Chiari-like malformation in the Cavalier King Charles Spaniel is a herniation of the cerebellum and brainstem into or through the foramen magnum. This condition predisposes to Syringomyelia; fluid filled syrinxes within the spinal cord. The resulting pathology in spinal cord and cerebellum create neuropathic pain and changes in gait. This study aims to quantify the changes in gait for Cavalier King Charles Spaniel with Chiari-like malformation and Syringomyelia.n = 9) and without (n = 8) Syringomyelia to Border Terriers (n = 8). Two video cameras and manual tracking was used to quantify gait parameters.We compared Cavalier King Charles Spaniel with Chiari-like malformation with  contains supplementary material, which is available to authorized users. The Cavalier King Charles Spaniel (CKCS) is an example of a dog selectively bred for distinct conformational traits. The breed shows a high degree of juvenile behaviour and brachycephalic traits associated with a juvenile morphology (paedomorphic) when compared with the wolf and other companion dog breeds . This seCoordination has been defined as “an ability to maintain a context-dependent and phase-dependent cyclical relationship between different body segments or joints in both spatial and temporal domains” . CoordinThe cerebellum is known to adjust and control balance and locomotion, incorporating autonomous sensorimotor feedback from the spinal cord and feed-forward from the cerebral cortex and other primary and secondary motor centres of the brain . The cerDogs were recruited into three groups; CKCS with CM only (Group CM), CKCS with CM and SM (Group CMSM) and Border Terriers. CKCS were recruited to the study if they had a neurological examination and an MRI report showing CM (<1 year) or CM and SM and the owner would consent to the dog participating in the study. Border Terriers were chosen as control breed (Group Control) due to their similar height and conformation to CKCS as described by the UK Kennel Club Breed Standards. Border Terriers were recruited for the study if they visited the QMH for routine veterinary work or elective procedures or through members of staff. Both CKCS and Control groups were examined and excluded from the study if the gait assessment showed clinical signs of lameness or systemic disease. The control group dogs were excluded if the owners reported concern of neurologic disease or if the gait evaluation showed ataxia. Measurements of distance between the shoulder joints were obtained and showed no difference between the CKCS and the control breed. The study was preapproved by the ethics committee of the Royal Veterinary College, approval number URN 2012 1139. All owners volunteering their dogs to the study signed a consent form explaining the details of the study.Motion capture was performed in the structure and motion lab at the Royal Veterinary College as well as at the homes of participating dog owners unable to travel to the college campus. The dogs were walked over-ground along an 8 m runway with black electrical tape marking up 0.5 × 0.5 m squares into a 0.5 × 5 m grid made up of black tape on a flat surface.Two digital stills set to film in full HD (1920 × 1080 pixels) and a frame-rate of 60 fps and were used in the study . The cameras were set up more than 3 m away from the grid . The image processing and analysis tool allowed for a scale to be set at 0.5 m matching the grid. Based on the reference frame ipsilateral distance, stride length, pelvic distance and thoracic distance were measured using the ‘straight line tool’. To ensure measurements were from the same point on the foot for each individual, measuring started at the point in between the 2nd and 3rd digit of the foot when moving towards the camera. The middle of the plantar ball of the paw that is visible when the dog is moving away from the camera, and when in lateral view the measurement was taken from carpal joint down to the middle of the foot.The videos were processed with video analysis software . A frame-by-frame advancement was used in the software to calculate step cycle and stance time of each subject. In order to measure the other 4 parameters, the video analysis software (Quintic) was then used to obtain still images each time the subject placed a paw on the ground. The still images were saved as JPEG format. The still images were processed using an image processing and analysis tool  and for the control group compared to CMSM (p = 0.01). When comparing the three groups and the CV of stride length, there is a significant difference between control group compared to either CKCS with CM (p = 0.003) or with CMSM (p = 0.001) as well as CM and CMSM pooled together (p < 0.001). When comparing the three groups and the CV of the ipsilateral paw distance, there is a significant difference between control group compared to either CKCS with CM (p = 0.04) or with CMSM (p = 0.02) as well as CM and CMSM pooled together (p = 0.01).The descriptive statistics are summarised in Table Firstly, we demonstrate increased variability through a significant increase in CV for the spatio-temporal characteristics for the CM group and the CMSM group compared with the control breed Table . In partMotor control theories such as the uncontrolled manifold hypothesis and synergies are the current prevailing theories for how quadrupeds and bipeds maintain a tight control of coordination. The uncontrolled manifold theory suggests that the extremities and head aim to keep the centre of mass (CoM) as stable as possible by varying the trajectory of the extremities . In a reChildren and adult humans with cerebellar ataxia and normal children show increased gait variability , 25–27. The increased variability of ipsilateral distance between paws and stride length in both the CM and CMSM groups is similar to that documented in humans with cerebellar ataxia  as well The human preference for juvenile traits in the CKCS pedigree has had a significant impact on behaviour  where thWe have not investigated the spatio-temporal characteristics across multiple breeds as this is necessary to fully support the argument of cerebellar ataxia in CKCS with CM and CMSM and further studies should be performed of the breed to breed variations and effect of age and cerebellar disease on gait in dogs. We also recognise there is a correlation between the size of a dorsal syrinx and clinical signs of neuropathic pain  and thiFurther the lack of control for gait speed when the dogs are walking along the runway could contribute to the stride-to-stride variation however this effect should not have a systematic bias between the groups. Future studies should include a neurologic examination of control dogs to improve the comparability.We conclude that Cavalier King Charles Spaniels with CM and CMSM have an increased variation of the ipsilateral distance between paws and length of the stride and a significantly wider base of support in the thoracic limbs but not in the pelvic limbs compared to Border Terriers. This could be an effect of cerebellar disease or cranial spinal cord disease however further breed-to-breed gait studies and objective quantification of hypermetria are necessary to further this conclusion. The increased spatial variation from CM and CMSM compared to control dogs could be used in rehabilitation and tracking of gait over time as well as monitoring for effect of treatment on gait changes and coordination.Additional file 1: Table S1.Table showing all information of the dogs allocated to each of the three groups used in the proposed study. Grading is used to define the presence and stage of syringomyelia. (DOCX 14 kb)Additional file 2: Figure S1.Representation of the camera setup and paw placements within the 0.5 × m grid. Two cameras are placed at the front and perpendicular to the grid, allowing video capture of both the forward and away movement as well as the lateral movement of the dog. Freeze frames were taken as each paw was placed flat on the ground and could then be used to calculate the parameters described. This figure displays how the pelvic distance is calculated. First the right pelvic paw was measured from the centre of the paw to the external grid line (blue line). The same was measured from the left pelvic paw to the same external grid line (green line). The two distances were then subtracted to result in the distance between the two pelvic paws (red line). (JPG 37 kb)


The first part of the recording Fig.  shows coIt is important to realise that, in contrast with the RV tip to ring configuration default depicted on the marker channel, this biventricular implantable cardioverter-defibrillator was programmed RV tip to RV coil. AF sensing in the RV lead is proven by the synchronous atrial and ventricular sensing in Fig. In this case, the key problems were the position of the RV lead and the coil being used as a proximal sensing electrode. Once the device was reprogrammed to tip to ring, the problem was solved. Patient had a good clinical response to biventricular pacing and is doing well.Although this is a rare phenomenon, the lesson to be learned is that, when we change the sensing vector to an integrated bipolar setting, we must make sure that the coil is well within the right ventricle.


