Louis Clavel
email: clavel.louis@gmail.com

Valérie Attali

Isabelle Rivals

Marie-Cécile Niérat

Pierantonio Laveneziana

Philippe Rouch

Thomas Similowski

Baptiste Sandoz

Lori Ann Vallis

Decreased respiratory-related postural perturbations at the cervical level under cognitive load

Keywords: Breathing, Centre of pressure, Cognitive dual task, Joint kinematics, Postural control, Optoelectronic plethysmography, Posturo-ventilatory interaction, Cervical spine Amp Signal amplitude AP Antero-posterior BF Breathing frequency BMI Body mass index CI Confidence interval COG Cognitive

Purpose In healthy humans, postural and respiratory dynamics are intimately linked and a breathing-related postural perturbation is evident in joint kinematics. A cognitive dual-task paradigm that is known to induce both postural and ventilatory disturbances can be used to modulate this multijoint posturo-ventilatory (PV) interaction, particularly in the cervical spine, which supports the head. The objective of this study was to assess this modulation. Methods With the use of optoelectronic sensors, the breathing profile, articular joint motions of the cervical spine, hip, knees and ankles, and centre of pressure (CoP) displacement were measured in 20 healthy subjects (37 years old [29; 49], 10 females) during natural breathing (NB), a cognitive dual task (COG), and eyes-closed and increased-tidal-volume conditions. The PV interaction in the CoP and joint motions were evaluated by calculating the respiratory emergence (REm). Results Only the COG condition induced a decrease in the cervical REm (NB: 17.2% [7.8; 37.2]; COG: 4.2% [1.8; 10.0] p = 0.0020) concurrent with no changes in the cervical motion. The CoP REm (NB: 6.2% [3.8; 10.3]; COG: 12.9% [5.8; 20.7] p = 0.0696) and breathing frequency 18.7]; COG: 18.6 min-1 [16.3; 19.4] p = 0.0731) tended to increase, while the CoP (p = 0.0072) and lower joint motion displacements (p < 0.05) increased. Conclusion This study shows stable cervical spine motion during a cognitive dual task, as well as increased postural perturbations globally and in other joints. The concurrent reduction in the PV interaction at the cervical spine suggests that this "stabilization strategy" is centrally controlled and is achieved by a reduction in the breathing-related postural perturbations at this level. Whether this strategy is a goal for maintaining balance remains to be studied.

Introduction

Postural and respiratory dynamics are intimately linked and exhibit strong temporal relationships in healthy humans [START_REF] Dally | An inquiry into the physiological mechanism of respiration with especial reference to the movements of the vertebral column and diaphragm[END_REF][START_REF] Gurfinkel | The compensation of respiratory disturbances of the erect posture of man as an example of the organization of interarticular interaction[END_REF][START_REF] Perry | The coupled evolution of breathing and locomotion as a game of leapfrog[END_REF]. Ribcage movements during breathing disturb an individual's balance by modifying the costo-vertebral articulation position, thoracic spinal curvature and postural alignment [START_REF] Dally | An inquiry into the physiological mechanism of respiration with especial reference to the movements of the vertebral column and diaphragm[END_REF][START_REF] Attali | Compensation of respiratoryrelated postural perturbation is achieved by maintenance of headto-pelvis alignment in healthy humans[END_REF] and by inducing ribcagerelated centre of mass (CoM) displacements. Breathingrelated postural perturbations during natural breathing are partially counteracted by phasic contractions of paravertebral [START_REF] Kantor | Does body stability depend on postural chain mobility or stability area?[END_REF]) and pelvic floor muscles [START_REF] Hodges | Postural and respiratory functions of the pelvic floor muscles[END_REF][START_REF] Talasz | Phase-locked parallel movement of diaphragm and pelvic floor during breathing and coughing-a dynamic MRI investigation in healthy females[END_REF]. The interaction between the breathing-related postural perturbations and these counteracting muscle contractions that occurs to maintain balance is defined as the posturo-ventilatory (PV) interaction. From a stabilometric point of view, the PV interaction is characterized by minimal rhythmic displacements of the centre of pressure (CoP-the whole body CoM vertical projection on the ground) induced by natural breathing at rest, whereas this displacement almost disappears during apnoea [START_REF] Kantor | Does body stability depend on postural chain mobility or stability area?[END_REF][START_REF] Caron | Effects of ventilation on body sway during human standing[END_REF]) and increases with CO2induced hyperventilation [START_REF] David | Postural control and ventilatory drive during voluntary hyperventilation and carbon dioxide rebreathing[END_REF] or voluntary deep breathing [START_REF] Hamaoui | Respiratory disturbance to posture varies according to the respiratory mode[END_REF]. The PV interaction is centrally integrated [START_REF] Gurfinkel | The compensation of respiratory disturbances of the erect posture of man as an example of the organization of interarticular interaction[END_REF], as evidenced by the perturbations that occur in patients with stroke [START_REF] Manor | Posturo-respiratory synchronization: effects of aging and stroke[END_REF] or by the anticipated increases-i.e., those involving cortical resources-in the role of the diaphragm in postural control during rapid postural adjustments [START_REF] Hodges | Contractions of specific abdominal muscles in postural tasks are affected by respiratory maneuvers[END_REF].

The PV interaction acts on a multijoint kinetic chain with several degrees of freedom at the spine, hip, knee and ankle levels [START_REF] Gurfinkel | The compensation of respiratory disturbances of the erect posture of man as an example of the organization of interarticular interaction[END_REF][START_REF] Hodges | Coexistence of stability and mobility in postural control: evidence from postural compensation for respiration[END_REF] to control breathing-related postural instability [START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF] . Compared to other joints in the postural chain, the cervical spine directly supports the head and is highly involved in the regulation of its position in space. It plays a crucial role in the maintenance of balance, as suggested by its role in sit-to-stand tasks [START_REF] Hamaoui | Influence of cervical spine mobility on the focal and postural components of the sit-to-stand task[END_REF] and by the presence of impaired stabilometric parameters in patients with a history of moderate neck trauma [START_REF] Gandelman-Marton | Postural stability in patients with different types of head and neck trauma in comparison to healthy subjects[END_REF] or neck pain [START_REF] Quek | An exploratory study examining factors underpinning postural instability in older adults with idiopathic neck pain[END_REF]) . In addition, changing the orientation of the head during tracking requires a synergy between the most inferior joints and the cervical spine for an individual to maintain balance [START_REF] Park | Functional synergies underlying control of upright posture during changes in head orientation[END_REF]. Permanent adjustments of the cervical curvature during standing are needed to preserve an adequate head-to-pelvis alignment, which is essential for balance [START_REF] Vital | Anatomical bases of the study of the constraints to which the cervical spine is subject in the sagittal plane. A study of the center of gravity of the head[END_REF][START_REF] Dubousset | Three-dimensional analysis of scoliotic deformity[END_REF][START_REF] Amabile | Invariance of head-pelvis alignment and compensatory mechanisms for asymptomatic adults older than 49 years[END_REF] , as well as to maintain the head position and a horizontal gaze [START_REF] Hasegawa | Standing sagittal alignment of the whole axial skeleton with reference to the gravity line in humans[END_REF], which are important for the transmission of the appropriate visual, vestibular and neck proprioceptive afferent feedback signals [START_REF] Bove | Interaction between vision and neck proprioception in the control of stance[END_REF][START_REF] Williams | Use of neck torsion as a specific test of neck related postural instability[END_REF][START_REF] Malmström | Disturbed cervical proprioception affects perception of spatial orientation while in motion[END_REF] . These adjustments of the cervical curvature are anticipated during tasks requiring attention, such as complex visual [START_REF] Boulanger | Interaction between the oculomotor and postural systems during a dual-task: compensatory reductions in head sway following visually-induced postural perturbations promote the production of accurate double-step saccades in standing human adults[END_REF] or motor tasks [START_REF] Hamaoui | Influence of cervical spine mobility on the focal and postural components of the sit-to-stand task[END_REF] , as evidenced by prefrontal cortex recruitment, which indicates their central nature [START_REF] Jahn | Brain activation patterns during imagined stance and locomotion in functional magnetic resonance imaging[END_REF][START_REF] Taube | Spinal and supraspinal adaptations associated with balance training and their functional relevance[END_REF][START_REF] Mihara | Role of the prefrontal cortex in human balance control[END_REF]. Therefore, even though it is a part of the postural chain involved in the PV interaction, the cervical spine is centrally modulated by predominant "nonrespiratory" cortical adjustments, which may compete with the central control of the PV interaction. Indeed, compared to other joints in the postural chain, the cervical spine exhibits less breathing-related mobility during natural breathing [START_REF] Hodges | Coexistence of stability and mobility in postural control: evidence from postural compensation for respiration[END_REF][START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF] and higher variability when the breathing amplitude increases [START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF].

