise in Adolescence.

A total of 852 adolescents between 12 and 17 years of age were evaluated (M = 14.86, SD = 1.67), randomly selected among a population of secondary school and Baccalaureate students. We applied an "ad hoc" questionnaire on sociodemographic data and the Self-Report of Reasons for the Practice of Physical Exercise (AMPEF) and Self-Report of Barriers to the Practice of Physical Exercise (ABPEF) questionnaires. The results obtained show a prevalence of competition, social recognition, and challenge as motivational factors, and fatigue or laziness as barriers to physical exercise among adolescents. With respect to gender, boys show a greater presence of motivations towards doing physical exercise, while girls show more barriers. In conclusion, the motivational factors and barriers interact and support each other for a better predisposition towards physical exercise.

The use of Polar Coordinates in the analysis of motor interaction in football according to the result / El uso de Coordenadas Polares en el análisis de las interacciones motrices en el fútbol en función del resultado / O uso de coordenadas polares na análise de interações motoras no futebol com base no resultado

The interactions of a Spanish football team of the Second A (10 official games) are analyzed, evaluating possible behavioral patterns that appear in a regular way in high level football. Observational methodology was used, by Polar Coordinates Analysis, to discover and evaluate the relationships generated between a considered focal behavior and the different conditioned categories, describing behavioral masses among the players. The matches were observed and recorded with an ad hoc observation instrument. The relations of dual character between the players taken as (focal behaviors) right midfielder and forward and the other players (conditioned conducts) were analyzed. The results show differences in the relationship established based on the outcome of the match. Matches that end with a favorable result, the right midfielder takes center stage, as a node of intermediation between the right centre back, left center back, left midfielder, and second striker. In these, the forward is clear receiver in the actions of completion. With the result of the unfavorable match, the connection networks change, generating a network of reciprocal interaction wider and different between the aforementioned player, right midfielder and the rest of the components, with special relation in the players that occupy the right back, left back, right centre back, right winger, left winger, second striker and forward. In these games the striker acquires a role of greater collaboration in the creation in offensive phases, participating as a node in the game network with intermediation functions

Se analizaron las interacciones de un equipo del fútbol español de la Segunda A (Liga 123) (10 partidos oficiales), evaluando posibles patrones de conducta que aparecen de forma regular en el fútbol de alto nivel. Se utilizó metodología observacional, mediante Análisis de Coordenadas Polares, para descubrir y evaluar las relaciones generadas entre una conducta considerada focal y las distintas categorías condicionadas, describiendo mapas conductuales entre los jugadores. Los partidos fueron observados, y registrados con un instrumento de observación ad hoc. Se analizaron las relaciones de carácter dual entre los jugadores tomados como (conductas focales) medio centro derecho y delantero centro y los demás jugadores (conductas condicionadas). Los resultados muestran diferencias en la relación establecida en función del resultado del partido

As interações de uma equipa de futebol espanhol do segundo A (Liga 123) (10 jogos oficiais) foram analisadas, avaliando possíveis padrões de comportamento que aparecem regularmente no futebol de alto nível. Foi utilizada uma metodologia observacional, pela Análise de Coordenadas Polares, para descobrir e avaliar as relações geradas entre um comportamento focal considerado e as diferentes categorias condicionadas, descrevendo mapas comportamentais entre os atores. As partidas foram observadas e registadas com um instrumento de observação ad hoc. Foram analisadas as relações de caráter dual entre os jogadores, tomadas como (comportamentos focais) centro médio direito e centro da frente e os outros jogadores (condutas condicionadas). Os resultados mostram diferenças no relacionamento estabelecido com base no resultado da partida

Effects of work-interval duration and sport specificity on blood lactate concentration, heart rate and perceptual responses during high intensity interval training.