Much of the novel physics predicted to be observable in the ultrastrong light–matter coupling regime rests on the hybridisation between states with different numbers of excitations, leading to a population of virtual photons in the system’s ground state. In this article, exploiting an exact diagonalisation approach, we derive both analytical and numerical results for the population of virtual photons in presence of arbitrary losses. Specialising our results to the case of Lorentzian resonances we then show that the virtual photon population is only quantitatively affected by losses, even when those become the dominant energy scale. Our results demonstrate most of the ultrastrong-coupling phenomenology can be observed in loss-dominated systems which are not even in the standard strong coupling regime. We thus open the possibility to investigate ultrastrong-coupling physics to platforms that were previously considered unsuitable due to their large losses. Phenomena linked with ultrastrong coupling rely on the presence of virtual photons in the system’s ground state. In this article, using exact diagonalization, De Liberato shows that the population of ground state virtual photons is only quantitatively affected by losses. Interest in this novel regime has been fuelled by its rich phenomenology, including quantum phase transitions20, modification of energy transport22 and optical properties27, and the possibility to use it to influence chemical and thermodynamic processes32.The study of the interaction between light and matter has been one of the cornerstones in the development of quantum mechanics. In most cases the light–matter coupling is weak enough to be intuitively described in terms of the emission and absorption of photons, while the matter system jumps between two of its quantised eigenstates. When the resonant coupling of an optically active transition with a mode of the electromagnetic field is larger than the losses determining their respective linewidths, it becomes possible to spectroscopically resolve the splitting due to the interaction. The system is then said to be in the strong light–matter coupling regime. Contrary to the weak coupling case, here the interaction between light and matter cannot be described in terms of emission and absorption of photons, but it is necessary to consider the dressed light–matter excitations of the coupled system. Finally, if the coupling becomes even larger, comparable with the bare frequencies of the excitations, we enter a third regime, called ultrastrong coupling. Such a regime, described33. Higher-order perturbative effects due to the antiresonant terms in the Hamiltonian, which do not conserve the number of excitations, are then able to hybridise states with different numbers of excitations. Such an hybridisation is at the origin of much of the ultrastrong-coupling phenomenology27 and one of its most striking consequences is that the ground state 45, a process which presents strong analogies with the dynamical Casimir effect50 and with the Hawking radiation53. Non-adiabatic modulation of the parameters of a light–matter coupled system has been experimentally achieved in dielectric systems by modifying the dipole density with a femtosecond laser pulse54, and in superconducting circuits by applying an external flux bias56. Another promising proposal in this direction is the use of the superconducting to classical transition to alter the resonator response57.The relevant dimensionless parameter in a perturbative treatment of the light–matter interaction is the ratio between the coupling and the bare excitation frequencies. In the ultrastrong-coupling regime such a parameter becomes non-negligible, with values larger than one recently achieved60, their impact on the structure of the ground state and on the presence of virtual photons has been for the moment almost totally neglected. One of the reasons is that interest in ultrastrong coupling has historically emerged from the study of strongly coupled systems. Its achievement is usually demonstrated by fitting the resonant splitting of the coupled resonances to measure the coupling strength. Any system in which ultrastrong coupling has been demonstrated was thus a fortiori also in the strong coupling regime. But in this situation the loss rate is the smallest frequency scale of the problem, and perturbative methods that neglect its impact on the structure of the ground state are totally justified. Nevertheless strong and ultrastrong coupling depend on different figures of merit, and they are thus a priori independent regimes. Systems in the ultrastrong but not in the strong coupling regime could still have large couplings, as well as large losses, both comparable with the bare frequency of the optical transition. The ground state of the system would then also hybridise with its environment, modifying its structure. A thorough investigation of the effect of the losses on the virtual photon population in those systems then becomes necessary to ascertain whether ultrastrong-coupling phenomenology can still be observed or if it is completely quenched by the effect of the environment.While the best way to correctly model losses in the ultrastrong-coupling regime has been object of much attention17, strong coupling has not yet been achieved. Another example are hybrid quantum systems which were recently highlighted as ideal platforms for some quantum vacuum emission scheme34. These have only very recently61 achieved strong coupling, as they are characterised by large losses63.In this article we calculate through a non-perturbative procedure the virtual photon population in presence of arbitrary losses. Specialising our results to the case of Lorentzian light and matter resonances we prove that losses do have an impact on the virtual photon population, but only a quantitative one. Even in presence of dominant losses a sizeable fraction of virtual photons remains. Ultrastrong-coupling phenomena can thus be observed in systems with very large couplings, in which losses have impeached the observation of strong coupling for intrinsic, or technological reasons. Prime examples could be graphene single and bilayers in which, notwithstanding different theoretical calculations predicting very large dipolesω), we show in the Methods section that the number of ground-state virtual photons in the mode k is given byj are the solution of the dispersion equationNk also represents the number of photons with wavevector k emitted upon an instantaneous switch-off of the interaction1: after the switch-off the ground state would be the standard, empty vacuum and all the virtual photons would be free to radiate. Notice that this identification remains valid in presence of losses because without light–matter coupling there can be no absorption and all the virtual photons in the ground state are emitted.The quantity of interest for us will be the photonic population in the ground state ω0, coupling strength ωc and linewidth γL. It is well known that the spectrum of a medium described by Eq. (γL > 2ωc) or in the strong (γL > 2ωc) coupling regime, as shown in Fig. 2 the threshold of ultrastrong coupling is usually assumed to be ωc ≥ 0.2ω0. The poles of the dielectric function in Eq.  to γmax (dotted red line). We see that, while losses do have a clear effect upon Nk, even in the case of an overdamped oscillator the virtual photon population is only diminished by roughly 25% when compared with the nondissipative case.In Fig. γP is present, obtained from the general approach developed in the Supplementary Note γL = γP = γmax and the light and matter resonances are both overdamped. Moreover from Supplementary Fig. Nk essentially depends on the total linewidth γL + γP. The results in Fig. The results in the case in which also a photonic linewidth ωc is varied from 0 to ω0 we expect the system, at least for the small and intermediate values of γL, to transition from the weak to the strong coupling regime. Still no discontinuity is observed in Nk showing that strong coupling has no direct effect on the virtual photon population. This can be confirmed from Fig. γL = ω0, as ωc is varied, identifying with different symbols specific values marked in Fig. 2a. We can clearly see a transition from the weak to the strong coupling regime as the two eigenfrequencies transition from having different loss rates but similar frequencies to the opposite case.As the coupling ck = ω0. In order to ascertain if this also remains true out-of-resonance, in Fig. Nk as a function of ωc = 0.5ω0 and values of the dissipation covering all the range between 0 and γmax. We verify again that dissipation does not have any qualitative impact, and also its quantitative effect is negligible for a plot over multiple orders of magnitude of the wavevector. More important, we do not see any sign of resonant enhancement of virtual photon population. This can be understood from the fact that the mixing of the vacuum state with states containing photonic excitations is due to the antiresonant terms of the Hamiltonian, and thus no resonance condition should be expected. Performing a perturbative development in inverse powers of k  from of the dissipationless version of Eq.  can be found in the Supplementary Note k asThe linear transformation used to diagonalise the system can then be inverted, allowing us to express the photonic operators as linear combinations of the mentclass2pt{minim27.Comparing Eq.  to Eq.  we can vωc, which quantifies the light–matter coupling, can be arbitrarily large. Our approach in fact takes into account the diamagnetic A2 term, forbidding the onset of superradiant phase transitions in polarisable media70. In order to verify our results, in the Supplementary Note As detailed below, the expression in Eq.  can be ck from Eq. (65j is a solution of Eq. (j to have the brach cut on the positive real axis, we arrive at65The total number of photons in the mode from Eq.  can be cThe data that support the findings of this study are available from the author upon request.Supplementary Information


SBF), total blood volume of the finger (TBVF), pulse pressure (PP), heart rate (HR), systolic, and diastolic blood pressure (BP) were analyzed in 10 healthy young subjects in supine and upright positions. By lowering the arm in supine position, SBF decreased compared to its basal measurement, PR increased, and PP contracted, indicating arterial vasoconstriction that rise BP. TBVF increased, demonstrating an increment in venous volume. HR did not change, reflecting no action of the baroreceptor reflex. In upright position with lowered arm, there was an additional increase in BP variables, demonstrating vasoconstriction. Moreover, BP and HR showed oscillations at 0.1 Hz reflecting the entrance of the baroreceptor reflex. The action of gravity in a dependent limb in supine position induces a regional vasoconstriction and an increase of BP due to activation of the myogenic response, while the baroreceptor reflex or other neural factors do not appear to operate. In the upright position with the arm dependent, there is a further increase in regional vasoconstriction and BP with reciprocal changes in HR, indicating the entrance of the baroreceptor superimposed to the myogenic response. This study demonstrates that the myogenic and baroreceptor vasoconstriction can be separated in vivo.Myogenic vascular response is a form of systemic and regional vasoconstriction produced increasing the intra‐arterial pressure by gravity. Here, the vasoconstriction due to the myogenic response, induced by the gravitational action in a dependent limb, is separated from that caused by the baroreceptor reflex. Regional changes of skin blood flow ( It is widely accepted that when a normal subject assumes the upright posture there is a redistribution of the blood volume; from 600 to 800 mL are displaced to the abdomen and the lower extremities due to the action of gravity. As a consequence of