The dual-task paradigm is known to divert cortical resources and induce an increase in cognitive load [START_REF] Stelzel | Contribution of the lateral prefrontal cortex to cognitive-postural multitasking[END_REF], and it has been validated for the investigation of the interactions between cognitive processes and other cortical functions such as postural control [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF][START_REF] Stelzel | Contribution of the lateral prefrontal cortex to cognitive-postural multitasking[END_REF]) and the cortical control of breathing [START_REF] Mador | Effect of alterations in mental activity on the breathing pattern in healthy subjects[END_REF][START_REF] Grassmann | Respiratory changes in response to cognitive load: a systematic review[END_REF]. Cognitive load is indeed known to be independently associated with modulations in postural control [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF][START_REF] Stelzel | Contribution of the lateral prefrontal cortex to cognitive-postural multitasking[END_REF]) and breathing patterns [START_REF] Mador | Effect of alterations in mental activity on the breathing pattern in healthy subjects[END_REF][START_REF] Grassmann | Respiratory changes in response to cognitive load: a systematic review[END_REF]. To date, no specific modulations in the PV interaction under a cognitive load have been described; one study reported reduced postural sway, which was partly related to a reduction in breathing amplitude, in young adults [START_REF] Hagio | Effects of breathing movement on the reduction of postural sway during postural-cognitive dual tasking[END_REF]. This result suggests that central postural perturbations induced by a cognitive task may be compensated by a reduction in breathing-related postural perturbations, indicating a strong interaction between cognition and the PV interaction.

Therefore, we hypothesized that cognitive processes can modulate the PV interaction and its distribution along the multijoint postural chain, with less important breathing-related postural perturbations occurring at the cervical level compared to other joints. To test these hypotheses, we used a cognitive dual-task methodology, and we assessed the global effect of the PV interaction on CoP displacement, as well as on the joint motions in the postural chain, with a focus on the cervical spine. The PV interaction is a delicate phenomenon that is easily altered, and the choice of our cognitive task was driven by the necessity to prevent the occurrence of any respiratory changes or postural perturbations linked to the cognitive task itself, particularly at the cervical level. Hence, motor tasks such as eye tracking (which induces head movements) and vocal counting (which involves the respiratory system) were not appropriate. Therefore, purely mental tasks were chosen, while the recall task (to verify that the subjects had truly completed the mental task) was performed only when the registration ended. Both cognitive tasks that were used in this study tested working memory, and one task additionally tested the subject's calculation ability and attentional capacities.

Materials and methods

Subjects

Twenty-one healthy adult subjects (age ≥ 18 years) were included in the study (10 F, age: 37 years [29; 49], BMI: 22.9 kg/m 2 [20. 6; 25.5], data presented as the median [Q1; Q3]). None of the subjects had a history of postural or respiratory disease, and all subjects had normal pulmonary function test results. This study was approved by the ethics committee Ile-de-France VI (Paris, France) (CPP IDF VI 06036) and recorded in the ISRCTN registry under the number ISRCTN56129394. All subjects gave their written consent to participate.

Protocol

A total of 65 retroreflective markers were placed on the subjects; four were placed on the head, 41 were placed on the trunk, seven were placed on each leg, and three were placed on each foot (Fig. 1a). Motion data were captured with an optoelectronic system (Vicon Nexus 1.8.5, Oxford, UK) using 12 cameras (Vero 1 MPa) that were placed around the subject and a sampling rate of 100 Hz (Fig. 1c); the motion data were used to determine the breathing profile and joint motions (cervical spine, hips, knees and ankles). One force plate (BP 4051040-2 k, AMTI, Watertown, USA) measuring the subject's CoP displacements was synchronized with the optoelectronic system at the same sampling frequency. The motion and CoP displacement data were simultaneously recorded with Nexus software.

The subjects were instructed to stand relaxed and barefoot on the force plate with the feet located shoulder-width apart and the arms placed alongside the body. The first set of several 45-s recordings was taken randomly under four different conditions: (1) a natural breathing reference condition (NB); two cognitive dual-task conditions (COG), which consisted of (2) listening and remembering 8 words, then repeating them once the previous task was completed (COG1) and (3) listening to five multiplication problems, performing them mentally, then giving the answers once the registration had ended (COG2); and (4) an eyes-closed condition (EC). During these conditions, no specific instructions were given regarding breathing. An additional thirty-second recording was performed under an increased voluntary tidal volume (ITV) condition, during which the subjects were asked to increase their tidal volume but not their breathing frequency. For all conditions except for EC, the subjects were instructed to focus on a landmark on the wall to maintain horizontal gaze.

Signal processing

CoP displacements

Subject-specific antero-posterior (AP) and medio-lateral (ML) axes were defined from the four foot markers (Fig. 1b). The AP axis was defined as the line joining the middle of the anterior markers and the middle of the posterior markers projected in the horizontal plane. The ML axis was defined as the line perpendicular to the AP axis in the horizontal plane. The output of the force plate allowed the computation of the CoP time series in the AP direction. The amplitude (Amp) of the CoP displacements was estimated by the mean of their linear envelopes.

Breathing profile

From the 41 markers placed on the trunk, the chest wall (CW) volume was computed at each time point as the volume enclosed in the surface of the triangular mesh delimited by the spatial marker locations (Fig. 1a), following the method developed in 1996 by [START_REF] Cala | Chest wall and lung volume estimation by optical reflectance motion analysis[END_REF] using Green-Ostogradski's theorem. In the following equation, S is the surface, V is the volume enclosed by S i , F i is an arbitrary vector considered as the unit vector (F), n i is the normal unit vector at the different points of S i , and ∇ is the divergence operator, which is considered the unit divergence. The analytical expression of the theorem becomes:

∫ S ⃗ F.�� ⃗ n i dS i = ∫ V ∇ ⃗ FdV i = ∫ V dV i
In the discrete form, the CW volume can be computed by summing the areas of the regions in the K triangle constituting the chest wall mesh, with A i corresponding to the area of each region:

With the signal of the variation in the CW volume, the start of each inspiration and expiration event was located. The inspiration/expiration events were defined as the local maxima and minima obtained from the volume signal: the end of an inspiration event corresponded to a local maximum, and the end of an expiration event corresponded to a local minimum. A respiratory cycle was defined as the duration between the start of two successive inspiration events. The global breathing frequency (BF) was calculated as the ratio between the number of respiratory cycle integers (n C ) and the elapsed time (i.e., 45 s). The mean inspiratory (TI) and expiratory (TE) times were computed over the whole breathing period. The amplitude (Amp.) of variation in the CW volume was estimated in litres by the mean of its linear envelopes.

CW = K ∑ i=i ⃗ F.�� ⃗ n i .A i

Joint motions

Seven specific 3D vectors were considered in a subjectspecific frame (F sub ) and defined with in the AP, ML and vertical axes. The head (V Head ), thoracic (V Thor ), hip (V Hip ), thigh (V Thigh ), tibial (V Tib ) and foot frames (V Foot ) were then defined using the local segment markers. V Head and V Hip were defined as the normal of the mean-squared plane defined by the four markers on each segment. V Thor was defined as the cross product of the vectors defined; the vector between the right and left acromion markers and the vector defined by the K4-manubrium markers were calculated first, then V Thor was calculated as the cross product of them (more simply, V Thor is orthogonal to the two other vectors defined by the acromion marker on one side and the K4 and manubrium markers on another side). V Thigh , V Tib and V Foot were defined by the markers located on the proximal and distal parts of each segment. These seven vectors were then projected in the sagittal plane (defined by the AP and Z axes), and the motions at the four joint levels (cervical spine, hip, knee and ankle) were computed as the angles between the projected vectors (Fig. 1d). Cervical spine motion was computed as the angle between V Head and V Thor , pelvic motion was computed as the angle between V Hip and the AP axis, knee motion was computed as the angle between V Thigh and V Tib and ankle motion was computed as the angle between F Tib and F Foot . The amplitudes (Amps) of the joint motions (cervical spine, hip, knee and ankle angles) were estimated by the means of their linear envelopes.