The aim of this study was to examine the impacts on blood lactate concentration, measured heart rate and assessment of perceived exertion during split sessions of equal relative load, as also their relationship to the specific sport practised. Nineteen regional-level athletes (nine middle and long-distance runners (cyclic group) and ten field-sport team players (acyclic group)) performed four high-intensity interval training (HIIT) sessions with work-interval durations of 10 s, 50 s, 90 s and 130 s. The sessions were carried out at their usual training sites with a separation of at least 48 hours. Blood lactate concentration was measured at rest and 3 min after the completion of each protocol. Heart rate was measured continuously during all sessions with a sampling rate of 1 s, and rating of perceived exertion (RPE) was requested at the end of the trial. The results showed an increase in blood lactate concentration, peak heart rate and rating of perceived exertion during long protocols as compared with short ones. No differences were observed in dependent variables between cyclic and acyclic groups. Significant but moderate correlations were observed between post-exercise blood lactate concentration, peak heart rate and RPE.

RELATIONSHIP BETWEEN TRAINING VOLUME AND BONE MINERAL DENSITY CHANGES IN ELDERLY WOMEN / RELAÇÃO ENTRE O VOLUME DE TREINAMENTO E MUDANÇAS DE DENSIDADE MINERAL ÓSSEA EM MULHERES IDOSAS / RELACIÓN ENTRE VOLUMEN DE ENTRENAMIENTO Y CAMBIOS EN LA DENSIDAD MINERAL ÓSEA EN MUJERES MAYORES

ABSTRACT Introduction: Several studies have analyzed the relationship between physical activity and bone density. However, the prescription of exercise is not entirely clear as to the type, quantity and intensity. Objective: The objective of this study was to determine if there is a relationship between the amount of exercise and changes in bone mineral density. Methods: Fifty-two women, members of the Municipal Program of Physical Activity for Seniors, voluntarily underwent two ultrasonographies of the calcaneus within a 6-month interval. During this period, all physical activity was recorded. Afterwards, a lineal correlation study was carried out between the amount of exercise and bone changes, expressed as T-Score variation, first in total number of participants and then in groups. Considering the average body weight obtained for all women, two groups were created ("light" < 69 kg and "heavy" > 69 kg). Later, women who had participated in less than 72% of the targeted program were excluded from both groups, and the differences between the groups "light and trained" and "heavy and trained" were analyzed. To do so, the nonparametric Mann-Whitney U test was used. Results: A significant relationship of r= -0.59 was found between the total amount of exercise and the T-Score variation in the group of women above 69 kg. Significant differences were found between the "light and trained" group and the "heavy and trained" group with respect to the variation of T-Score. Conclusion: The effect of exercise on bone mineral density is determined, somehow, by body weight. This interaction is due, possibly, to mechanical demands difference.

RESUMO Introdução: Vários estudos analisaram a relação à quantidade de atividade física e densidade óssea. No entanto, a prescrição de exercício físico não é totalmente clara, em relação ao tipo, quantidade e intensidade. Objetivos: o objetivo deste estudo foi determinar se existe relação entre a quantidade de exercício físico e alterações na densidade óssea. Métodos: 52 mulheres, membros do programa municipal de atividade física para idosos participaram voluntariamente na realização de dois ultrassonografias de calcâneo, separado 6 meses. Durante este período, toda a atividade física foi registrada. Em seguida, foi realizado um estudo de correlação linear entre a quantidade de exercício físico e alterações ósseas, expressos como variação do T-Score, primeiro de maneira conjunta e despois em grupos. Considerando-se o peso corporal médio obtido para todas as mulheres, dois grupos foram criados ("magro" < 69Kg e "pesado" > 69Kg). Mais tarde, as mulheres que tinham participado em menos do 72% do programa alvejado foram excluídas de ambos os grupos, e foram analisadas as diferenças entre o grupo "magro e treinado" e o grupo "pesado e treinado". Para alcançar este objetivo, foi utilizado o teste não paramétrico da U de Mann-Whitney. Resultados: foi encontrada ama relação significativa de r = -0,59 entre a quantidade total do exercício físico e da variação do T-Score, no grupo das mulheres de mais do que 69Kg. Foram encontradas diferenças significativas entre o grupo "magro y treinado" e o grupo "pesado e treinado", em relação à variação do T-Score. Conclusão: o efeito do exercício físico na densidade mineral óssea é determinado de alguma forma, pelo peso corporal. Esta interação é possivelmente devida à diferente demanda mecânica.