the volume displacement, there is an abrupt fall in the blood pressure (BP) of about 40 mmHg (5.33 kPa) that is rapidly compensated, in about 20 to 25 sec, by the vasoconstriction of the arteries below the heart. Concomitantly, there is an increase in the heart rate (HR) of about 15 beats per minute, which is induced by the withdrawal of the vagal action and subsequently maintained by sympathetic discharges to the sinus node, thus increasing the cardiac output. There is a simultaneous increase from 80 to 100 mmHg (10.66–13.33 kPa) in venous pressure in the veins below the heart. This change is directly proportional to the distance from the veins of the lower limbs and the abdomen to the heart, specifically to the right atrium. The venous return to the right side of the heart is aided by the changes induced by the negative intrathoracic pressure , produced by inspiration and by the muscle contraction of the abdomen and muscles of the lower extremities (Wieling and van Lieshout The vasoconstriction of the arteries has been attributed to an activation of the baroreceptor reflex (BRR) that induces, as a consequence of the fall of BP at the carotid sinus and aortic receptors, a sympathetic discharge to the sinus node (cardiosympathetic BRR) and to the arterial and arteriolar vessels by the BRR (vasosympathetic reflex) Rowell . The comHowever, this classical model does not take into consideration the increase in BP induced by the pressure of gravity on the walls of the arteries and arterioles . Subjects underwent a complete physical examination by a medical doctor. They were healthy control subjects without a history of hypertension, diabetes mellitus, cardiovascular or peripheral vascular disease, peripheral, or central neurological disorders. They did not take antihypertensive, antidiabetic, sympathetic, anticholinergic, or adrenergic medications. They all had blood glucose levels below 92 mg/dL and normal muscle stretch reflexes.All the subjects provided written informed consent after the procedure was explained to them, emphasizing its noninvasive nature. The study was performed according to the Helsinki and Tokyo recommendations for human investigations and was approved by our institutional review board.°C. The skin temperature on the arm was kept between 34 to 36°C measured with an infrared thermometer .The room temperature was kept at 23Pulsatile arteriolar skin blood flow (SBF) and nonpulsatile total blood volume of the index finger (TBVF) were recorded with a red‐infrared photoplethysmograph. Red‐infrared photopletismography has been used in several studies for a long time Burton . Pulsati® sphygmomanometer model CE0297, México City, Mexico).Basal BP of each subject was assessed by routine sphygmomanometry after a 5‐minute rest both in a supine and in an upright position  to monitor multiple hemodynamic parameters, including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean average blood pressure (MBP), pulse pressure (PP), heart rate (HR), interbeat interval (IBI), and total peripheral resistance (TPR). The signals were obtained with the probe positioned on the middle finger. The height sensor was disconnected from the device and the distance from the hand to the heart was measured manually with a measuring tape.Noninvasive short‐term measurements of BP were taken at each heartbeat using a PortapresIn humans, in the supine position, BP is the same at feet, head, and heart levels , and h is the distance from the heart  below the heart level and supported by a lever. Subjects were asked to make no muscle effort with the dependent arm.Supine position with one arm dependent. One arm was passively lowered by the researcher 45Standing position with one arm dependent. Subjects were asked to stand up as rapidly as possible, keeping the arm in the dependent position and with the same inclination as in the supine position.Standing position with the hand at heart level. Subjects were asked to return the previously dependent limb to the heart level.Supine position with the hand at heart level .sk), and the kurtosis (κ) , standard deviation (SD), skewness  region from 0.04 to 0.15 Hz, and in the high‐frequency (HF) region from 0.15 to 0.4 Hz. We calculated the slope of the power spectral density (PSD) by using a linear minimum squares approximation and the associated parameters LF/HF, P < 0.05.Differences occurring between maneuvers were assessed by ANOVA with the subsequent Sidak's multiple comparisons test. Statistical significance changes were defined as ® are shown in Figure Typical records of the noninvasive short‐term time series measurements of the PortapresThe photopletysmographic noninvasive temporal records of nonpulsatile TBVF and PP of a typical young subject during the five maneuvers are plotted in Figure Numerical results for the parameters of HR variability for the different maneuvers are summarized in Table P = 0.05 level  of the lowered arm from the heart. Even when in supine position, the PSD slope of SBP at a P = 0.05 level, statistically is not significantly different from the basal conditions . The pulse pressure widened from 40 ± 3 to 70 ± 4 mmHg , and in all the BP variables, in particular SBP increases 60 mmHg with respect to basal values. SBP becomes less correlated, as can be seen in the PSD Fig. , 6 and 7All the variables indicating vasoconstriction  returned in 3–4 beats to the previous value (before lowering the arm) and the TBVF also returned to its previous value of basal oscillation. When the subject is upright with his arm at the heart level, the amplitude of the pulsatile skin blood flow immediately increases, whereas the nonpulsatile blood volume decreases Fig. , going tWhen reassuming the supine position with the hand at the heart level, the pulsatile skin blood flow has some rebound increase in amplitude and the venous volume return to basal values making the return of all the variables to their basal values, including those reflecting vasoconstriction and those reflecting changes of BP and HR.h of the dependent arm to the heart level considered as the hydrostatic neutral point;the increase of BP was directly proportional to the distance the increase of BP was not associated with changes of HR and therefore not likely to be secondary to BRR activation or central command;there were no oscillations of BP at 0.1 Hz (characteristic of BRR activation). The peripheral resistance increased, the amplitude of the pulsatile SBF decreased, and the PP widened, indicating vasoconstriction. It was unlikely that an adrenergic stimulation could induce the arterial vasoconstriction because intensive study of this response has shown that it persists in the lower extremities with epidural blockade , the isolated regional changes of BP and the SBF induced by the gravity stress in the dependent arm. Assuming the upright position with the arm down (third maneuver), we analyzed the generalized vasoconstrictive changes of BP and SBF induced by the BRR superimposed to the gravity‐induced myogenic vasoconstriction, as the arm was kept lowered during the upright position. In all subjects, lowering the arm in the supine position induced a vasoconstriction response with an increase in SBP, DBP, and an important rise of regional peripheral resistances, and an increase of the PP. We also demonstrated that there was a decrease in the amplitude of the arterial pulsatile SBF due to vasoconstriction and an increase in the total volume of blood in the cutaneous vessels of the finger. In the second position, all these changes were regional as the BRR was not activated. This is suggested by the fact that the subjects remained in the supine position, and the changes of BP were not associated with reciprocal changes of the HR. The central command was not activated because the arm was lowered passively and the HR did not increase. Therefore, vasoconstriction was likely to be due to a local myogenic response induced by the pressure of gravity. The facts that militate for a myogenic response induced by gravity are:there is a further increase of BP that occurs when the subject stands up. It is known that in assuming the upright position, there is a generalized redistribution of the blood volume below the heart and a sudden fall of BP followed by BRR activation and generalized vasoconstriction Berne ,there are oscillations at 0.1 Hz that are characteristic of BRR activation, the BP fluctuates in a characteristic fashion on visual inspection (Mayer waves), and there is more variability of BP typical of reflex control, andthe HR increases in a reciprocal way to the increment of BP. There was an additional increase in peripheral resistances and a decrease in the amplitude of the pulsatile SBF indicating an increment of vasoconstriction.When the subject is upright with the arm dependent, there is a further increment of BP that is related to BRR activation. This response is superimposed to the already existing gravity‐induced myogenic vasoconstriction. The facts that support that there is BRR activation and sympathetic vasoconstriction when standing with the arm dependent are:All these changes were readily observed in our subjects. This is also supported by the last phase of the experiment: when the subject, while still standing, elevates his arm to the heart level, the BP in the arm abruptly descends due to the withdrawal of the gravity‐induced myogenic local response, whereas the BRR maintains the BP slightly above the basal level. The HR continues to increase with respect to its basal levels and the low‐frequency oscillations at 0.1 Hz persists. The regional peripheral resistance decreases and the amplitude of the SBF increases, demonstrating a reduction in arterial and arteriolar vasoconstriction. Lastly, when the subject lies down, with his arm at the heart level, the BP, HR, peripheral resistances return to basal values, as the BRR is not active and there is no gravity stress on the arm and finger at the heart level. The increase in venous pressure, arterial vasoconstriction, and in BP seems to be due, at least in part, to the action of gravity, as both, venous and arterial pressure are increased by gravity. The venous pressure at the feet has been shown to increase from 5 mmHg in supine position to 100 mmHg (13.33 kPa) in the upright position, whereas the SBP increases from 120 mmHg (15.99 kPa) in the supine position to 200 to 220 mmHg (26.66 to 29.33 kPa) in the upright position. Meanwhile, supine BP is approximately the same at the heart level, the head, and the feet because the gravitational vector does not act upon the arterial and venous system either below or above the heart Berne ., in healthy young humans. MVR was discovered by Bayliss The important finding of our experiment is the demonstration that it is possible to separate or segregate the myogenic vascular response (MVR) induced by gravity and the vascular response induced by the BRR, in vivoLimitation of the study: Although the generalized BRR was assessed by HR variability, we also used the changes on the SBP and the spectral analysis that relates the sympathetic activity with baroreflex activation at 0.1 Hz (Mayer waves). One limitation of this study is that, as a noninvasive approach was taken, we could not record the muscle sympathetic nerve activity. This can be done in a future study, in order to characterize with some other method, the entrance of the BRR.We are bipeds, with our heads above our heart that have to live two‐thirds of our lives with our circulatory system counteracting the action of gravity. In fact, it is likely that the BP, in microgravity conditions, is the same in all regions of the body, no matter the position of the body, as the BP under this condition, is a function only of the cardiac output as all the vessels are vasodilated (Norsk et al. The vascular myogenic response is thought to be present in the kidney, the brain, and the coronary arteries (Davis and Hill The authors have no conflict of interest to report.