For each kinematic signal, the power spectral density was computed from the signal, which was downsampled to 10 Hz, using Welch's periodogram over 512 points with a 128-point Hamming window and 66-point overlap (1 point every 0.0196 Hz).

Posturo-ventilatory interaction

The posturo-ventilatory interaction was estimated by respiratory emergence (REm) [START_REF] Hamaoui | Respiratory disturbance to posture varies according to the respiratory mode[END_REF]) and by timelocked averaging (TLA) (Kuznetsov and Riley 2012) variables, which both require a calculation using the respiratory signal and synchronized motion signal as follows. The REm is a measure of the weight of the respiratory component during movement and was assessed following a method that was previously described [START_REF] Hamaoui | Respiratory disturbance to posture varies according to the respiratory mode[END_REF]. In brief, fast Fourier transformation was performed on the motion signal, the REm was computed in the frequency domain as the ratio of the average power of a band of 0.08 Hz centred on the mean breathing frequency and the overall average power of the motion signal. A REm close to 100% indicates that the respiratory component is a major component of the motion signal. In this study, the REm was assessed in the CoP displacement projected on the AP axis and cervical spine, hip, knee and ankle motions in the sagittal plane. For the TLA, each breathing cycle was resampled to obtain 100 equally spaced points. Changes in the signals were then computed over the duration of each breathing cycle as the deviation in a signal from the value at the beginning of the cycle (Fig. 2). For the four joint angles (cervical spine, hip, knee and ankle), the presence of significant movements repeated throughout the breathing cycle was determined following a method that was previously described [START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF]: first, the 95% confidence interval (95% CI) over the breathing cycle (step 1 to 100) was computed; then, a movement was considered significant if for over 20% of the breathing cycle, the value 0 was not included in the 95% CI. Significant movements with a 95% CI entirely less than 0 were considered flexion movements, and those entirely greater than 0 were considered extension movements (Fig. 3).

Data analysis

For the knees and ankles, the Amp, TLA and REm values were averaged across the left and right sides. The results for the COG1 and COG2 conditions were also averaged (COG). The majority of the variables were non-normally distributed (Shapiro-Wilk test), and all of them are described by the median and interquartile range [Q1; Q3]. The continuous variables were compared with Wilcoxon's test, and the proportions were compared with Fisher's exact test on the 2 × 2 corresponding contingency tables, considering a significance level of 0.05. The change in PV interaction along the multijoint postural chain with a cognitive load was assessed by comparing the REm values derived from the CoP displacement and cervical spine, hip, knee and ankle motions and the occurrences of changes in the TLA (flexion, extension or no Fig. 2 Left: beginning of each cycle (-); middle: the variation in each signal; right: 95% confidence interval for the signal variations change) for the four joint motions (cervical spine, hip, knee and ankle) between the COG and NB conditions (analysis 1). The change in PV interaction induced by a cognitive load at the cervical level was compared to the change induced by the EC and ITV conditions and was assessed by the REm derived from the cervical spine motion (analysis 2). Figure 4 summarizes the different computed variables and performed analyses in a flowchart.

Results

Data from one subject were excluded due to data recording issues, as two pelvic markers were missing. The data from twenty subjects were included in the analysis. The subjects' baseline characteristics are presented in Table 1.

Modulation of the PV interaction along the multijoint postural chain during a cognitive load (analysis 1)

For COG1 and COG2, 7/8 words [6; 8] ankle + 0.12° [0.03; 0.60] (p = 0.0152) (Fig. 5). Regarding the PV interaction, the REm increased nonsignificantly for the CoP (p = 0.0696) but significantly decreased at the cervical level (p = 0.002), and it remained unchanged at the other joints (Fig. 6). The TLA analysis showed a significant decrease in the occurrence of breathing articular motion at the cervical spine and ankle levels but no change at the knee and hip levels. This phenomenon is illustrated in one subject (Fig. 7a), showing that the breathing-related perturbation manifested predominantly by the extension of the cervical spine, which was observed during the NB condition and was counteracted during the COG condition. At the cervical level, breathing-related extension and flexion movements were reduced globally by a similar magnitude; however, different profiles of cervical motion were observed between subjects (Fig. 7b; Table 2). Table 2 presents changes in the PV interaction between the NB and COG conditions for the REm and TLA analyses.

Cervical PV interaction variations by conditions (analysis 2)

With respect to the NB condition, only the COG condition induced a decrease in the breathing-related postural perturbation at the cervical level, as evidenced by a significant reduction in the REm (p = 0.002) (Table 3). Moreover, only the COG condition altered the ventilatory profile, as evidenced by a decrease in the TE and an increase in the BF (ns), while no change was observed under the ITV and EC conditions (Table 3). The amplitude of the CoP displacement increased during the COG and ITV conditions and remained unchanged during the EC condition. The motion of the cervical spine remained unchanged during the COG condition, while it decreased during the EC condition and increased during the ITV condition (Table 3).

Discussion

This study shows that a cognitive load induces specific changes in the PV interaction, as attested by a change in the breathing-related postural perturbation along the postural chain, with a reduction at the cervical level only. This result

Posturo-ventilatory interaction and cognition

Cognitive dual-task paradigms allow us to investigate interactions between cognitive processes and other cortical functions, such as postural control [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF] or the cortical control of breathing [START_REF] Grassmann | Respiratory changes in response to cognitive load: a systematic review[END_REF]. A cognitive load is known to perturb the control of balance by diverting attentional resources [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF]). These interactions between the mental process and postural control are mostly modulated by visual or motor stimuli, the difficulty of the cognitive task, postural constraints, sensorimotor expertise (for example, in experts in gymnastics), and ageing [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF].

The subjects in our study were all healthy and had no sensorimotor expertise. They were in a relaxed standing position without any postural stimulation, and the orientation of their gaze was constant across the conditions. Therefore, we may anticipate that the postural impact of a cognitive task on individuals' balance depends mostly on their age and on the intensity of the cognitive load [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF] . A cognitive load is generally associated with improved balance parameters in subjects in their twenties [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Hagio | Effects of breathing movement on the reduction of postural sway during postural-cognitive dual tasking[END_REF] , with no change or impaired balance in older subjects [START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF], and/ or during a high cognitive load [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF]. As expected, considering the median age of the subjects in this study (39 years) and the high cognitive load to which they were subjected, we observed impaired balance during the COG condition. We observed a relative

