Optimization of multi-environment trials for genomic selection based on crop models.

KEY MESSAGE: We propose a statistical criterion to optimize multi-environment trials to predict genotype × environment interactions more efficiently, by combining crop growth models and genomic selection models. Genotype × environment interactions (GEI) are common in plant multi-environment trials (METs). In this context, models developed for genomic selection (GS) that refers to the use of genome-wide information for predicting breeding values of selection candidates need to be adapted. One promising way to increase prediction accuracy in various environments is to combine ecophysiological and genetic modelling thanks to crop growth models (CGM) incorporating genetic parameters. The efficiency of this approach relies on the quality of the parameter estimates, which depends on the environments composing this MET used for calibration. The objective of this study was to determine a method to optimize the set of environments composing the MET for estimating genetic parameters in this context. A criterion called OptiMET was defined to this aim, and was evaluated on simulated and real data, with the example of wheat phenology. The MET defined with OptiMET allowed estimating the genetic parameters with lower error, leading to higher QTL detection power and higher prediction accuracies. MET defined with OptiMET was on average more efficient than random MET composed of twice as many environments, in terms of quality of the parameter estimates. OptiMET is thus a valuable tool to determine optimal experimental conditions to best exploit MET and the phenotyping tools that are currently developed.

Skeletal muscle changes after endurance training at high altitude.

The effects of endurance training on the skeletal muscle of rats have been studied at sea level and simulated high altitude (4,000 m). Male Wistar rats were randomly assigned to one of four groups: exercise at sea level, exercise at simulated high altitude, sedentary at sea level, and sedentary at high altitude (n = 8 in each group). Training consisted of swimming for 1 h/day in water at 36 degrees C for 14 wk. Training and exposure to a high-altitude environment produced a decrease in body weight (P less than 0.001). There was a significant linear correlation between muscle mass and body weight in the animals of all groups (r = 0.89, P less than 0.001). High-altitude training enhanced the percentage of type IIa fibers in the extensor digitorum longus muscle (EDL, P less than 0.05) and deep portions of the plantaris muscle (dPLA, P less than 0.01). High-altitude training also increased the percentage of type IIab fibers in fast-twitch muscles. These muscles showed marked metabolic adaptations: training increased the activity levels of enzymes involved in the citric acid cycle (citrate synthase, CS) and the beta-oxidation of fatty acids (3 hydroxyacyl CoA dehydrogenase, HAD). This increase occurred mainly at high altitude (36 and 31% for HAD in EDL and PLA muscles; 24 and 31% for CS in EDL and PLA muscles). Training increased the activity of enzymes involved in glucose phosphorylation (hexokinase). High-altitude training decreased lactate dehydrogenase activity. Endurance training performed at high altitude and sea level increased the isozyme 1-to-total lactate dehydrogenase activity ratio to the same extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle mass as a factor limiting physical work.

Maximal exercise has been performed by eight men and eight women, using four types of ergometer (2-leg, 1-leg, arm + shoulder, and arm) while breathing room air and while breathing 12% O2. Results have been related to anthropometric estimates of muscle mass in the active limbs. Although significant sex differences of O2 transfer and power output are shown, the sex-specific aerobic performance was roughly proportional to active muscle volume (both when comparing individuals on a given type of ergometer and when comparing average scores of the several types of ergometer). However, the relationship was closer for steady power output than for peak O2 intake (where the scores for arm work were boosted by the use of accessory muscles and by hyperventilation). When breathing 12% O2, the 2-leg performance was substantially reduced (an average of 28.7% for O2 transport and 19.2% for power output). This effect dropped to 9.1% for O2 transport and 12% for power output in one-leg ergometry and was negligible for arm or arm plus shoulder work. It is argued that because of difficulty in perfusing small muscles, arm work is limited largely by the intrinsic power of the active muscles, that single-leg ergometry is limited rather equally by central circulatory and muscular factors, and that two-leg ergometry is almost entirely dependent on the central circulatory transport of O2.

Contractile properties of old rat muscles: effect of increased use.