Previous studies regarding the comparative costs of laparoscopic and open surgery for rectal cancer provide ambiguous conclusions, and there are no large randomized trials or long-term follow-up.n = 105). Sick leave information was provided by the Swedish social insurance agency. Unit costs were collected from Swedish sources. The primary outcome was the difference in mean cost between laparoscopic and open surgery.A prospective cost-minimization analysis was carried out by using data of clinical resource use from the randomized controlled trial COLOR II. Some data needed for the health economic evaluation were not collected in the clinical trial; therefore, a retrospective data collection was made for COLOR II-patients operated at the largest participating Swedish hospital  and 3 years  after surgery. There were, however, no differences in long-term costs to society between laparoscopic and open surgery .Though the study found short- and long-term cost differences for the healthcare sector, there was no difference in regard to the long-term societal perspective. Future research is suggested to investigate the effects of sick leave costs using material from a greater number of patients. Several smaller series and one large randomized trial, the COLOR  II-trial , have shUncertainties remain regarding the relative costs of laparoscopic and open rectal cancer surgery. Several studies performed alongside randomized trials comparing the costs of laparoscopic and open surgery for rectal cancer have had short time perspectives –9 or havThe aim of the present study was to evaluate the cost of laparoscopic versus open resection for rectal cancer from both the healthcare and the societal perspective, based on the randomized COLOR II-trial. The health economic method employed was a cost-minimization analysis . The costs were assessed at 28 days  and 3 years . The hypothesis was that laparoscopic surgery would be more costly when assessed at 28 days after the primary operation but not at 3 years.The COLOR II-trial provided the clinical data for the present cost study . The stuHealth economic evaluations such as cost-effectiveness analysis are based on the incremental cost for an incremental unit of a clinically relevant outcome  or a QALY  as a measure of treatment or program effectiveness . SurvivaThe cost analysis comprises the health care and the societal perspective, where the latter adds the cost of sick leave to the direct healthcare cost. The study period was set from inclusion into the clinical trial up to 3 years postoperatively, including the short- and long-term clinical endpoints of the COLOR II-study. Censoring and missing data can cause bias in economic studies conducted alongside clinical trials . In thisData on resource use were collected prospectively through CRF’s in the COLOR II-trial. Details of the use of resources that were needed for this study, but had not been collected within the trial , were determined using other sources.n = 105). The factor between time in anesthesia and skin-to-skin time was established for the Sahlgrenska patients and applied on all COLOR II-patients. The average time in the recovery room for COLOR II-patients operated on at Sahlgrenska University Hospital was extrapolated to all study participants.The basic equipment required for laparoscopic surgery, as well as the type and quantity of instruments required for laparoscopic and open surgery, was determined by conclusions drawn from the health economic evaluation of laparoscopic versus open colon cancer resection within the framework of the randomized trial COLOR  and in cSick leave was retrieved from the Swedish Social Insurance Agency for Swedish COLOR II-patients, and the observed average number of days on sick leave per surgical technique was calculated. To be able to analyze the total cost at the patient level, the average number of sick leave days per surgical technique observed in the Swedish cohort was applied to the non-Swedish COLOR II-population using random selection. It was ascertained that the proportion of patients on sick leave in the Swedish cohort, and the non-Swedish COLOR II-populations was the same.Unit costs for basic equipment and surgical instruments were obtained from regional procurement records in Region Västra Götaland, Sweden. The cost per minute in the operating room, time in anesthesia and time in the recovery room were derived from the health economic evaluation of laparoscopic versus open surgery in the COLOR trial . The uniThe cost of sick leave was calculated by using the average monthly wage (provided by Statistics Sweden) with addition of the social security and supplementary pension fees. All prices were inflation adjusted for 2013 SEK using the consumer price index provided by Statistics Sweden. Costs were converted from SEK to the average value of the US dollar in 2013 ($1 = 6.51 SEK).The distribution of cost data is non-negative and right skewed due to the low number of patients incurring particularly high costs, a common phenomenon in studies involving resource items with high unit costs such as hospital care, reoperations and sick leave. The average cost will consequently be higher than the cost of the average patient, but it is still meaningful as it enables the calculation of the total cost of treating all patients with the new therapy . Due, hop value for any of the results in Table For the health economic study, 699 patients in the laparoscopic group and 345 in the open group were available for analysis Fig. . InformaThe mean healthcare cost per patient Table  during tFrom the societal perspective, the mean cost per patient Table  at 28 daSensitivity analyses can be found in Table The incidence of colostomy had considerable impact on the study result and so did the number of days on sick leave. A detailed analysis of the data showed that patients in the open surgery group were older, and therefore, fewer were eligible for sick leave compared with patients in the laparoscopic group. Numerically more patients on sick leave in the laparoscopic group died earlier compared to the open group which resulted in a lower cost of sick leave in the laparoscopic group. As survival did not significantly differ between the groups, this was most likely a random finding due to sick leave only having been examined in a small sub-group of the COLOR II-trial. The mean number of days with a colostomy was higher in the laparoscopic group, partly due to numerically more laparoscopic patients subject to abdominoperineal resection at the index operation and partly due to longer survival time in the sub-group of colostomy patients in the laparoscopic group. Additional sensitivity analyses were, therefore, performed and the results showed that excluding the costs of stoma material the difference in mean cost per patient to the health care sector   was similar to the base case analysis at 28 days after the index surgery. At 3 years the difference in mean health care cost per patient   was lower compared to the base case analysis ($3854). Laparoscopic surgery was numerically less costly per patient for society than open surgery −$926  (95 % CI −7261 to 5409) at 3 years after primary operation. In the short time perspective, it made little difference  .This health economic evaluation of laparoscopic and open surgery for rectal cancer in the framework of the randomized trial COLOR II showed that laparoscopy was significantly more costly from the societal perspective at 28 days, but no statistical significance was detected at 3 years. From a healthcare perspective, laparoscopy was more costly than open surgery at both 28 days and 3 years.n = 251), which increased the risk of random findings. This is a common problem for costly resource use items that may vary without relation to the studied interventions. In this study, the finding was a disadvantage to the results in the open surgery group. On the other hand, patients with a cancer in the lowest part of the rectum, who received a colostomy due to abdominoperineal resection, lived longer (n.s.) and were numerically more frequent in the laparoscopic group [The one-way sensitivity analyses showed that variations in sick leave and the incidence and days with colostomy had a large impact on the results. Data on sick leave were elicited for the Swedish sub-group only  , althougIn the present study, the length of hospital stay was considerable in both groups, although 1 day shorter (median) in the laparoscopic group. The trial protocol prescribed that the same local principles for discharge should be applied for both groups and did not include the enhanced recovery after surgery (ERAS) concept . The difThe strengths of our study include that it is based on clinical results from a large randomized trial with a multicenter design and that the principles for the health economic analysis were outlined before the start of the randomized trial. Thus, the clinical record forms included variables of importance for the economic analysis. The study also had a high rate of returned clinical record forms.A limitation is that the present health economic analysis was a secondary objective within the randomized trial COLOR II and the sample size, thus, not calculated for the health economic outcomes. Further, some of the resource units are for a sub-group of patients of the COLOR II-trial, which adds to the uncertainty of these variables.In conclusion, laparoscopic surgery for rectal cancer is more costly than open surgery from the health care perspective. It is important, however, that the cost of sick leave is taken into account to ensure inclusion of all costs arising as a consequence of the surgical method chosen. In the present study, sick leave was investigated in the Swedish cohort only which was too small to detect a true difference between the treatments. Future research is suggested to investigate the sick leave costs of rectal cancer surgery.


Erpetoichthys calabaricus, has been noted being capable of capturing prey on land. We now identified the mechanism of terrestrial prey‐capture in reedfish. We showed that this species uses a lifted trunk and downward inclined head to capture ground‐based prey, remarkably similar to the mechanism described earlier for eel‐catfish. Reedfish similarly use the ground support and flexibility of their elongated body to realize the trunk elevation and dorsoventral flexion of the anterior trunk region, without a role for the pectoral fins. However, curving of the body to lift the trunk may not have been an option for the Devonian tetrapodomorphs as they are significantly less elongated than reedfish and eel‐catfish. This would imply that, in contrast to the eel‐like extant species, evolution of the capacity to capture prey on land in early tetrapods may be linked to the evolution of the pectoral system to lift the anterior part of the body.Due to morphological resemblance, polypterid fishes are used as extant analogues of Late Devonian lobe‐finned sarcopterygians to identify the features that allowed the evolution of a terrestrial lifestyle in early tetrapods. Previous studies using polypterids showed how terrestrial locomotion capacity can develop, and how air ventilation for breathing was possible in extinct tetrapodomorphs. Interestingly, one polypterid species, the reedfish  Two specimens were kept in a large aquarium with a wooden perch above the water level and were fed pieces of Atlantic cod fillets (Gadus morhua). The other four individuals were kept in a two‐compartment aquarium connected by a horizontal terrestrial area that can be reached by surfaces inclined at 45 degrees on both sides  were recorded during terrestrial capture of prey . High‐speed videos at 500 frames per second were recorded for benthic, aquatic capture of prey . As the animals displayed more activity in low‐light conditions and at night, we used infrared‐LED panels for lighting. Out of the daily recording session during 4 months, we managed to record two feeding events with sufficient image sharpness and contrast from a lateral view on the head, four from a posterior–dorsal view on the head, and a large number of terrestrial excursions and searches for prey. In the aquatic environment, thirteen feeding events were recorded and analyzed from a lateral view.E. calabaricus (N = 6) and for the other terrestrially feeding fish Anableps anableps . Anguilliform taxa displayed in this book with a folded tail were replaced by measurements on pictures from the scientific literature. These data were compared to the ratios from