**

Fig. 6 Variations in respiratory emergence (REm) for the centre of pressure (CoP) displacement amplitude and joint motions (cervical spine, hip, knee, ankle) during the natural breathing (NB) and cognitive conditions (COG) in the standing position in N = 20 subjects. (Wilcoxon's test: *p < 0.05; **p < 0.01; ***p < 0.001) increase, though the difference was not significant, in the breathing frequency during the cognitive load, which was also previously reported [START_REF] Grassmann | Respiratory changes in response to cognitive load: a systematic review[END_REF]. This modulation in the breathing pattern induced by a dual task is not surprising, as in addition to automatic commands, cortical commands transmitted from the primary cortex [START_REF] Similowski | Facilitation-independent response of the diaphragm to cortical magnetic stimulation[END_REF], premotor cortex and supplementary motor area [START_REF] Raux | Electroencephalographic evidence for pre-motor cortex activation during inspiratory loading in humans[END_REF]) control human respiration. To the best of our knowledge, however, our study is the first to demonstrate a specific modulation in the PV interaction under cognitive load. This result was shown in the subjects in our study by an increase in respiratory emergence close to statistical significance (p = 0.0696) and by significant changes in the TLA. This result indicates the central modulation of the PV interaction and suggests that postural control partly depends on central interactions between the cortical control of breathing and cognition in healthy humans. Therefore, as dual tasks are common in real-life settings, our results lead us to hypothesize that postural control may be less efficient in situations associated with a pathological adaptive increase in awake cortical respiratory drive, such as in healthy individuals during experimental dyspnoea [START_REF] Raux | Electroencephalographic evidence for pre-motor cortex activation during inspiratory loading in humans[END_REF] or in some patients with obstructive sleep apnoea syndrome (OSAS) [START_REF] Launois | Cortical drive to breathe during wakefulness in patients with obstructive sleep apnea syndrome[END_REF], chronic obstructive pulmonary disease (COPD) [START_REF] Nguyen | Inspiratory pre-motor potentials during quiet breathing in ageing and chronic obstructive pulmonary disease[END_REF] or hyperventilation syndrome [START_REF] Dubois | Neurophysiological evidence for a cortical contribution to the wakefulness-related drive to breathe explaining hypocapnia-resistant ventilation in humans[END_REF]. In these settings, the pre-inspiratory potential observed in the motor cortex and the supplementary motor area during resting ventilation [START_REF] Raux | Electroencephalographic evidence for pre-motor cortex activation during inspiratory loading in humans[END_REF]) has been associated with a perturbation of attentional resources [START_REF] Sharman | The cerebral cost of breathing: an fMRI case-study in congenital central hypoventilation syndrome[END_REF], which suggests that the balance between cognition, postural control and the control of breathing under cognitive load is impaired. This concept has been demonstrated in healthy subjects by a negative impact of experimental dyspnoea on both cognition and locomotion, as measured by the "timed up-and-go" test and its imagery version [START_REF] Nierat | When breathing interferes with cognition: experimental inspiratory loading alters timed up-and-go test in normal humans[END_REF]. This concept has also been shown in patients with OSAS by a reduction in cognitive performance to maintain a balance performance similar to that of controls [START_REF] Baillieul | Continuous positive airway pressure improves gait control in severe obstructive sleep apnoea: a prospective study[END_REF] , but it remains to be specifically addressed.

Preservation of the cervical spine from breathing-related perturbations and cognitive load

Our analysis at the joint level confirmed a significant reduction in the PV interaction at the cervical level and no change or increase at the other joints. Our results suggest that the PV interaction in humans cannot be modelled using a simple inverted pendulum model; rather, the model must take into account multiple degrees of freedom, as previously reported [START_REF] Hodges | Coexistence of stability and mobility in postural control: evidence from postural compensation for respiration[END_REF][START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF] . Several joints (spine, hip, knee and ankle), each acting as a degree of freedom on the postural body chain, counteract the breathing-related postural perturbation and provide flexibility in central control [START_REF] Hodges | Coexistence of stability and mobility in postural control: evidence from postural compensation for respiration[END_REF][START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF] . In our study, we confirmed that this postural chain was altered under cognitive load. In addition, our study supports the hypothesis that the maintenance of stability of the neck is related to the protection of the cervical spine from ventilatory perturbations, as attested by the reduction in respiratory emergence at the cervical level. It has been previously reported that the breathing-related component of CoP displacements, i.e., the breathing-related postural perturbation, almost disappears during voluntary apnoea in healthy humans [START_REF] Caron | Effects of ventilation on body sway during human standing[END_REF]) . Indeed, in healthy humans, during standing, even the breathing-related rhythmic perturbation is continuously counteracted, and this counteraction is not complete, which implies that natural breathing "normally perturbs" the balance along the multijoint postural chain [START_REF] Kuznetsov | Effects of breathing on multijoint control of center of mass position during upright stance[END_REF]. Therefore, when a subject voluntarily holds his or her breath, the effect of the suppression of the breathing-related postural perturbation on the CoP displacements [START_REF] Caron | Effects of ventilation on body sway during human standing[END_REF]), as well as on the cervical spine motion [START_REF] Hodges | Coexistence of stability and mobility in postural control: evidence from postural compensation for respiration[END_REF], may be linked to a "stabilization strategy". Similarly, in the subjects in our study, the reduction in the breathing-related perturbation, the unchanged amplitude of the motion of the cervical spine during cognitive load, and the increased amplitudes of postural sway and knee and ankle motion probably correspond to a "local stabilization strategy" of the cervical spine that may have limited the impairment of balance; however, we acknowledge this postulation remains to be addressed. Actually, the stabilization of the neck may help maintain the head position and a horizontal gaze [START_REF] Hasegawa | Standing sagittal alignment of the whole axial skeleton with reference to the gravity line in humans[END_REF] . This compensatory adaptation may stem from the necessity to improve visual, vestibular and neck proprioceptive afferent feedback efficiency [START_REF] Bove | Interaction between vision and neck proprioception in the control of stance[END_REF][START_REF] Williams | Use of neck torsion as a specific test of neck related postural instability[END_REF][START_REF] Malmström | Disturbed cervical proprioception affects perception of spatial orientation while in motion[END_REF]) during a high-load cognitive task [START_REF] Huxhold | Dual-tasking postural control: aging and the effects of cognitive demand in conjunction with focus of attention[END_REF][START_REF] Lacour | Posture control, aging, and attention resources: models and posture-analysis methods[END_REF] , which is known to compromise balance by diverting attentional prefrontal resources [START_REF] Mihara | Role of the prefrontal cortex in human balance control[END_REF][START_REF] Stelzel | Contribution of the lateral prefrontal cortex to cognitive-postural multitasking[END_REF] that are primarily dedicated to the control of balance and anticipatory adjustments of the cervical curvature [START_REF] Hamaoui | Influence of cervical spine mobility on the focal and postural components of the sit-to-stand task[END_REF][START_REF] Boulanger | Interaction between the oculomotor and postural systems during a dual-task: compensatory reductions in head sway following visually-induced postural perturbations promote the production of accurate double-step saccades in standing human adults[END_REF]. Consequently, the stability of the cervical spine, which is known to play an important role in the maintenance of balance (see also Introduction section) [START_REF] Gandelman-Marton | Postural stability in patients with different types of head and neck trauma in comparison to healthy subjects[END_REF][START_REF] Hamaoui | Influence of cervical spine mobility on the focal and postural components of the sit-to-stand task[END_REF] , is probably crucial when cortical resources are challenged. Likewise, in COPD patients, who often recruit their respiratory neck muscles, pathologic breathing-related neck stabilization in dual-task settings may represent one of the physiopathological mechanisms of their specific postural dysfunction [START_REF] Janssens | Impaired postural control reduces sit-to-stand-to-sit performance in individuals with chronic obstructive pulmonary disease[END_REF]. This hypothesis needs to be investigated in future studies.

Methodological considerations and limitations

We acknowledge that the instruction to subjects to focus their gaze on a landmark may have encouraged them to stabilize their neck. The instruction to stand barefoot on the force plate and focus their gaze on a landmark on the wall was used to minimize the variability of their posture and to avoid additional postural perturbations between subjects and between the NB and COG conditions. Therefore, the instruction's effect on balance was comparable in the reference NB and COG conditions. In addition, we acknowledge that this instruction may induce an additional cognitive load. Regardless of whether it induced an additional cognitive load, our results demonstrate that a higher cognitive load during the dual task was accompanied by a reduced respiratory postural perturbation at the neck joint level, no change in the neck motion, and increases in the COP displacement and lower joint motions. This strategy seems specific to a central modulation, as it was not observed during "non-cortical" postural perturbations, such as the EC or ITV conditions, while the breathing-related postural perturbation at the neck level remained unchanged.

In our study, the relatively small number of subjects, particularly that of subjects aged under 25 years (n = 3), limits our interpretation of the age effects present in our results. This factor remains to be addressed in future studies.

Balance in the standing position is a complex phenomenon that varies with postural alignment [START_REF] Amabile | Invariance of head-pelvis alignment and compensatory mechanisms for asymptomatic adults older than 49 years[END_REF] , postural tone and metabolic cost [START_REF] Van Emmerik | On the functional aspects of variability in postural control[END_REF][START_REF] Houdijk | Relation between postural sway magnitude and metabolic energy cost during upright standing on a compliant surface[END_REF] . Therefore, some changes in postural sway are not associated with perturbations in balance. However, postural sway increases when balance tends to be compromised. This relation is the reason why we considered, by analogy, the amplitude of the motions of the cervical spine and other joints to be stability parameters of a given segment. We acknowledge that electromyography of the neck muscles can be useful; however, we are confident that the divergence between the cervical spine and other joints in terms of the breathing-related postural perturbation supports our "stabilization hypothesis" at the cervical level.