RESUMEN Introducción: Diversos estudios han analizado la relación entre la actividad física y la densidad ósea. Sin embargo, la prescripción de ejercicio físico no está completamente clara en relación al tipo, cantidad e intensidad. Objetivo: El objetivo de este estudio fue determinar si existe relación entre la cantidad de ejercicio y los cambios en la densidad mineral ósea. Métodos: Cincuenta y dos mujeres, integrantes del Programa Municipal de Actividad Física para Mayores, participaron voluntariamente en la realización de dos ultrasonografías de calcáneo, con intervalo de 6 meses. Durante este periodo, se registró toda la actividad física. A continuación, se realizó un estudio de correlación lineal entre la cantidad de ejercicio y cambios óseos, expresados como la variación del T-Score, primero de forma conjunta y posteriormente por grupos. Teniendo en cuenta el peso corporal medio obtenido para el total de mujeres, se crearon dos grupos ("ligero" < 69 Kg y "pesado" > 69 Kg). Más tarde, las mujeres que habían participado en menos del 72% del programa dirigido fueron excluidas de ambos grupos y se analizaron las diferencias entre el grupo "ligero y entrenado" y el grupo "pesado y entrenado". Para lograr este objetivo, se utilizó la prueba no paramétrica de la U de Mann-Whitney. Resultados: Se encontró una relación significativa de r= -0,59 entre la cantidad total de ejercicio físico y la variación del T-Score en el grupo de mujeres de más de 69 Kg. Se encontraron diferencias significativas entre el grupo "ligero y entrenado" y el grupo "pesado y entrenado" con respecto a la variación del T-Score. Conclusión: El efecto del ejercicio físico sobre la densidad mineral ósea está determinado, de alguna manera, por el peso corporal. Esta interacción es debida posiblemente a la diferente demanda mecánica.

Capacidade de repetição da força: efeito das recuperações interséries / Ability to repeat strength: effects of recovery between repetitions

Para estimular uma melhor resposta adaptativa aos objetivos específicos do treinamento de força, a sobrecarga progressiva é a estratégia mais utilizada. A quantidade de trabalho realizada no treino com sobrecarga é aspecto determinante para a melhora do rendimento. Porém, a sobrecarga resulta em desgaste muscular mecânico, o que desencadeia o aparecimento da fadiga. A fadiga é proporcional à característica das cargas (volume, intensidade e recuperação). Neste sentido, a manipulação do intervalo de recuperação é fundamental para evitar a fadiga excessiva, possibilitando uma menor influência do desgaste mecânico na execução da série, auxiliando, assim, na manutenção do volume e intensidade adequadas. A estratégia frequentemente empregada é aumentar o tempo de recuperação entre séries. No entanto, evidências existentes na literatura científica indicam que a maneira mais eficiente de conseguir estes objetivos (diminuir a fadiga e manter o volume de treinamento necessário) é incorporar curtas recuperações no interior da série ("intra-set" ou ISR). Pode-se alcançar tal objetivo, incorporando a recuperação entre repetições ("inter-repetition rest" ou IRR), ou a cada bloco de repetições (agrupamento ou "cluster"). Estas estratégias metodológicas permitem conservar as características mecânicas do gesto (velocidade, força e potência) durante as repetições por série.

The progressive training sessions with overloads are the most used strategies to elicit greater adaptive response to specific aims in strength performance. The total work with overload is a relevant factor in performance improvement. However, overload training affects mechanical muscular response, which triggers the onset of fatigue. The muscle fatigue reflects the loads characteristics (volume, intensity and recovery). In order to avoid the excessive fatigue while maintaining appropriate mechanical techniques, it is necessary to manipulate the recovery. Add more time between sets is the most applied strategy. According to the current researches, it seems that the best way to get both goals (less muscle fatigue and equal efficiency volume), is due to intra-set rest (ISR), which may be achieved by adding inter-repetition rest (IRR) in each repetition or cluster of repetitions within sets. These methodological approaches allow us to maintenance of technical characteristics (speed, strength, power) within many repetition-sets.

Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection.

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.

Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

Comparison of real-time PCR detection chemistries and cycling modes using Mon810 event-specific assays as model.

The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3' flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes.

Interlaboratory transfer of a real-time polymerase chain reaction assay for quantitative detection of genetically modified maize event TC-1507.

A real-time polymerase chain reaction (QPCR) assay was developed for quantitative detection of a genetically modified (GM) maize event TC-1507 and modification to conventional PCR for qualitative purposes. Sequences 5'-flanking TC-1507 full-length insert were characterized and showed multiple rearrangements involving insert and maize chloroplast fragments. The event specificity of the TC-1507 assays was based on the detection of transgene and plant rearranged sequences found to 5' flank the insertion site. They were fully specific and exhibited a limit of detection below 10 target copies, allowing consistent detection of 0.1% GM levels. The QPCR was highly linear and efficient and proved adequate for quantification of GM contents, aiming at the fulfillment of legal requirements established in the European Union (i.e., compulsory labeling of TC-1507 levels >0.9%). It satisfactorily determined TC-1507 contents on different matrixes and was successfully transferred a different laboratory.

A new PCR-CGE (size and color) method for simultaneous detection of genetically modified maize events.

We present a novel multiplex PCR assay for simultaneous detection of multiple transgenic events in maize. Initially, five PCR primers pairs specific to events Bt11, GA21, MON810, and NK603, and Zea mays L. (alcohol dehydrogenase) were included. The event specificity was based on amplification of transgene/plant genome flanking regions, i.e., the same targets as for validated real-time PCR assays. These short and similarly sized amplicons were selected to achieve high and similar amplification efficiency for all targets; however, its unambiguous identification was a technical challenge. We achieved a clear distinction by a novel CGE approach that combined the identification by size and color (CGE-SC). In one single step, all five targets were amplified and specifically labeled with three different fluorescent dyes. The assay was specific and displayed an LOD of 0.1% of each genetically modified organism (GMO). Therefore, it was adequate to fulfill legal thresholds established, e.g., in the European Union. Our CGE-SC based strategy in combination with an adequate labeling design has the potential to simultaneously detect higher numbers of targets. As an example, we present the detection of up to eight targets in a single run. Multiplex PCR-CGE-SC only requires a conventional sequencer device and enables automation and high throughput. In addition, it proved to be transferable to a different laboratory. The number of authorized GMO events is rapidly growing; and the acreage of genetically modified (GM) varieties cultivated and commercialized worldwide is rapidly increasing. In this context, our multiplex PCR-CGE-SC can be suitable for screening GM contents in food.

Efficient sequence-specific purification of Listeria innocua mRNA species by triplex affinity capture with parallel tail-clamps.

Parallel clamps can interact in a sequence-specific manner with homopyrimidine DNA and RNA oligonucleotides to form triplexes. For longer nucleic acids, we have previously demonstrated the inhibitory effect of DNA-target secondary structures on triplex formation. We further designed a modification of these molecules-that is, tail-clamps formed by addition of a tail sequence to the parallel clamp-and proved efficient binding of the molecules with structured single-stranded DNA targets. Here we explore the possible application of the tail-clamp strategy for triplex formation with RNA targets, which are typically found as strongly folded single-stranded molecules. Efficient and specific binding of a tail-clamp designed to form a parallel triplex with Listeria innocua iap mRNA sequences has been verified by UV melting curves and triplex affinity capture techniques. Furthermore, we show for the first time the formation of stable complexes of mRNA with tail-clamps not only under acidic but also under neutral and slightly basic pH conditions. These results signify a further step towards the possible applications of triplexes with mRNA molecules; research, analytical, and therapeutic uses can be envisaged. As an example, our tail-clamp-based triplex affinity capture assay allowed the specific capture and recovery of iap mRNA molecules from an L. innocua total RNA solution with 45 % yield.