reconstructions of four Upper Devonian fossils from the stem tetrapod lineage that are sufficiently complete to allow this quantification: Gooloogongia loomesi  was measured on preserved specimens for 3E. calabaricus emerges from the water and propels itself on to the shore, with its head slightly pitched downward . Once the snout makes contact with the prey, the mouth is opened, and this elevates the skull to some extent as the mandible pushes against the wooden ground surface . The pectoral fins are moved backward to become adjacent to the body, seemingly without making contact with the ground. The body is then propelled forward toward the prey, the anterior part of the trunk is lifted, and the head increases its nose‐down tilting angle . Next, the jaws are closed over the prey, and the lifted pectoral region falls back down to the substrate . The prey is held between the jaws while the reedfish moves back into the water. The dorsoposterior views on the head show that the opercular slits open to release air during terrestrial prey‐captures. While searching for prey in the terrestrial environment, the head angle is often similarly tilted downward to about 20°  pivot about the pectoral fins to bring their mouth toward terrestrial prey  uses jaw rotation and protrusion to orient the oral jaws downward  and four‐eyed fishes (0.19 ± 0.02) in this respect. If we assume from this that the capacity to lift the trunk without needing pectoral fins or limbs is unlikely to work in stem tetrapods because of their relatively short body and tail with respect to the anguilliform fishes, development of weight‐bearing capacity by the pectorals is probably essential for the evolution of terrestrial capturing of prey. This would mean that the selective pressure to exploit ground‐based terrestrial prey may have been an important factor in the evolution of pectoral fins to limbs in the earliest tetrapods.However, while the reedfish's and eel‐catfish's elongated body and tail allow a controlled and stable lifting of the trunk by curling their anterior part of the body, the stem tetrapods may lack the body length needed to successfully lift the trunk in the same way  without losing stability against swaying or rolling sideways. Our analysis of the head length‐to‐total length ratios showed that the stem tetrapods have values between 0.190 and 0.216 (Figure Although the pronounced elongatedness of the reedfish and eel‐catfish may make these species less suitable as model species to infer the capacity and mechanics of terrestrial feeding in tetrapodomorphs, other early tetrapod taxa do show similar or even higher levels of elongatedness. Aistopoda, a group of limbless lepospondyls that lived from early Carboniferous to Early Permian, are extremely elongated Baird, . Based oNone declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


The penetration of D–D neutrons in water has been studied through measurements of first collision dose. A relative measurement of first collision dose as a function of distance from the neutron source was made at 0°. At large penetrations the results appear to approach asymptotically the slope predicted by the theoretical calculations of Goldstein et al. [0] for a monoenergetic, isotropic 4.0-Mev neutron source in water. However as expected, the measurements close to the source where the average neutron spectrum is relatively soft indicate a steeper slope than the theoretical calculation. Further calculations will be required to obtain an explicit check of this experiment. Experiments such as these are needed to check the calculation methods and input data used for neutron shielding and reactor physics.This experiment is a continuation of a series of experiments performed at this laboratory pertaining to the slowing down of neutrons in hydrogenous media –3.1 The The first collision neutron dose  at 0° wThe absolute neutron dose was not measured in this experiment because it was not possible to measure accurately the beam current or the pressure of the gas target. The associated particle method of obtaining an absolute measurement was undesirable due to the duct effect of the associated equipment. Consequently, only the slope of the experimental curve can be compared to the calculations of Gold- stein.d,n)He3 reaction using an analyzed deuteron beam from a 2-Mev Van de Graaff accelerator. The deuteron beam impinged upon a modified gas target of the type described by Richardson [Neutrons were produced by the DHe3 reaction. The foil was cooled by continuously circulating the deuterium gas. This permitted the use of 2-μa current for as long as 30 hr without burning out the foil. The pressure was maintained at 760 mm Hg in the cell. This target differed from the one described by Richardson in that a thick gold backing was used instead of an exit foil. The gold backing was rotated frequently in order to reduce the buildup of a “drive-in” target.The gas target had a cell 1.24 cm long and used a 0.1 mil nickel entrance foil. The entrance aperture was The deuteron beam incident energy was 1.36-Mev. Using the data of Whaling , the aveA polyethylene-ethylene proportional counter dosimeter modeled after the secondary counter described by Hurst  was usedThe signal was brought out of the dosimeter by a 3-ft RG–59/U coaxial cable passed through a The signal was bought from the dosimeter to a gain-of-sixteen, transistorized preamplifier powered by batteries. The signal was then carried by a 40-ft cable through a variable attenuator to an RCL linear amplifier and 256-channel analyzer. A constant pulse from a mercury-relay pulser could be fed into the input of the preamplifier thus permitting a quick check on the overall gain of the electronics and the gain could be quickly adjusted to the desired level by means of the variable attenuator.n)He3 neutron spectrum to zero and then adjusting electronic gains so that this intercept would fall in channel 17. The dosimeter was then exposed to the 87-kev gamma ray of Cd109 and to the higher energy gamma rays of radium. All the pulses from the 87-kev gamma ray fell below the 0.1-Mev bias but a few pulses from the radium source fell as high as six channels above the bias. The dose falling above the 0.1-Mev bias was measured for the 4.0-Mev monoenergetic neutron source and for the radium source and these were compared when the total energy deposition in the dosimeter was the same for each. This neutrongamma ray discrimination ratio was found to be about 120. The 0.1-Mev cutoff as determined experimentally was reproducible to within one channel although the absolute accuracy was estimated to be no better than two or three channels.It was necessary in the computing of neutron doses to reject small pulses which were caused by electrons ejected by gamma rays. Previous experience with this dosimeter  had indiThe dosimeter was checked for possible drift by periodically measuring the dose in air from a plutonium-beryllium source. A slight drift was noticed when the energy calibrations were finished and the dosimeter was first placed in the water. The data were corrected for this drift and the resultant error due to dosimeter drift is estimated to be not greater than 5 percent.3 counter placed at 100° to the incident deuteron beam and approximately 40 cm from the target. The measured dose is given in R, as given in r is the distance from the target-water boundary to the center of the dosimeter.The D–D neutron first collison dose was measured as described by promenading the dosimeter at 2.5 cm intervals from 15 to 45 cm at 0° and then calculating the dose using a 0.1-Mev bias. The neutron flux was monitored during t hese runs by a cylindrical BFR and r due to (1) the finite size of the neutron source and the variation in yield along its length, (2) the finite size of the dosimeter, and (3) the anisotropy of the neutron flux incident upon the dosimeter. In the ideal situation of a point source of neutrons located at the center of a void sphere of radius r1 and at a distance r2 from a point dosimeter, R would be given exactly by r2 while r would be given by r2—r1. In our experiment, the upper limit of the difference between the nominal values of R and r chosen above and the values of R while the second and third effects opposed each other and would cause r.There was some uncertainty in determining the best value of Y=MX+B where Y is the counts per channel and X is the channel. M and B are adjusted so that Y=0 for X=256 and IX=channel 18, fX=channel 256, and The statistical errors for the first collision dose were computed by approximating the dosimeter spectrum with one of the form A calculation to compare directly to this experiment is not yet available. However the 4- and 2-Mev monoenergetic, isotropic calculation of Gold stein  should bIt is desirable that a calculation be made of the dose distribution at 0° with exactly the D–D neutron spectrum and angular distribution and the experimentally observed dosimeter response  of figur


Caulobacter crescentus where past events are expected to only be informative for one of the two cells emerging from division, the sessile cell that remains in the same microenvironment and does not migrate.The ability to form a cellular memory and use it for cellular decision-making could help bacteria to cope with recurrent stress conditions. We analyzed whether bacteria would form a cellular memory specifically if past events are predictive of future conditions. We worked with the asymmetrically dividing bacterium Time-resolved analysis of individual cells revealed that past exposure to low levels of antibiotics increases tolerance to future exposure for the sessile but not for the motile cell. Using computer simulations, we found that such an asymmetry in cellular memory could be an evolutionary response to situations where the two cells emerging from division will experience different future conditions.conditionally in situations where it is beneficial.Our results raise the question whether bacteria can evolve the ability to form and use cellular memory The online version of this article (doi:10.1186/s12862-017-0884-4) contains supplementary material, which is available to authorized users. In bacteria, as in every cellular organism, the current state and composition of a cell is determined by events in the past –4. FactoThis question is motivated by the fact that the degree of cellular memory is shaped by biological processes that are encoded by genes, and that can thus undergo evolutionary change in response to natural selection. Cellular memory depends on the half-lives and turnover of cellular components—for example RNAs and proteins—and on the architecture of gene-regulatory networks , 5–7. Imconditional cellular memory.The ‘adaptive value’ of cellular memory—its effect on survival and reproduction of individual cells—is expected to depend on the specific situation. For some environmental factors the conditions in the past might be indicative of the conditions in the future, and cellular memory for phenotypic traits that determine the response to these environmental factors might thus be beneficial; for other factors this might not be the case. This raises the question whether bacteria (and other organisms) evolved high degrees of cellular memory specifically in traits and in circumstances where this is beneficial, and low degrees of cellular memory when it is not. We refer to this possibility as Caulobacter crescentus—where one cell emerging from division remains in the same microenvironment, while the other migrates to a different microenvironment [We investigated if organisms memorize past events specifically in situations where past events have predictive value about the future. To address this question, we used an experimental system where cellular memory is expected to be beneficial to some cells in a population, and less beneficial to other cells. Specifically, we worked with an asymmetrically dividing bacterium—ironment . For theironment ).C. crescentus to address this question of conditional cellular memory because it is a well-established model organism to study asymmetric cell division. A surface attached cell divides asymmetrically into a sessile stalked cell and motile swarmer cell. We refer to the sessile stalked cell as ‘mother’ and the motile swarmer cell as ‘daughter’ as for example in [We chose ample in , 11. Thiample in .Bacterial memory has been studied in different contexts such as changing nutrient conditions , 13–16 oC. crescentus to the antibiotic ampicillin [C. crescentus carries six genes that confer—when