Conclusion

This study shows that the cervical spine is protected from the breathing-related postural perturbation during a cognitive dual task, while the postural perturbation occurs globally and at inferior joints. This result represents a cortical adaptation of the posturo-ventilatory interaction to stabilize the neck and possibly to limit the cognitive-induced postural perturbation. This adaptation may eventually become impaired in patients suffering from chronic respiratory disease. More generally, this adaptation supports the existence of a strong interaction between cognition, the control of breathing and postural control.

Fig. 1 a

 1 Fig. 1 a Locations of the retroreflective markers and thoracic mesh used to define the body segment frame; b definitions of a specific subject's antero-posterior and medio-lateral axes according to the foot markers; c positions of the cameras located around the subject; d segment vector projected in the sagittal plane and the definition of the articular joint angle (ATMP anterior temple bone, LOCC lateral occipital bone, MAN manubrium, ACR acromion, K4 fourth kyphosis

Fig. 3 Fig. 4

 34 Fig.3Representative cervical spine changes over a breathing cycle with respect to the beginning of each cycle for three subjects under different conditions, with the mean value shown as the black line and the 95% confidence interval (CI) shown as the grey shaded

Fig. 5

 5 Fig. 5 Variations in the amplitude (Amp.) of a the centre of pressure (CoP) displacement; b joint rotations (cervical spine, hip, knee, ankle) during the natural breathing (NB) and cognitive conditions (COG) in the standing position in N = 20 subjects. (*p < 0.05; **p < 0.01; ***p <

Fig. 7 a

 7 Fig. 7 a The cervical spine, hip, knee and ankle signal changes over a breathing cycle during natural breathing (NB) and the cognitive task (COG) in one subject (black line: mean value; grey shaded region: 95% confidence interval); b motion distribution (flexion, no change and extension) measured in the 20 subjects for the cervical spine,

Table 1

 1 Anthropomorphic characteristics and pulmonary function testing results

	Sex (M/F)	10/10
	Age (years)	39 [29; 50]
	Height (cm)	171 [163; 175]
	Weight (kg)	69 [56; 78]
	BMI (kg/m 2 )	23.2 [20.5; 25.6]
	SVC (% predicted)	114 [106; 124]
	FRC (% predicted)	105 [96; 116]
	TLC (% predicted)	109 [99; 117]
	RV (% predicted)	90 [82; 103]
	FEV 1 (% predicted)	110 [103; 118]
	The results are presented as the median [Q1; Q3]
	SVC slow vital capacity, FRC functional residual capacity measured
	by helium dilution, TLC total lung capacity, RV residual volume,
	FEV 1 forced expiratory volume in one second

Table 2

 2 Change in the posturoventilatory interaction during the cognitive load condition TLA time locked averaging, REm: respiratory emergence, CoP centre of pressure TI inspiratory time, TE expiratory time; Amp. Amplitude, REm respiratory emergence, CoP centre of pressure, NB natural breathing condition, COG cognitive condition, EC eyes-closed condition, ITV increased tidal volume condition

	N = 20	CoP	Cervical spine motion Hip motion	Knee motion Ankle motion
	TLA
	No change		+ 22.5	+ 2.5	-12.5	+ 17.5
	Flexion		-12.5	-10	+ 10	-20
	Extension		-10	+ 7.5	+ 2.5	+ 2.5
	p		0.0024	0.2818	0.1384	0.0059
	REm
	(%)	+ 7 [-1; + 12] -14 [-35; -1]	+ 1 [-3; + 10] 0 [-4; + 3]	+ 1 [-3; + 5]
	p	0.0696	0.0020	0.2943	0.8695	0.5217
	Table 3 Posturo-ventilatory interaction at the cervical level during
	the cognitive load, eyes-closed and increased tidal volume conditions
	compared to natural breathing

Service des Explorations Fonctionnelles de la Respiration, de l'Exercice et de la Dyspnée, Département "R3S", Assistance Publique des Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière Charles Foix, 75013 Paris, France

Service de Pneumologie, Médecine Intensive et Réanimation (Département R3S), AP-HP, Groupe Hospitalier Universitaire APHP-Sorbonne Université, Site Pitié-Salpêtrière,

Paris, France

Funding This work was funded by the Chancellerie des Universités de Paris (Grant number: Legs Poix (LEG 1604)); ENS Cachan (Grant number: Ph.D. fellowship); Assistance Publique -Hôpitaux de Paris (Grant number: Grant "poste d'accueil APHP/Arts et Métiers" délégation à la Recherche Clinique et à l'Innovation (DRCI)); Paristech (Grant number: BiomeCAM chair).

Compliance with ethical standards

Author contributions LC, VA, BS and TS contributed substantially to the study design, data analysis and interpretation, and the writing of the manuscript. IR, MCN, PL and PR contributed substantially to the data analysis and interpretation, and the writing of the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work.

Conflict of interest

The authors have no conflict of interest for this study.

Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Eric Maréchal
email: eric.marechal@cea.fr

From a free-living cyanobacteria to an obligate endosymbiotic organelle: early steps in lipid metabolism integration in

Keywords: Paulinella, Galactolipid, Chromatophore, Plastid, Primary endosymbiosis

diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In this issue, Sato et al. (2020) have performed the first comprehensive analysis of glycerolipids in Paulinella micropora, a unicellular organism containing a photosynthetic organelle called the chromatophore, acquired by a primary endosymbiosis about 100 million years ago. Based on lipidomic analyses and metabolic labeling experiments, the provision of fatty acids and/or fatty acid precursors to the host cell, synthesized photosynthetically and rapidly by the chromatophore, is likely one of the early steps in the integration of this recent organelle. The capacity to produce membranes independently of any supply of organic carbon may be one of the critical benefits the heterotrophic host may get from its photosynthetic guest, and this may have been a fitness advantage for this slow growing species. Further work using Paulinella as a model may help understand the early steps of membrane lipid integration in the primary chloroplast, deriving from a more ancient endosymbiosis event, more than 1 billion years ago.

Main text:

Endosymbiosis events are key milestones in the evolution of eukaryotic cells, from the acquisition of the mitochondrion during eukaryogenesis [START_REF] Imachi | Isolation of an archaeon at the prokaryote-eukaryote interface[END_REF] to the emergence of the most sophisticated cellular architectures, following primary, secondary or serial endosymbioses. To our knowledge, the acquisition of photosynthesis by eukaryotes has always derived from the engulfment of a photosynthetic cell, prokaryotic or eukaryotic, leading to the emergence of primary or secondary photosynthetic organelles, respectively (for review, [START_REF] Fussy | Complex Endosymbioses I: From Primary to Complex Plastids, Multiple Independent Events[END_REF][START_REF] Marechal | Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events[END_REF]). The engulfment of a symbiotic cell inside a host cell is necessary to comprehend how novel subcellular compartments can appeared, but is not sufficient to understand the transition to a fully integrated organelle transmitted through generations.