To examine how different kinds of activity affect the composition and contractile properties of aging skeletal muscle, old male rats were strength and swim trained. The mass of weights lifted during the strength training increased by 85 +/- 9% (P less than 0.05), which was accompanied by an increase by 32 +/- 5% (P less than 0.05) of the estimated force developed. The wet muscle weight of the soleus and the plantaris decreased significantly with age. The phenomenon was counteracted but not neutralized by the strength training. Twitch and tetanic tension also decreased significantly with age in both the soleus and plantaris muscle. This was avoided by the strength training. This training also significantly decreased time to peak tension and half-relaxation time of both muscles. The swim training increased the heart-to-body weight ratio by 21 +/- 5% (P less than 0.05) and the endurance of the soleus muscle. Time to peak tension and triosephosphate dehydrogenase activity of the plantaris muscle were strongly correlated (P less than 0.001) with myosin adenosinetriphosphatase activity. The results show that the composition and contractile properties of old skeletal muscle are considerably affected by strength training repeated during a substantial period of old age, whereas swim training only affects the endurance of the skeletal muscle.

Morphological and biochemical changes in old rat muscles: effect of increased use.

Male Wistar rats were strength and swim trained during a substantial period of old age to determine the influence of aging and activity on the histochemical and metabolic characteristics of a predominantly slow (soleus) and a predominantly fast (plantaris) skeletal muscle. Strength training counteracted the age-related atrophy of the fibers and the age-induced changes in fiber-type distribution of both muscles. Swim training, on the other hand, was without any effect on these parameters. The activity of both mitochondrial and cytoplasmic enzymes became lower with aging in the soleus muscle, whereas only the activity of the cytoplasmic enzymes became lower in the plantaris. Strength training reduced the aerobic capacity of both muscles, whereas swim training had the opposite effect. Aging induced a lower glycogen concentration of the lateral gastrocnemius muscle. This was avoided by swim training. The phosphocreatine and adenosine 5'-triphosphate concentrations were unchanged with aging but became higher with strength training. The activity pattern, therefore, seems to have a considerable influence on the age-related modification of the histochemical and metabolic characteristics of skeletal muscles of the rat. The effect, however, is related to the recruitment pattern of the fiber populations and the form of activity.

Sex-linked differences in equilibrium reactions among adolescents performing complex sensorimotor tasks.

Equilibrium reactions were compared between male and female adolescents (prepuberal and puberal), classified into two groups: those who had previously learned complex motor tasks (dance or acrobatics) and those with no particular training. Subjects stood (eyes open or eyes closed) on a free seesaw platform, the displacements of which were calculated from accelerometer measures. They were instructed to maintain a vertical position with their frontal plane either parallel (to measure antero-posterior oscillations) or perpendicular to the axis of the platform (to measure lateral oscillations). Girls had a better stability than boys as shown by the smaller displacement of their center of gravity. Untrained subjects, irrespective of sex, were the least stable. Subjects trained in acrobatics were more stable than dancers. Differences related to sex can be attenuated by physical training involving equilibrium exercises which suggests that moderate sustained training could reduce the incidence of falls in aged persons and in professionally exposed workers.

The contribution of vision in dynamic spontaneous sways of male classical dancers according to student or professional level.

We investigated the involvement of vision in the regulation of dynamic equilibrium in male children and young adults performing a physical activity requiring a high level of spatial skill: self-induced body sways of ballet dancers on a free unstable platform, 45 professional male dancers (Paris Opera) participated in the study. They included two student groups (beginners and confirmed) and two performer groups (adolescent and adult). They maintained their equilibrium on the platform under different visual and position conditions. The displacement of the seesaw platform were calculated from accelerometer measures. Fast Fourier transform processing of stabilograms allowed spectral frequency analysis. The total spectrum energy and the energies of the three frequency bands (0-0.5 Hz, 0.5-2 Hz, 2-20 Hz) were determined. For all groups, ANOVA indicated that values were higher for eyes-closed than for eyes-open conditions. The visual dependence differed according to age: for 14-year-old students the postural control for dynamic equilibrium was less visually dependent than for 11-year-old students. The 18-year-old dancers, although professional, were more dependent on vision than 14-year-old student dancers. These 18-year-old dancers were still adolescent because they had recently undergone growth acceleration which could disturb their proprioceptive references and internal body representations. Thus, visual input may dominate over the other sensory inputs in the regulation of postural control.

Standard anaerobic exercise tests.