expressed—a certain degree of resistance to ampicillin, a beta-lactam antibiotic [C. crescentus is potentially exposed to naturally occurring beta-lactams in its freshwater environment [We used a combination of single-cell experiments and computer simulations to address these questions. We experimentally exposed picillin . C. crestibiotic . Resistatibiotic –27. C. cironment . AntibioThese experimental results motivated us to ask whether such asymmetry in how past events influence future behavior could indeed be an evolved response to a situation where the predictive value of past events differs for the two cell types emerging from division. We used computer simulations to test whether asymmetric memory is expected to evolve in a situation where one individual emerging from division remains in the same environment while the other individual migrates to a different environment. These simulations support the notion that the asymmetry in cellular memory that we observed in the single-cell experiments could be the result of differential selection on the sessile and motile cell type. More generally, our experimental and theoretical results suggest that analyzing what types of past events are stored in different cell types allows formulating hypotheses about the adaptive nature of bacterial memory.C. crescentus in microfluidic devices . By exposure to a sub-MIC .We used the bacterium YE broth ) of ampictamases . We refeC. crescentus exposed to sodium chloride [We recorded and analyzed time-lapse movies of the initial cohort of sessile stalked cells and the subset of their daughters that remained in the microfluidic chip after cell division, differentiated to stalked cells and could be observed Fig. : Fig. S2chloride , we founWe used this system to test the main idea put forward above: that the sessile mothers—but not the motile daughters—would retain a cellular memory of past events. To do so, we compared how survival under antibiotic exposure of the two groups of cells depended on a past warning. The first group  was the cohort of stalked cells that were already present at the time of the warning; the second group  was the cohort of cells that were produced after the warning, and that stayed in the microfluidic device and could thus be observed. It is important to note that many of the daughters had already differentiated to stalked cells at the onset of the ‘stress event’. While the time-point of differentiation for individual cells cannot be determined in our experimental set-up, the swarmer cells that remain in the microfluidic device, and that can thus be observed, only take about 20 min longer to reach the next cell division than the stalked cells  cellular components upon cell division that make their stalked counterpart more tolerant to antibiotics following after the warning, as will be discussed further down. We interpret these results thus not as conclusive evidence for a beneficial asymmetric memory, but rather as an interesting observation that raises new questions. One of the questions that it raises, and that we addressed in the next part, is: do we indeed expect asymmetric memory to evolve in response to a situation where the predictive value of past events differs for the two cell types emerging from division? We used computer simulations to analyze how cellular memory evolves in situation where the predictive value of past events differs for the two cells emerging from division.This outcome is Motivated by the experimental results shown above, we used an agent-based simulation to analyze the evolution of cellular memory in simulated bacterial populations. While our main goal here was to investigate the evolution of asymmetric memory, we first asked a simpler question: under what circumstances do we expect evolution of the ability to form a cellular memory? Building on these results, we then analyzed the distribution of memory among asymmetrically dividing cells.We simulated individual cells with evolvable traits that were genetically encoded. These traits determined the formation of a cellular memory that can provide protection against an external stressor. The level of protection of a given cell was a continuous trait that could change over time, and that determined its survival upon exposure to stress. The current level of protection, and its modulation by the environment, depended on three genetically encoded traits: ‘basal protection’ determined the minimal protection a cell maintained during all times; ‘protection increase’ determined the absolute amount of protection that was added to the current protection level when warning or stress conditions were sensed; ‘protection decrease’ referred to the absolute amount of protection that was subtracted from the current protection level during each time step. If an addition or subtraction of protection to the current level of protection resulted in a protection level higher than 1 or lower than 0, it was corrected in order to ensure that the protection level was always between 0 and 1. When a cell divided, these genetically encoded traits were copied to the two cells emerging from division. At cell division the protection level, which is a phenotypic property of the dividing cell, was divided into two equal parts, and both cells emerging from division received one of these parts .In the context of our model, we interpreted the phenotypic trait of increasing the protection level in response to a warning or stress event as the ability to form a cellular memory, and the phenotypic trait of decreasing the protection level under favorable conditions as the ability to erase this memory. The current protection level of a cell then characterized the current state of the cellular memory; it was influenced both by the environment the cell was exposed to in the past as well as by the cell’s genotype that determined how these past events influenced the cellular state. The current protection level determined the probability that a cell would survive a stress event.The environment was modeled as a series of distinct time periods, with each period belonging to one of three types of environmental states: ‘favorable’, ‘warning’ or ‘stress’. A warning period had no negative impact on the cell, but was sometimes  preceding a stress period. In repeated simulations we evolved a population of 10’000 individuals for 100’000 time steps Fig. . During history-dependence in stress response strategies.To identify environmental patterns that promote the evolution of the ability to form a cellular memory, we evolved cells in two different types of environments Fig. . In the The main outcome of this model was that the ability to form memory evolved in an informative but not in a non-informative environment, in line with our expectations. In non-informative environments cells evolved elevated levels of basal protection Fig. . These tWe extended the simulation framework to investigate the evolution of memory in organisms where the two cells emerging from division had different fates. Specifically, we were interested in situations where one cell would remain in the current environment, and the other could migrate and potentially colonize a different environment. We refer to the first cell as the ‘mother’, and the second as the ‘daughter’, as above. We then tested the idea developed above, that such a situation could select for organisms where the mother cell carries a memory, while the daughter cell would not keep a memory. The basis of this hypothesis was that the daughter cell would migrate to a new environment, so that past events in the birth environment were not informative. To address this question with our simulation framework we introduced an additional evolvable trait that we called ‘memory distribution factor’. The memory distribution factor controlled how memory  was distributed between the mother and the daughter at cell division. A memory distribution factor of 0 corresponded to a situation where the mother kept the entire memory , while a distribution factor of 1 corresponded to a situation where the daughter cell received the entire memory . In the previous simulations memory was split equally between these two cells. This corresponded to fixing the memory distribution factor to 0.5. By permitting the memory distribution factor to evolve we expected it to deviate from 0.5 when the past was only informative to the mother but not to the daughter cell.between environments. Upon cell division the sessile mother cell remained in the same environment, while the motile daughter cell was randomly placed in one of the two environments. That way, the mother cell always stayed in the same environment, while the daughter cell had a 50% chance to leave the current environment. Once the daughter cell was placed in one of the two environments it differentiated to a mother cell and initiated reproduction.To achieve the situation where the mother would stay in the current environment and the daughter would potentially migrate, we carried out simulations using two instead of one environment Fig. . Each ofWe expected that this situation would select for organisms where memory, i.e. protection, would be distributed asymmetrically at cell division: for the mother, past events were informative about the future, and thus the mother was expected to maintain memory. For the daughter, past events were less predictive about the future (since it only stays in the same environment in 50% of the cases), and we thus expected that it would keep less memory. In the context of our model, such asymmetric distribution of memory would manifest as a memory distribution factor smaller than 0.5. As a control, we ran the simulations with the same evolvable traits in a single environment where both cell types emerging from a cell division were staying in the same environment Fig. .orange trajectory in Fig. When populations of cells were evolved in two environments Fig. , we obseorange) to either mother or daughter across the 20 replicate populations , within most of the 20 replicate populations there was a clear tendency for protection to be either passed on to the mother cells (memory distribution factor close to 0) or to the daughter cells (memory distribution factor close to 1). This means that most of these 20 replicated populations evolved a type of asymmetric memory where cellular protection was passed on to one of the two cells emerging from division, rather than distributed to both types equally . We note that this outcome is not equivalent to the situation with two environments, where cellular protection consistently is passed on to the mother cell that stays in the same environment . This enabled us to test 8 conditions in one experiment . The chromosomal setup suggests that blaA (CC2139) is part of an operon with three other genes  [gfp (green fluorescent protein). The amplified region was inserted into pMR10 background. The resulting plasmid was transformed into wildtype C. crescentus CB15 [blaA  . We ampltus CB15 , ATCC 19Bacteria were grown overnight in culture tubes  in 5 mL PYE complex medium  shaking For the experiment two pumps  with 8 syringes in parallel were used. This ensured constant growth conditions, keeping the sessile cells in exponential phase and preventing the formation of biofilm. 