One of the most important challenges for evolutionary science in the 21 st century, is therefore to identify those ancestral partners at the origin of membrane bound compartments, and to reconstruct the transitions from free-living cells to obligate endosymbiotic organelles. This ambitious objective, considered sometimes unattainable [START_REF] Koskela | Looking for the Last Universal Common Ancestor (LUCA)[END_REF], relies on multidisciplinary approaches, combining comparative and functional genomics, phylogenetics, molecular and biochemical analyses and cell biology evidence to propose reasonable scenarios ultimately testable in appropriate experimental systems. For organelles such as the mitochondrion or the primary chloroplast, ancestral endosymbionts are not clearly established yet [START_REF] Ball | Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?[END_REF][START_REF] Cenci | Biotic Host-Pathogen Interactions As Major Drivers of Plastid Endosymbiosis[END_REF]Huang andGogarten 2007, 2008;[START_REF] Marechal | Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events[END_REF][START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Sato | Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan[END_REF]. Models of eukaryogenesis include both mitochondrial-early and mitochondria-late scenarios [START_REF] Poole | Eukaryotic origins: How and when was the mitochondrion acquired? Cold Spring Harbor perspectives in biology[END_REF]. In the most recent model, mitochondria may have emerged early, deriving at least in part from the association of a facultatively aerobic organotrophic bacterium with an Archae host, belonging to the 'Asgard Archae' superphylum [START_REF] Imachi | Isolation of an archaeon at the prokaryote-eukaryote interface[END_REF]). Acquisition of mitochondria may have occurred from 1.9 to 1 billion years ago [START_REF] Marechal | Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events[END_REF]. Concerning primary chloroplasts, molecular, biochemical and ultrastructural evidence support that this photosynthetic organelle derives from a single endosymbiotic event occurring 1.5 to 1 billion years ago [START_REF] Jensen | Chloroplast evolution, structure and functions[END_REF]. Although an ancestral photosynthetic cyanobacteria, seemingly unrelated to present phyla of cyanobacteria, is hypothesized to be at the origin of the primary chloroplast, major components, including plastid synthesizing enzymes, are of non-cyanobacterial origin [START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Sato | Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan[END_REF]. Contribution of other 'partners' have been suggested, including pathogenic bacteria such as Chlamydiales, via additional lateral gene transfers, but cellular relicts of these additional contributors have been lost and are no more visible in the ultrastructure of the organelle [START_REF] Huang | Did an ancient chlamydial endosymbiosis facilitate the establishment of primary plastids?[END_REF]2008;[START_REF] Ball | Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis?[END_REF][START_REF] Cenci | Biotic Host-Pathogen Interactions As Major Drivers of Plastid Endosymbiosis[END_REF]. Having clues on the identity of endosymbiotic cells is not sufficient to understand how novel compartments have remained from cell division to cell division. Over evolutionary time, interdependency reaches such a level that the endosymbiont is no more able to live by its own, becoming a so-called semi-autonomous organelle, when still containing a functional genome, or a completely integrated organelle, when all necessary genes have been transferred to the host nucleus and the organellar DNA is eventually lost. The transfer of genes from the endosymbiont to the host genome, and the reduction of the endosymbiont genome are well-known markers of this integration. Less is known on the driving forces 'rewiring' the endosymbiont and host metabolic networks, optimizing cooperative pathways, developing systems transferring precursors from one partner to the other, eventually reducing redundant pathways that have become futile. In the case of cyanobacteria integration, the two-membrane envelope limiting the organelle, thought to derive from the two limiting membranes of the cyanobacteria ancestor, needs to be scrutinized as it is the interface where transporters facilitating novel exchanges of metabolites will be embedded, and because this membrane system needs to be transmitted from cell division to cell division.

Glycerolipids make the bulk of biological membranes. They consist of a glycerol backbone, esterified by two fatty acids (FA) and harboring a polar head group. In a non-photosynthetic eukaryote, FA are synthesized by a multi-enzymatic fatty acid synthase of type 1 (FAS1) in the cytosol (Figure 1A). The FAS1-generated FA are then used, schematically, at the level of the endoplasmic reticulum to generate glycerolipids, such as phosphatidylcholine (Figure 1A). Cyanobacteria rely on a dissociated fatty acid synthase of type 2 (FAS2) for the assembly of their membrane glycerolipids (Figure 1B). Cyanobacteria membranes are enriched in specific glycerolipids including two major galactolipids (MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol), a sulfolipid (SQDG, sulfoquinovosyldiacylglycerol) and a phospholipid (PG, phosphatidylglycerol). In addition, a glucolipid (MGlDG, monoglucosyldiacylglycerol) is also present, being the precursor of MGDG by epimerization of its polar head group. MGDG, DGDG, SQDG and PG are conserved in cyanobacteria and primary plastids, whereas MGlDG is not. In spite of this conservation of lipid classes, numerous chloroplast proteins involved in their biosynthesis are of non-cyanobacterial origin [START_REF] Boudiere | Glycerolipids in photosynthesis: composition, synthesis and trafficking[END_REF][START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF][START_REF] Sato | Diverse origins of enzymes involved in the biosynthesis of chloroplast peptidoglycan[END_REF]. Reconstructing the early steps of lipid metabolism rewiring is therefore extremely difficult in such an integrated organelle as the chloroplast.

The study of Paulinella chromatophora has allowed the discovery of an independent primary endosymbiosis involving the integration of a cyanobacterium into a Rhizarian amoeba, 100 to 60 million years ago, leading to the emergence of a photosynthetic organelle resembling primary plastids, termed the chromatophore [START_REF] Marin | A plastid in the making: evidence for a second primary endosymbiosis[END_REF][START_REF] Nowack | Chromatophore genome sequence of Paulinella sheds light on acquisition of photosynthesis by eukaryotes[END_REF]Singer et al. 2017). Being more recent, the emergence of the chromatophore is therefore a unique opportunity to study early stages in the transition from a free-living -cyanobacteria (here related to Synechococcus and Prochlorococcus) into an obligate endosymbiotic organelle. It must be stated that the chromatophore is not a chloroplast, and there is no reasonable argument to assess that both organelles should follow the same evolutionary sequences. Nevertheless, converging trends seem to exist, including the transfer of endosymbiont genes to the host nuclear genome, where addressing sequences were added allowing nuclear-encoded proteins to be imported 'back' to the photosynthetic organelle [START_REF] Archibald | Evolution: Protein Import in a Nascent Photosynthetic Organelle[END_REF]Singer et al. 2017).

In this issue, Sato et al. (2020) have performed the first comprehensive analysis of glycerolipids in Paulinella micropora. The authors have confirmed the conservation of cyanobacteria lipids, i.e. MGDG, DGDG, SQDG, PG, as well as MGlDG, which is absent from primary plastid. This lipidomic analysis is consistent with the detection of cyanobacteria lipid synthesis genes in the chromatophore genome [START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF]. Pulse-chase experiments with radiolabeled substrates show that MGlDG is the precursor of MGDG in P. micropora, like in cyanobacteria. The authors also found that the nuclear genome of P. micropora encoded two forms of FAS1, whereas the chromatophore genome contained homologues of FAS2 genes. Both FAS1 and FAS2 systems seem therefore to coexist (Figure 1C). Based on the realistic hypothesis that FAS2 is located in the stroma of the chromatophore, this machinery is expected to provide FA for the biosynthesis of plastid lipids, characterized as enriched in palmitic and hexadecenoic acids, having 16-carbon chain lengths and being saturated (16:0) or monounsaturated (16:1), respectively. Glycerolipids expected to be synthesized at the ER were enriched in longer-chained FA, with more unsaturations, such as 18:1, 20:4, 20:5 and 22:6. Based on metabolic labelling kinetics of photosynthetically fixed carbon, authors hypothesize that FAS2-generated FA may be exported and used for host-cell glycerolipid synthesis at the ER (Figure 1C, arrow 4). Alternatively, an acyl-CoA precursor (Sato et al. 2020) or possibly shorterchained FA, may be exported from the chromatophore and feed cytosolic FA biosynthesis by FAS1 (Figure 1C, arrow 4').