Anaerobic tests are divided into tests measuring anaerobic power and anaerobic capacity. Anaerobic power tests include force-velocity tests, vertical jump tests, staircase tests, and cycle ergometer tests. The values of maximal anaerobic power obtained with these different protocols are different but generally well correlated. Differences between tests include factors such as whether average power or instantaneous power is measured, active muscle mass is the same in all the protocols, the legs act simultaneously or successively, maximal power is measured at the very beginning of exercise or after several seconds, inertia of the devices and body segments are taken into account. Force-velocity tests have the advantage of enabling the estimation of the force and velocity components of power, which is not possible with tests such as a staircase test, a vertical jump, the Wingate test and other long-duration cycle ergometer protocols. Maximal anaerobic capacity tests are subdivided into maximal oxygen debt test, ergometric tests (all-out tests and constant load tests), measurement of oxygen deficit during a constant load test and measurement of peak blood lactate. The measurement of the maximal oxygen debt is not valid and reliable enough to be used as an anaerobic capacity test. The aerobic metabolism involvement during anaerobic capacity tests, and the ignorance of the mechanical efficiency, limit the validity of the ergometric tests which are only based on the measurement of work. The amount of work performed during the Wingate test depends probably on glycolytic and aerobic power as well as anaerobic capacity. The fatigue index (power decrease) of the all-out tests is not reliable and depends probably on aerobic power as well as the fast-twich fibre percentage. Reliability of the constant load tests has seldom been studied and has been found to be rather low. In theory, the measure of the oxygen deficit during a constant load test is more valid than the other tests but its reliability is unknown. The validity and reliability of postexercise blood lactate as a test of maximal anaerobic capacity are probably not better than that of the current erogmetric tests. The choice of an anaerobic test depends on the aims and subjects of a study and its practicability within a testing session.

Respiratory, muscular, and overall perceptions of effort: the influence of hypoxia and muscle mass.

Overall, respiratory and peripheral muscular perceptions of exercise have been examined in 16 subjects (eight men and eight women), each performing four types of exercise (two-leg, one-leg, arm + shoulder, and arm ergometry) under both normoxic and hypoxic (12% oxygen) conditions. Subjects could distinguish ratings for the three types of sensation. Both overall and peripheral muscular perceptions associated with a given oxygen intake or power output increased more than respiratory perceptions as the volume of active muscle was decreased. Hypoxia tended to increase both overall and respiratory perceptions for a given absolute oxygen consumption. Cross-modal comparisons suggested an average overall RPE of close to 13 units at 70% of a task and environment-specific peak oxygen intake, irrespective of exercise conditions, but the SD of individual responses varied by at least +/- 2 units about this average. Peripheral muscular sensations dominated small muscle tasks, but the peripheral muscular RPE became relatively consistent when related to external work rate per liter of active muscle. Respiratory perceptions provided some guide to the intensity of physical activity with a given mode of exercise and fixed environmental conditions. However, if respiratory RPE is used to "fine-tune" cross-modal exercise prescriptions, account must be taken of the distorting influence of active muscle volume and of hypoxia.

Is the exhaustion time at maximal aerobic speed an index of aerobic endurance?

The velocity of the last stage of the Montreal Track Test (MTT) has been measured in fifteen well trained runners. This velocity (vMTT) was assumed to be close to maximal aerobic running speed. In three different sessions, the subjects ran up to exhaustion at velocities corresponding to 95, 100 and 105% vMTT. The exhaustion time at 100 % vMTT (tlim100) was assumed to be an estimation of the exhaustion time corresponding to maximal aerobic speed. The relationship between exhaustion time (tlim) and distance (Dlim) in the case of running exercises at constant velocity until exhaustion can be described by a linear relationship (Dlim = D + b*tlim). The slope of the relation corresponds to a velocity (vcrit) which can be sustained for a long time. The values of vcrit were calculated from the results of running exercises performed at 95, 100 and 105% of vMTT. The present study showed that tlim at 100% vMTT (tlim100) was negatively correlated with vMTT and vcrit but that D and ratio vcrit/vMTT were independent of vMTT. A theoretical study based on models previously proposed for oxygen kinetics during supramaximal exercises (exponential model and Margaria's model) demonstrates that this negative relationship between vMTT and tlim100 can be explained by the kinetics of the accumulation of oxygen deficit (O2 def). tlim100 should also depend on VO2max, maximal oxygen deficit (Max O2 def) and the relative importance of anaerobic energy when a VO2 plateau is reached. Moreover, the value of tlim100 largely depends on the accuracy of the assessment of vMTT. Consequently, the exhaustion times corresponding to the different estimations of maximal aerobic speed on a track or a treadmill cannot be considered as valid indices of aerobic endurance.