50 mL syringes (Pic Solution) were loaded with 50 mL PYE and used for the non-stress periods. For the ampicillin warning and stress events, 10 mL syringes (Soft-Ject) were loaded with 2 mL of PYE + ampicillin . Tubing  was connected to the syringes using 20G needles . Smaller tubing  was then inserted into the bigger tubing (Tygon S54HL), and directly connected to the inlet hole in the PDMS chip. Medium change was performed by disconnecting and reconnecting the tubing from the PYE to PYE + ampicillin. Pumping speed during the experiments was set to 2 mL/h.Microscopy was performed using an Olympus IX81 inverted microscope system with automated stage, shutters, and laser based autofocus system. Several positions were monitored in parallel on the same device, and phase contrast images of every position were taken every 5 min. Images were acquired using an UPLFLN40x phase contrast objective (Olympus) and a cooled CCD camera (Olympus XM10). For image acquisition, the Xcellence Pro software package  was used. To keep temperature at 30 ° C the microscope was placed in an incubated box . Fluorescence images were acquired using a 120 W mercury short arc lamp (Xcite 120PC Q) and the U-N49002 EGFP filter set .CellCounter, Kurt de Vos, University of Sheffield, http://rsb.info.nih.gov/ij/plugins/cell-counter.html) was adapted such that cells could be marked across all time frames. Division frames of each cell were stored in a XML file upon visual inspection. Simulations were programmed in C++. Matlab and R were used for data analysis. The complete code of the computational model can be obtained from the corresponding author upon request.Images were analyzed with ImageJ. A plugin Additional file 2: Figure S2.N = 763 and 742 for exposure to 0 and 10 µg/mL ampicillin during the warning event). (B) The influence of the warning and stress event on the interdivision time (time since last division before event + time to next division after event) was analyzed. The area of the grey circles corresponds to the number of cells  found in the experimental data. In color the median is shown for unwarned (green) and warned (yellow) cells. The warning event had no detectable effect on the interdivision time . In contrast, the stress event had a clear effect on the interdivision time (middle and right panel): Mother cells for which the last division had been less than 50 min ago divided only after around 150 min after the onset of the stress event. The first division of a daughter cell takes longer due to differentiation into a stalked mother cell. This delay can be observed when comparing the middle and the right panel. (PNG 868 kb)The warning event had no detectable influence on cell division timing, while the stress event delayed cell divisions of mother and daughter cells and led to cell cycle synchronization. (A) The number of cell divisions per 5-min time interval during the course of the experiment is shown for unwarned cells (green trajectory) and cells that were exposed to a warning event (yellow). The favorable/warning period is represented in grey , the stress period in red (2000 µg/mL ampicillin during 1 h) areas respectively. In different colors the mean number of cell divisions per 5-min interval for each warning condition event is shown  For the number of cells that had already divided before (A) and cells that were in the process of dividing for the first time (B) cell cycle position at the time of onset of stress is depicted. (C and D) Survival per cell cycle position and cell type is shown in fractions and was modeled with a second-degree polynomial. For the model the filled bars in panel A and B were used (cell cycle position 5–70 for mother cells and 5–90 for daughter cells). (PNG 510 kb)For each cell, cell cycle position was estimated at the time-point when cells were exposed to the stress event (2000 µg/mL ampicillin for 1 h). Since the time period between warning and stress event exceeds the time to the first division of daughter cells, some of the daughter cells in our analysis had already divided. These daughter cells, while being daughters of mothers that had experienced the warning event, are staked cells that had already divided once. To correct for the cell cycle state we therefore needed to correct the daughter cells that had already divided differently from the daughter cells that had not yet divided. For the daughter cells that had not yet divided we used a cell-cycle position correction that accounted for their longer interdivision time . The cells that had already divided were corrected the same way as the mother cells that were born before the warning event since their cell cycle timing is the same. For both types of cells, cell cycle position was approximated by the time that had passed since the last division. Additional file 4:Figure S8.1. Trait distributions from single simulations in non-informative and informative environments. Trait (columns) distributions of the 20 simulation runs (rows) in a non-informative environment  distributions of the 20 simulation runs (rows) in one informative environment . But we did no longer observe the evolution of a memory genotype . Interestingly a phenotype that segregated cellular protection only to one of the two cells emerging from division still did evolve in the case of two environments . The simulation trajectories were comparable to the reference when lambda was increased from 0.2 of 0.4 . Qualitatively we observed the same simulation outcome when decreasing the mutation rate from 0.01 to 0.0001, although a slower convergence was observed . Simulation results that were run with an increased mutation rate (0.1) diverged from what we observed in the reference simulations . Note the bimodal distributions of both basal protection and the memory distribution factor in Figure S9.4c and S9.4d. Decreasing the fraction of individuals that are killed randomly in each simulation round from 0.05 to 0.01 led to the evolution of a basal protection genotype independent of information content and number of environments . In these simulations the carrying capacity of the population  was almost always exhausted, there was not enough ‘room’ for evolutionary mechanisms in the 100’000 time steps. When increasing the killing rate to 0.1 the mean basal protection that evolved in a random environment was significantly lower compared to the reference . A high population turnover favors a genotype with an intermediate basal protection to increase probability of reproduction. The simulation results observed when increasing random killing of individuals from 0.05 to 0.1 were comparable to the reference . A set of simulations was run with the same parameters as shown in Fig. 9b, but daughter cells were not randomly moved to one of the two environments. Instead, the daughter cells were always moved to the environment where they were not ‘born’. This had an impact on the evolution of the memory distribution factor . As expected we did not observe asymmetric memory when simulating two informative environments, where daughter cells were forced to stay in the environment they were born . Increasing the number of informative environments from 2 to 5 had no noticeable impact on the evolution results . (ZIP 5.56 mb)To assess the sensitivity of the simulation outcomes to varying simulation parameters, we changed a single simulation parameter at a time and rerun the simulations shown in Fig. Additional file 6: Figure S6.blaA (CC2139) gene is a major contributor to ampicillin resistance in C. crescentus [blaA using a transcriptional reporter  and after (t2) the warning event (0 or 10 µg/mL ampicillin for 2 h). After background correction the intensity level at t2 was subtracted from the intensity level at t1. (A) The mean of the differences is represented by the bars (error bars denote standard errors of the mean). For both conditions the intensity levels at t1 was compared to the intensity levels at t2 using a paired t-test statistic (N = 117 and 119 for 0 and 10 µg/mL ampicillin exposure). When cells were exposed to 10 µg/mL ampicillin for 2 h (yellow), the increase of the measured GFP intensity was significant (p < 0.01), while for the unwarned cells (green) no significant changes were observed (p = 0.3). (B) Numbers of surviving and dead cells are reported for small intervals of GFP intensity values (20 bins from -1.5 to 2.5 with size 0.2). No statistically significant association of GFP levels with survival after stress (logistic regression p = 0.68) could be established. (PNG 272 kb)It has been reported that the escentus . We measrter see . GFP


A new method has been developed for creating localized in-plane fibre waviness in composite coupons and used to create a large batch of specimens. This method could be used by manufacturers to experimentally explore the effect of fibre waviness on composite structures both directly and indirectly to develop and validate computational models. The specimens were assessed using ultrasound, digital image correlation and a novel inspection technique capable of measuring residual strain fields. To explore how the defect affects the performance of composite structures, the specimens were then loaded to failure. Predictions of remnant strength were made using a simple ultrasound damage metric and a new residual strain-based damage metric. The predictions made using residual strain measurements were found to be substantially more effective at characterizing ultimate strength than ultrasound measurements. This suggests that residual strains have a significant effect on the failure of laminates containing fibre waviness and that these strains could be incorporated into computational models to improve their ability to simulate the defect. This resulted in symmetric laminates consisting of eight plies, with an average ply thickness of 0.37 mm after curing. Each level of waviness was produced using a milled aluminium former. The profile of each former had an arc at its centre from which flat surfaces extended on either side; a photograph of one former is shown in lbottom. This is because the stiffness of the uncured plies in the fibre direction is much higher in tension, where the stiffness of the fibres dominates, than it is in compression, where the uncured resin allows the fibres to buckle, resulting in the neutral axis being close to the bottom surface of the laminate. The nominal waviness is therefore equivalent to the percentage excess length of the fibres in the top ply and can be expressed as follows:r is the radius of the arc section of the former, θ is the arc central angle. By substituting equations (2.2) and (2.3) into (2.1), the nominal waviness can be expressed in terms of the radius of the former and the thickness of the uncured laminate as follows:Quasi-isotropic laminates were produced with six levels of waviness severity. Six specimens were produced at each level to explore the variability of the defect generation technique. This resulted in a large set of 36 specimens. The laminates were manufactured using RP507UT210  unidirectional prepreg with a [0lbottom, can also be varied by changing the central angle using equation (2.3). The nominal length of the defects after flattening was kept constant at 20 mm. The six levels of nominal waviness chosen were: 0%, 10%, 15%, 17.5%, 20% and 25%. The arc radii and arc central angles for the corresponding five formers are listed in As the thickness of the uncured laminate is typically the same for every specimen, the nominal waviness can be varied by changing the arc radius of the former. The nominal length of the waviness defect, which is equal to the length of the bottom of the curved section of the unflattened laminate, −1 to a temperature of 130°C while being subjected to 2.5 bar pressure, and were held at this temperature and pressure for 45 min. Once the laminate was cured, the hot press was left to cool to room temperature over 6 h before the pressure was released and the specimen removed. The laminates were then cut into 40 mm by 220 mm coupons using a wet diamond saw . Care was taken to ensure the long edge of the specimens was parallel to the 0° direction of the fibres and that the wavy area was close to the specimen centre. This resulted in the 0° fibres in the top and bottom plies running parallel to the X-direction, as indicated in Then, the laminates were cured between flat plates in a hot press  according to the manufacturer's instructions. The rate of heat and pressure application was controlled by the machine and thus did not vary between specimens. The plates were heated at a rate of 10°C minTo examine the effect of high levels of waviness, two specimens were produced with the top 0° plies replaced with 90° plies. This corresponds to the worst possible case of fibre waviness, as it represents the complete misalignment of fibres and thus was used to determine how much the ultimate bending moment can be reduced due to fibre waviness. These specimens were cured using the same process described previously and cut to the same size coupons as the other specimens.X-direction, to observe out-of-plane waviness; and the defective ply surface, to observe in-plane waviness. The sectioned specimens were mounted in epoxy and then polished until the individual fibres could be observed. Images were then captured using an inverted microscope .Additional waviness specimens were produced with a nominal waviness of 0% and 20%. These were sectioned in order to perform microscopy on: the cut edge running in the 2.2.a. The power spectrum obtained had a roughly elliptical shape at its centre with its major axis in the transverse fibre direction, as shown in figure 3b,c. Thus, by measuring the orientation of the power spectrum ellipse, the local fibre orientation was obtained. This technique, of using ultrasound to measure fibre orientation, is