The provision of FA and/or FA precursors to the host, synthesized photosynthetically and rapidly by the chromatophore, are highlighted by Sato et al (2020) as one of the early steps in the integration of this recent organelle. The capacity to produce membranes independently of any supply of organic carbon may be one of the critical benefits the heterotrophic host gets from its photosynthetic guest, and this may have been a fitness advantage for this slow growing species. Question marks remain, such as the precise localization of glycerolipids within P. micropora cell, and the precise nature of the precursor(s) provided by the chromatophore to the ER for host membrane glycerolipid production. Further work using Paulinella as a model may help understand the early steps of membrane lipid rewiring following the acquisition of a photosynthetic organelle in other systems as well, including the primary chloroplast. Primary plastids show a much higher level of integration: FAS2-generated FA are first used in the plastid for the production of glycerolipids via a pathway termed 'prokaryotic'. Whereas FAS1 has been lost in the cytosol of Archaeplastida, plastid FA are exported to the cytosol and used in the ER to generate glycerolipid molecular species termed 'eukaryotic'. In Viridiplantae, such as Angiosperms, some precursors with 'eukaryotic' structures are then imported 'back' into the chloroplast to be used for plastid glycerolipid biosynthesis (Figure 1D, arrow 5). Plant chloroplast lipids are therefore synthesized by a cooperation of plastid-localized 'prokaryotic' and ER localized 'eukaryotic' pathways. Over evolution, redundant components of the 'prokaryotic' pathway have become futile [START_REF] Marechal | Modeling of regulatory loops controlling galactolipid biosynthesis in the inner envelope membrane of chloroplasts[END_REF] and were lost independently in numerous clades of Angiosperms [START_REF] Mongrand | The C16:3/C18:3 fatty acid balance in photosynthetic tissues from 468 plant species[END_REF]. Interestingly, the 'prokaryotic' pathway relies on noncyanobacterial enzymes in primary plastids [START_REF] Sato | Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis[END_REF], whereas it does rely on cyanobacterial proteins in the chromatophore. If appropriate tools were available to efficiently engineer Paulinella nuclear genome, one could test the effect of the introduction of some noncyanobacterial genes producing galactolipids in the nuclear genome of green algae or plants and evaluate whether their presence could lead to the loss of cyanobacterial genes, in the same way this is hypothesized to have occurred in chloroplasts. Figure 1. Integration of fatty acid and glycerolipid metabolism following the primary acquisition of a cyanobacteria by a eukaryotic host cell. Two independent primary endosymbiosis events have led to the emergence of either a chromatophore (chr) in Paullinelidae or a primary plastid (pl) in Archaeplastida. A. In mitochondriate eukaryotes, a fatty acid synthase of type 1 (FAS1) generates fatty acids (FA) in the cytosol (red arrow, 1). These FA are utilized as substrates in the endoplasmic reticulum (ER) for the production of glycerolipids. ER-generated glycerolipids serve as building blocks for the biogenesis of cell membranes (blue arrow, 2). These include the endomembrane system, comprising the ER, nuclear envelope (nuc), the Golgi apparatus, trans-Golgi network, vesicles, vacuoles and plasma membranes, all connected by direct membrane bridges or vesicular trafficking. ER-generated glycerolipids can also be transferred to the mitochondria (mit), via inter-organellar contact sites. B. In cyanobacteria, a dissociated fatty acid synthase of type 2 (FAS2) generates FA (yellow arrow, 3) utilized for the production of membrane glycerolipids. These include cyanobacteriaspecific lipids such as galactolipids and the sulfolipid. C. In Paulinellidae, genomic evidence indicate that FAS2 and FAS1 systems coexist. Sato et al., 2020 have shown that FAS2-generated FA are likely used as substrates for the production of galactolipids and the sulfolipid, as well as glycerolipids usually synthesized in the ER, phosphatidylcholine and triacylglycerol. This suggests a transfer of chromatophore-generated FA to the ER (green arrow, 4) and/or precursors to cytosolic FAS1 (green arrow, 4'). D. In Archaeplastida, such as green algae and plants, the primary plastid, or chloroplast, is the site of FA synthesis for membrane glycerolipids. FAS2generated FA are either used for plastid glycerolipid synthesis (yellow arrow, 2) or exported to the cytosol (green arrow, 4). Some ER-synthesized glycerolipids are imported back into the chloroplast (purple arrow, 5), where they serve as precursors for a part of plastid lipid synthesis. In the course of the evolution of Archaeplastida, the dependence of plastid on ER-synthesized glycerolipids increased, leading to a tight cooperation between these two organelles for FA and glycerolipid metabolism. Singer, A., Poschmann, G., Muhlich, C., Valadez-Cano, C., Hansch, S., Huren, V., et al. (2017) Massive Protein Import into the Early-Evolutionary-Stage Photosynthetic Organelle of the Amoeba Paulinella chromatophora. Curr Biol 27: 2763-2773 e2765.

  Figure Legend

  .,Yoshitomi, T. and Mori-Moriyama, N. (2020) Characterization and biosynthesis of lipids in Paulinella micropora MYN1. Evidence for efficient integration of chromatophores into cellular lipid metabolism. Plant Cell Physiol.

Plant & Cell Physiology


J N Girard

P Zarka

M Tagger

L Denis

D Charrier

A Konovalenko

Antenna design and distribution for a LOFAR Super Station in Nançay

The Nançay radio astronomy observatory and associated laboratories are developing the concept of a "Super Station" for extending the LOFAR station now installed and operational in Nançay. The LOFAR Super Station (LSS) will increase the number of high sensitivity long baselines, provide short baselines and an alternate core, and be a large standalone instrument. It will operate in the low frequency band of LOFAR (30-80 MHz) and extend this range to lower frequencies. Three key developments for the LSS are described here: (i) the design of a specific antenna, and the distribution of such antennas (ii) at small-scale (analogphased mini array) and (iii) at large-scale (the whole LSS).

1 The LOFAR Super Station

Principle and general design

The Nançay Radio Observatory (www.obs-nancay.f r) hosts the LOFAR international station "FR606". Each LOFAR station is a subset of two arrays of antennas: the "Low Band Antenna" (LBA -[30-90] MHz) array and the "High Band Antenna" (HBA -[110-250] MHz) array, both connected to the two inputs of the station back-end (more details about LOFAR in [START_REF] De Vos | The LOFAR Telescope: System Architecture and Signal Processing[END_REF]). A third input to the cabinet's receivers exists, which was initially dedicated to a "Low Band Low" (LBL) antenna field. The main idea underlying the LOFAR Super Station (LSS) concept is to build and connect to this input a new field of 96 new antennas operating in the LBA range (and extend it to ≤ 15 MHz). Each of these new antennas will actually consist of an analog-phased mini-array of 10-20 antennas, increasing thus the sensitivity of the station in the LBA range, while remaining fully compatible with the whole LOFAR array.

Each LSS mini-array of 10-20 antennas (dual-polarized) must be analog-phased because only 96 dualpolarization inputs available. These mini-arrays are actually very similar in their principle to the HBA tiles of 16 (4×4 square grid) high frequency antennas, phased using delay lines. However, the mini-array layout will not be necessarily square. The signals beamformed at the tile level are the signals that are digitized and numerically combined in the cabinet's back-end, either in summation (phased array mode) or in correlation (interferometer mode). The LSS will consist of 96 mini-arrays distributed within ∼150 m of the LOFAR station cabinet (see Figure 1 right). Its layout must then be optimized at two different scales : mini-array and full LSS.

Interests of the LOFAR Super Station

Within the LOFAR array: The LSS will provide several improvements to the present LOFAR design and capabilities. First, the 10 -20 times improved sensitivity of the mini-array compared to a standard LBAs will increase the sensitivity of all long baselines involving the LSS. It will thus approximately double the number of long baselines with high sensitivity. Second, only station-to-station correlations were planned in the initial LOFAR project, which implies in the LBA range a minimum baseline length of one LBA field diameter (B min ∼60 m). This implies that LOFAR would be blind to structures larger than λ/B min at wavelength λ. With the LSS, tile-to-tile correlations will be performed, providing baselines as short as a mini-array diameter (10-15 m) and up to the LSS diameter (∼300 m) with 10-20 times better sensitivity than antenna-to-antenna correlations. The LSS will thus fill very efficiently a missing part of LOFAR present (u,v) coverage in the low band. Third, several LOFAR observation programs need large bandwidths and excellent calibration rather than high angular resolution and will consequently use only the stations of the core. In the meantime, remote and international stations may be correlated in parallel by the central computer and run other programs. By correlating the LSS to all remote and international stations, sensitive long baselines will be restored. The LSS can thus be viewed as an alternate core providing a decent (u,v) coverage and a good sensitivity. In this way, the LSS can contribute to "create" up to 30% of additional LOFAR observing time in parallel with core-only observations.