[Changes in blood pressure during static work. Practical implication]. / Modifications de la pression artérielle au cours du travail statique. Implications pratiques.

while the cardiocirculatory response to dynamic exercises (e.g. running, cycling, swimming) is the object of the physician's full attention, the cardiovascular repercussions of static exercises, such as weight-lifting, sustained postures in gymnastics or immobilization in judo, are often ignored. Yet any sustained isometric contraction is associated from the art with an increase in blood pressure which may be major and involves about equally the systolic and diastolic pressures. As soon as the strength developed by the muscle exceeds 20 p. 100 of its maximum voluntary strength (MVS) the increase of blood pressure continues until the contraction comes to an end. Whatever the muscle used, the intensity of a cardiovascular response is independent of the muscular mass in action, but it mostly depends on the MVS percentage This rise in blood pressure is mainly due to an increase in heart and cardiac output. The reactions are such that relatively moderate and localized static effort may result in a rapid and important increase in myocardial work, and this may cause serious accidents in subject with a cardiovascular weakness, either isolated or associated with such risk factors as age or arterial hypertension. In consequence, adults who are insufficiently trained and present with one or several risk factors should avoid sports which required frequent respiratory arrests and/or sustained postures. If such sports are nevertheless chosen, it would seem that practising also a dynamic sport relying on aerobic metabolism might lin , at least in part, the effects of increased blood pressure due to static activities.

Maximal power and force-velocity relationships during cycling and cranking exercises in volleyball players. Correlation with the vertical jump test.

BACKGROUND: The aim of this study was to propose a test battery adjusted to volleyball players and to study the links between dynamic (vertical jump, force-velocity relationships and maximal anaerobic power in cranking and cycling) and static (maximal voluntary force and rate of force development in isometric conditions) performances. METHODS: The relationships between braking force (F) and peak velocity (V) have been determined for cycling and cranking exercises in 18 male volleyball players of a district league. According to previous studies, these F-V relationships were assumed to be linear and were expressed as follows: V = V0(1-F/F0), where V0 should be an estimate of the maximal velocity at zero braking force whereas F0 is assumed to be a braking force corresponding to zero velocity. Maximal anaerobic power in cycling (Pmax leg) and cranking (Pmax arm) were calculated as equal to 0.25 V0F0. The same subjects performed a vertical jump test (VJ) and a strength test on an isometric leg press with the measurement of the unilateral isometric maximal voluntary force (MVF) and indices of rate of isometric force development (RFD): maximal rate of force development (MRFD) and the time from 25% to 50% of MVF (T25-50). RESULTS: Pmax leg (15.8 +/- 1.4 W.kg-1) and V0 arm (259.6 +/- 13.1 rpm) were high but similar to the results of elite athletes, previously collected with the same protocols and the same devices. VJ was significantly with F0 leg, Pmax leg and Pmax arm related to body mass. The performances of the dynamic tests were significantly correlated and especially the parameters (V0, F0, Pmax) of the force velocity tests in cycling were significantly correlated with the same parameters in cranking. The results of the isometric tests (MVF, MRFD) were not correlated with VJ, except T25-50 of the left leg. CONCLUSIONS: A vertical jump test and a force velocity test with the arms are proposed for a test battery in volleyball players.

Work-exhaustion time relationships and the critical power concept. A critical review.