described in detail in [Each specimen was inspected using immersion pulse-echo ultrasound to measure the fibre orientation. A multi-axis scanner  was used to produce C-scan amplitude images of the interface immediately below the 0° ply on the top surface of the laminate. The scanner had a focused probe with a natural frequency of 10 MHz, and the probe and coupon were immersed in water for ultrasonic coupling. The probe was attached to an ultrasonic flaw detector  that produced an A-scan of the laminate at the location of the probe. The amplitude in a gated portion of the A-scan, corresponding to the echoes from a 0.1 mm thick layer of the laminate at the first ply interface, was recorded. The echo amplitude was recorded at 100 µm increments along lines with a spacing of 200 µm, resulting in an amplitude C-scan. The fibre bundles in the 0° ply were visible in the texture within the C-scans, and thus the local orientation of the fibres could be measured. This was achieved by performing the two-dimensional discrete Fourier transform on square subsets of the C-scan. The size of these subsets was 75 pixels (7.5 mm) and the subsets had a spacing of 10 pixels (1 mm), resulting in a grid of orientation measurements with a pitch of 1 mm across the surface of the specimen. Prior to calculating the Fourier transform, a radial Hann window was applied; this limits spectral leakage, which would cause the peak at the centre of the spectral image to broaden . A windofigure 3etail in .Figure 2.3.DIC was used to measure strain on the surface of each coupon as it was loaded to failure. A speckle pattern was applied to a 60 mm by 40 mm section of each coupon at the location of the fibre-waviness defect on the opposite surface to the defect. A white base coat  was applied to the specimen and then black speckles  sprayed over the top. This resulted in speckles with a nominal diameter of 0.25 mm. A sprayed specimen is shown in −1. The specimens were loaded with the fibre-waviness defect on the top surface of the laminate in compression, and the lower surface, on which the speckle pattern was applied, in tension. The DIC system was set to capture images every 15 s; this resulted in a bending-rig displacement of 0.2 mm between each image. The test was stopped when the stiffness of the coupon was reduced to approximately half its initial value.For the tests, each specimen was initially rested on the lower pair of load noses of the bending rig. The reference images for DIC were captured at this stage so that the unloaded curvature of the specimens was measured. The lower half of the bending rig was then moved towards the upper load noses at a constant displacement rate of 0.8 mm min3.a,c. The two images in figure 4b,d are of the same features but for a 20% nominal waviness specimen. After the images in The waviness was observed in the two sectioned specimens using microscopy. The polished edge and ply surface of a 0% waviness specimen are shown in the two images in figure 4X-direction. This curvature was caused by residual strains in the laminate. As the curvature of the specimens was small, the residual strains in the specimens that caused the curvature were determined from the out-of-plane displacements [tc is the cured thickness of the laminate and w is the displacement of the defect-free surface from a flat plane. For each specimen, the DIC reference image was processed to measure the shape before any loads were applied, resulting in the data shown in figure 5a. The second partial derivative of these shape data was then calculated using the central difference method [figure 5b.After the specimens were cured and cut into coupons, it was observed that despite being cured between flat plates, the specimens were slightly curved along their length in the acements . The rese method  with a sx = −2 mm in the ultrasound data, and a corresponding area of high strain is visible in the strain field and the residual strain field. In the ultrasound data, there is not a corresponding strip of fibres orientated at +50°; but only small areas of fibres orientated at about +25° on either side at around x = −10 mm and x = +10 mm. This perhaps implies that the waviness is z-shaped, which would be consistent with other studies where complicated shapes have been observed [x = 17 mm.The fibre waviness was apparent in the ultrasound data, in the DIC strain data when the specimens were loaded, and also in the residual strain maps when no load was present. It was also possible to identify the same spatial distribution of the waviness defect in each specimen using the three distinct inspection techniques. Data from a specimen that had a nominal waviness of 25% are shown in observed . A less The overall waviness measured using ultrasound was quantified by calculating the root mean square (RMS) value of the fibre orientation fields. By calculating the RMS value of the ultrasound data, both local misalignment due to the waviness defect and any gross misalignment of the plies were quantified. When waviness was present, a correlation between the nominal waviness and the RMS of the measured waviness was observed and is shown in The ultimate bending moment of the specimens containing fibre waviness was also found to correlate with the RMS of the measured waviness, as shown in Strain fields on the surface of the fibre-waviness specimens were captured while loading the specimens to failure. These strain fields allowed the progression towards failure of the fibre-waviness defects to be observed. The load–displacement curve for a specimen that had a nominal waviness of 25% is shown in 4.Waviness defects in composite laminates can be caused by: misalignments in the unidirectional prepreg tape used to produce the laminate; the geometry of the tooling for the desired component shape; or the orientation of the reinforcement fibres . Thermalb, the deflection of the plies from the horizontal is minor compared to the in-plane waviness observed in figure 4d. Also, when the top ply was removed, the 90° ply beneath it was found to have no discernible level of in-plane waviness. These two observations suggest that the defect is only significant in the top 0° ply and that the rest of the specimen can be assumed unaffected by the process of creating the defect. Voids can be observed in the micrographs in Some waviness is always present in the prepreg prior to lay-up ; as all figure 4b and 6c. While the measured residual strains are significant, they are still small relative to the surface strains that can be reached when loading is applied. For example, on the 0% nominal waviness specimens, strains in excess of 10 000 µε were measured at ultimate load, while the largest residual strains measured on any of the specimens containing waviness was approximately 1200 µε. The shape data used for calculating the residual strain fields were measured by the same DIC system used to calculate surface strain in the loaded specimens. While the raw data for these two measurements were obtained from the same source, the processing of the data is completely different and similar measurements of residual strain would be obtained by any suitably sensitive shape measurement technique. Similarities in the spatial distribution of the defects detected by the three inspection techniques were observed for all specimens with significant fibre waviness.Three distinct techniques have been used to inspect the specimens. The first technique was the use of ultrasound to measure the fibre orientation in the top ply of the specimens, allowing the defects to be located and their severity quantified. While these ultrasound measurements provide information on the general orientation of the fibres, it is difficult to directly determine the shape of the buckled fibres due to the complex shape of the fibres and the limited spatial resolution of the measurement technique . The sec5b.A relationship was found between the nominal waviness and the ultrasound-measured waviness after the specimens were cured. The nominal waviness is a quantity derived from the shape of the formers used to create the specimens and should not be confused with the ultrasound-measured waviness which is a measurement of the actual waviness present after the specimens were cured. When the nominal waviness was over 10%, a linear correlation was observed between the nominal and measured waviness, with the data points being widely spread above and below the line-of-best-fit; this is shown in figure 7b, as well as the fibre-waviness defect acting as a stress concentrator, causing higher stresses at the defect than if the stiffness of the ply was uniformly reduced. It is likely that in-plane waviness in other plies had no influence, as waviness was not observed in the lower plies of the sectioned 20% nominal waviness specimen.A correlation is evident in figure 4The correlation between the ultrasound measurements and ultimate bending moment implies that strength predictions can be made using these measurements, as shown in y-axis at x = −7 mm and x = +7 mm. After strain field ‘a' was captured, cracks were observed forming through the 0° ply on the top surface at x = −7 mm, running parallel to the y-direction. When this specimen was inspected using microscopy, as shown in x = −18 mm and x = 8 mm. This area of high strain increased in size by growing further in the negative x-direction as the bending displacement was increased, as can be seen in strain field ‘d' in Finally, DIC has been used to observe the progression towards failure of all the specimens. A typical example of these data for a specimen with a nominal waviness of 25% is shown in By testing specimens across a wide range of nominal waviness levels, this study has shown that in-plane fibre waviness can result in significant reductions in the load-bearing strength of laminates. If similar defects occurred in an aerospace structure, this could result in the structure failing before it reached its design ultimate load. The shape of the buckled fibres that form the waviness defect, and the way the defect affects remnant strength is subject to high levels of variability due to the complex microstructure of the heterogeneous composite material. The technique for generating fibre waviness developed in this study allows the variability in the shape and severity of the waviness defects to be explored, with the average level of severity dependent on the shape of a rigid former. Computational studies of these defects can provide information about how they will behave, but it is not possible to confirm that a model satisfactorily describes the mechanics of a defect without experimental studies. For example, findings in this study suggest that residual strains may have a significant effect on the remnant strength of laminates containing fibre waviness, while computational studies often focus only on the orientation of fibres ,13. The 5.A method of creating in-plane fibre waviness in composite laminates has been developed and used to produce a large number of specimens at six levels of severity of waviness. This method allows the size, position and severity of the defect to be varied. The manufactured specimens were inspected using two non-destructive techniques and a novel assessment technique based on residual strains. By creating a large batch of specimens, the capabilities of the new residual strain-based inspection technique were explored and it was shown to be capable of both detecting and characterizing waviness defects. Two robust Bayesian linear regression models, capable of predicting the ultimate strength of the specimens in bending, were fitted to the ultrasound and residual strain data. The predictions of the ultimate bending moment based on the residual strain measurements were found to have an uncertainty that was 57% of that for the ultrasound-based predictions. This suggests that residual strains could be used, under certain conditions, to inspect laminates for waviness defects. It also indicates that residual strains appear to have a significant effect on the failure of laminates containing waviness defects and, thus, that incorporating residual strains into computational models of the defect could result in improved simulations of its behaviour. The use of these new techniques, for creating and characterizing in-plane waviness defects, has the potential to enhance our understanding of the influence of fibre-waviness defects on the behaviour of composite structures and to provide detailed experimental data for the development and validation of computational models.