As a stand-alone instrument: With N = 19 antennas (Figure 1 right), the LSS will have an effective area ≃ 96 × 19λ 2 /4 ≃ 40000 × (λ/10) 2 comparable to that or the Giant Meterwave Radio Telescope (GMRT) in India, ≃ 3× the Very Large Array (VLA) in New-Mexico, or ≃ 10× the Nançay Decameter Array (NDA). It will thus be a large instrument by itself, with relevant standalone use independent of LOFAR, with no loss for LOFAR when the Nançay FR606 is not included in ongoing obsevations.

Scientific objectives: Scientific programs of the LSS (within LOFAR or in standalone mode) will include low frequency surveys, detection of weak sources in time-frequency (coherent phased-array) mode, contribution to the study of large-scale diffuse structures, etc. and it will in addition provide a better calibration for VLBI imaging. A standalone LSS is also well adapted to student training purposes.

2 Three key design studies for the LSS LSS feasibility, design, cost and prototyping studies are presently ongoing. We present below the results of three design studies that are crucial for the LSS: (i) the antenna design, (ii) the antenna distribution in a mini-array, and (iii) the 96 mini-arrays distribution in the LSS.

Antenna design: Several low frequency ground-based instrument are presently under development : LOFAR [START_REF] De Vos | The LOFAR Telescope: System Architecture and Signal Processing[END_REF], the Long Wavlength Array -LWA [START_REF] Ellingson | The Long Wavelength Array[END_REF], the Murchison Widefield Array -MWA, the Giant Ukrainian Radio Telescope -GURT, etc. For each of them, specific antennas have been developed in order to meet their scientific and technical requirements.

Antenna design implies the determination of physical (geometrical) and electrical parameters of the antenna radiators. These parameters are the antenna beaming pattern in E & H planes (constraining the array's Field of View (FoV)), the level of side lobes, the frequency bandwidth, and the efficiency (related to electrical and ground losses). In a sky-dominated noise regime (T sky ≈ 23000K at 30 MHz), we can neglect the noise contribution from the antenna itself. To limit the noise of instruments beyond the antenna (cables, electronics, etc.), a low noise amplifier (LNA) is directly attached to the antenna radiators, forming a so-called "active" antenna. For a phased-array application, a large and smooth beam pattern is required, close to that of an isotropic antenna, as it will condition the final FoV of the array, which is expected to go down to an elevation of 20 • with LOFAR. We also wish to obtain a broadband antenna that can operate down to 15-20 MHz.

Considerations of cost effectiveness constrain the design space to linearly polarized dipoles, thus we carried out comparative studies of different geometry of radiators using the numerical electromagnetic code (NEC -www.nec2.org). This method of moments code, can derive among the simulated far field pattern and the electrical parameters of any antenna defined by a wire model and feed (characteristics in reception are obtained by application of the reciprocity theorem). We investigated the two classes of radiator designs ("butterfly" or "bow-tie", and "inverted-V" antennas) and the influence of a ground plane (metal grid), and we performed optimization studies of their parameters (height, length, droop and fork angles, grid mesh size and step, etc.).

These studies led us to select a "thick" [START_REF] Balanis | Antenna Theory: Analysis and Design[END_REF] inverted-V dipole similar to the LWA Fork [START_REF] Ellingson | The Long Wavelength Array[END_REF]. We found that -as for LOFAR and the LWA -a metallic ground screen is necessary for inverted-V antennas in order to reduce ground losses (efficiency > 50% -resp. > 80% -of that of a Perfect Electrical Conductive plane at 20 MHz -resp. 80 MHz), and to ensure the stability of the antenna impedance against variations of the ground characteristics (dry or wet ground). In parallel, various LNA architectures and matching have been considered. A first prototype of this thick dipole has been built in Nançay in order to test the relevance of our simulation results against measurements on the sky.

Distribution of antennas within a mini-array:

The role of the mini-array is to combine analog antenna signals to synthesize a single wide beam and coarsely point it toward the direction of interest. The fine pointing of the LSS beam (96 mini-arrays) will be done either by the LOFAR beamforming system in the station back-end or a stand-alone beamformer, and will be therefore tapered by the beam pattern of the mini-array. The constraints on the distribution of the antennas within a mini-array include : a smooth & symmetric primary beam, with a low level of side lobes, a large effective area (high sensitivity), a not too complex phasing scheme, a low mutual coupling between antennas, etc. We first performed an optimization study of the mini-array distribution aiming at the maximum reduction of side lobes. We used global minimization algorithms such as the simulated annealing [START_REF] Kirkpatrick | Optimization by Simulated Annealing[END_REF] based on a thermodynamically-controlled Monte-Carlo displacement of the positions of the antennas. The resulting distributions are dense arrays (whose compactness is limited by a threshold distance between antennas) displaying circular symmetries around the phase center but without any kind of periodicity. Such an axisymmetric and aperiodic shape guarantees the minimization of the side lobes (down to -30 dB attenuation or more) and the absence of strong grating lobes. But it is also very difficult and costly to phase by analog means in practice. Thus, we opted for an array of antennas presenting regularities along two orthogonal x and y directions (as in NDA or GURT). This implies large savings in cable lengths. A good compromise between beam characteristics and phasing complexity is shown in Figure 1. By using a triangular lattice, it is possible to find a balance between periodicity (which causes large grating lobes but cheaper phasing) and axisymmetry (give an axisymmetric primary beam).

Regular arrays also present the advantage to reduce the mutual coupling variability from an antenna to another because all embedded antennas (i.e. not at the edges of the mini-array) behave in a similar way. Conversely, in irregular/aperiodic arrays, the coupling may vary substantially from one antenna to the next.

Further studies of distribution, phasing, and coupling effects are ongoing.

Distribution of mini-arrays within the LSS: The LSS will consist of 96 mini-arrays distributed in an area of ∼300 m diameter around (or off-centered) the back-end of the FR606 station. It will work either in a phased-array mode or in interferometer mode. The digitization of each mini-array output gives a large flexibility in the distribution of the 96 mini-arrays. The main constraints that the LSS must fulfill are a low side lobe level and a good (u,v) coverage by the 96 × 95/2 baselines. As the filling factor of a disk of 300 m diameter by mini-arrays of 19 antennas is high (∼64% at 30 MHz), the LSS will be a rather dense array/interferometer at low frequencies. A pseudo-random homogeneous distribution of mini-arrays over the whole LSS area, taking into account specific constraints of the station environment (pond, building, other instruments, etc.) is an acceptable baseline solution. We are in the process of optimizing this first solution by using the algorithm described in [START_REF] Boone | Interferometric array design: Optimizing the locations of the antenna pads[END_REF]. It is a "pressure-driven" algorithm that optimize the (u,v) coverage (relatively to a target (u,v) model) of a "gas" of individual antennas, taking into account a "site mask".

Another major improvement of the LSS response is brought by rotating by a random angle each miniarray relative to a reference direction and rotating back the antennas within the mini-array in order to keep all antennas along the 2 main polarization axes, as it was done for the LOFAR antenna fields. We computed that this decreases by ∼8 dB the side lobes level in our case. But it come with an increase the complexity of its calibration and the mutual coupling variability. We are currently modeling the whole LSS in interferometer mode using the MeqTrees software package [START_REF] Noordam | The MeqTrees software system and its use for third-generation calibration of radio interferometers[END_REF]. It enables one to establish the Measurement Equation (product of "Jones" matrices) describing the instrument and to solve for its parameters.

Conclusion

LSS detailed design, prototype and tests studies (including the construction of 3 mini-arrays), and cost evaluation, will be pursued in the next ∼20 months. Its detailed scientific case is being developed in parallel (and inputs are permanently welcome). We expect LSS construction to start in 2013. If the concept is successful, it could be applied by other European participants to LOFAR, preparing a future "super LOFAR".

Figure 1 :

 1 Figure 1: Left: The LOFAR Super Station is a set of 96 mini-arrays (or tiles) of 10-20 antennas analogphased, spread around the Nançay international LOFAR Station within a range of ∼150 m. Right: Possible antennas distribution within a mini-array, here with N = 19 elements arranged on an hexagonal grid.

Acknowledgements : The authors acknowledge the support of the Observatoire de Paris, the CNRS/INSU, and the ANR (french "Agence Nationale de la Recherche") via the program NT09-635931 "Study and Prototyping of a Super Station for LOFAR in Nançay".