The present review is focused on the physiological meanings of the critical power concept proposed by Scherrer in 1954 and its applications to general exercises such as running, cycling and swimming. Since the first studies on the critical power of local exercises, many studies have found that critical power is correlated with indices which are related to aerobic endurance such as maximal oxygen uptake, ventilatory threshold, OBLA or maximal lactate steady state. In fact, the relationship between exhaustion time t(lim) and the Work Wlim (or Distance Dlim) performed at exhaustion is not exactly linear and, consequently, the power-t(lim) equation is not a true hyperbola. The effect of the range of t(lim), used in the calculation of the slope of the Wlim-t(lim) relationship (called critical power) are discussed. When critical power is calculated from short supramaximal exercises, this power is higher than the power output which corresponds to a lactate steady state (or an oxygen uptake steady state) and does not correspond to a power output which can be sustained a long time. The authors present experimental data collected during local (knee extension) and general (running and cycling) exercises which suggest that critical power could correspond to a steady state provided that critical power is calculated from heavy submaximal exercises only (t(lim) ranging between 6 and 30 min). It is difficult to predict exhaustion time from critical power or critical velocity because of the hyperbolic nature of the power-t(lim) relationship. On the other hand, a large error in the measure of t(lim) should have a small effect on the calculation of critical power or velocity. In contrast, the value of Y intercept of the Wlim-t(lim) (or Dlim-(t(lim)) relationship should be sensitive to errors in t(lim).

Sex differences in cardiac responses to successive apnea periods.

Cardiac response to breath-holding is generally described as a bradycardia, which is explained by a two-fold mechanism involving the pulmonary mechanoreceptors and the arterial chemoreceptors. This study was conducted to determine the cardiac effects of five successive apnea periods separated by 1 min of free ventilation (FV). Heart rate (HR) and ventilation (V, VT, f) were measured during this protocol in 12 young subjects (6 men, 6 women). Ventilatory responses during FV periods were similar in both sexes, but HR responses were different during the apnea periods. The mean exhibited a bradycardia and the women a tachycardia. Although the statistical significance of the results was weak, they showed a clear tendency which was interpreted as differences in central cardioventilatory interactions. Sex difference in the cardiac consequences of static work from respiratory muscle is also evoked.

Quantifying fatigue in working divers.

Thirty professional divers involved in training, bounce dives, working saturations, and deep experimental saturations were observed over a 1-month period. The subjects themselves performed simple twice-daily measurements of oral temperature, heart rate, breathhold time, handgrip strength, length and quality of sleep, and a subjective estimation of fatigue. The data were analysed with respect to the type of work done by the diver, separating diving from nondiving days. The results showed that subjective estimation of fatigue corresponded to the increment of evening oral temperature and heart rate over morning values. Both inspiratory and expiratory breathhold times decreased from morning to evening in those situations deemed most tiring by the divers. Sleep was generally of average duration and quality; however, diving during the daytime was associated with a decreased amount of sleep in the 24-h period including the following night. Sleep was also of poorer quality during periods of saturation diving. In addition to such group variations, individual divers showed significant performance changes on the various tests, demonstrating the value of this approach to the practical question of deciding when a man is too tired to dive safely. Extension of this method can, we hope, aid in the definition of safe working rotations for professional divers.

Complementary roles of central command and muscular reflex in the regulation of heart rate during submaximal isometric contraction.

During submaximal isometric contraction, the heart rate (HR) and the electromyographic activity (EMG) increase continuously. Although activation of the muscle and the cardiovascular center is placed partly under the common control of the central command, the nature of the relationship that may exist between HR and the integrated electromyogram (iEMG) is seldom studied. Seventeen healthy men, 22.4 +/- 0.5 years of age (M +/- SE), performed isometric contractions with the right elbow flexors. Forces of 25, 40, 50 and 65% of the maximum voluntary contraction (MVC) were used, and the contractions were sustained until (isotonic isometric contraction: IIC) and beyond exhaustion (anisotonic isometric contraction: AIC). During IIC, a linear relationship exists between HR and iEMG; the slope of this relationship is independent of the relative force developed, which is in favor of a predominant role played by the central command in HR increase. The increase in the ratio iEMG/HR at the approach of local muscular exhaustion would indicate that at the end of IIC there is an increase in the relative part furnished by the information of peripheral origin in HR regulation. During AIC, the force (F) decreases in an exponential manner and stabilizes at around 25% MVC from tAIC = 70 s on. The iEMG and HR change independently: iEMG decreases like F such that iEMG/F remains constant; HR continues to increase in the first phase corresponding to the rapid decrease in F and iEMG, then in a second phase, it decreases linearly with respect to time. Our results suggest that the action of the central command is dominant during stage 1 of AIC, while during stage 2 the relative part furnished by the muscle reflexes increases. Beyond tAIC = 70 s, there seems to be a certain degree of central fatigue.

Critical power as an endurance index.

The relationship between exhaustion time (t(lim)) and the work performed at the end of constant-power exercises can be described by a linear relationship (Wlim = a + b t(lim)) for work involving the whole body (eg cycling) or part of the body (eg knee extensions). The slope b in the equation is termed the critical power and has been proposed as an index of the capacity to perform work over a long period of time. The first objective of the present study was to compare the values of slopes b calculated from whole-body work of short duration, ie maximal and supra-maximal cycling exercises (slope b1), with the values calculated from the same work, the durations of which were between 3.5 and 35 min (slope b3), as in the protocols used by Scherrer and Monod (1960) for body-part work. Slope b1 was significantly higher than slope b3 in 10 subjects who performed 5 cycling exhausting exercises (60, 73, 86, 100 and 120% of maximal aerobic power (MAP) in watts). Exhaustion times corresponding to power outputs equivalent to b1 and b3 were equal to 29.0 +/- 19.1 min and 48.6 +/- 9.8 min respectively. Moreover, the exhaustion times at 60 and 73%,MAP were significantly correlated with slope b3 (expressed in %MAP) but not with slope b1. Consequently, slope b3 should be considered as the critical power instead of slope b1 as in some studies in the literature (Moritani et al, 1981). The second objective was to study the physiological significance of the critical power (slope b3) of whole-body work (cycling). The workload that corresponded to a lactate steady state was not significantly different from b3 (68.8 +/- 6.0 vs 68.7 +/- 6.3% MAP). Nevertheless, slope b3 represents a workload corresponding to a slight but significant drift of heart rate or oxygen uptake. These results probably explain why b3 is a power which can be maintained for a long time but not beyond about l h in an average subject.

[Relationship between blood zinc level and EEG changes under the influence of hyperpnea in normal subjects]. / Relations entre le traux du zinc sanguin et les modifications de l'E.E.G. sous l'influence de l'hyperpnée chez l'homme normal.

The red blood cell and serum zinc levels have been determined by atomic absorption spectrophotometry on 27 human subjects in apparent good state of health, tested monthly for five months successively. The subjects who present a decreased frequency of the E.E.G. pattern during hyperventilation, also show a diminished red blood cell Zn level, by comparison with the other subjects. This phenomenon suggests a possible relationship between red blood cell zinc concentration and E.E.G. signs of cortical hyperexcitability. These findings are discussed in the light of the recent literature.

[The physical fitness visit: a serious medical procedure for the surveillance of sportsmen]. / La visite médicale d'aptitude: un acte médical sérieux pour la surveillance des sportifs.

Assessment of physical capability is especially important in sports medicine. It allows the choice of the appropriate sport, and the biomedical follow-up of the health of the sportsman engaged in a monitored training. The so called aptitude examination is a responsible action which requires time, an accurate clinical sense and involves the responsibility both of the general practitioner and of the sport medicine specialist. During the medical follow up, with the help of specialized functional tests, the physician may detect a state of overtraining and start a dissuasive action against doping habits.

Maximizing the reliability of genomic selection by optimizing the calibration set of reference individuals: comparison of methods in two diverse groups of maize inbreds (Zea mays L.).

Genomic selection refers to the use of genotypic information for predicting breeding values of selection candidates. A prediction formula is calibrated with the genotypes and phenotypes of reference individuals constituting the calibration set. The size and the composition of this set are essential parameters affecting the prediction reliabilities. The objective of this study was to maximize reliabilities by optimizing the calibration set. Different criteria based on the diversity or on the prediction error variance (PEV) derived from the realized additive relationship matrix-best linear unbiased predictions model (RA-BLUP) were used to select the reference individuals. For the latter, we considered the mean of the PEV of the contrasts between each selection candidate and the mean of the population (PEVmean) and the mean of the expected reliabilities of the same contrasts (CDmean). These criteria were tested with phenotypic data collected on two diversity panels of maize (Zea mays L.) genotyped with a 50k SNPs array. In the two panels, samples chosen based on CDmean gave higher reliabilities than random samples for various calibration set sizes. CDmean also appeared superior to PEVmean, which can be explained by the fact that it takes into account the reduction of variance due to the relatedness between individuals. Selected samples were close to optimality for a wide range of trait heritabilities, which suggests that the strategy presented here can efficiently sample subsets in panels of inbred lines. A script to optimize reference samples based on CDmean is available on request.