Most studies on the adhesive mechanisms of climbing animals have addressed attachment against flat surfaces, yet many animals can climb highly curved surfaces, like twigs and small branches. Here we investigated whether tree frogs use a clamping grip by recording the ground reaction forces on a cylindrical object with either a smooth or anti-adhesive, rough surface. Furthermore, we measured the contact area of fore and hindlimbs against differently sized transparent cylinders and the forces of individual pads and subarticular tubercles in restrained animals. Our study revealed that frogs use friction and normal forces of roughly a similar magnitude for holding on to cylindrical objects. When challenged with climbing a non-adhesive surface, the compressive forces between opposite legs nearly doubled, indicating a stronger clamping grip. In contrast to climbing flat surfaces, frogs increased the contact area on all limbs by engaging not just adhesive pads but also subarticular tubercles on curved surfaces. Our force measurements showed that tubercles can withstand larger shear stresses than pads. SEM images of tubercles revealed a similar structure to that of toe pads including the presence of nanopillars, though channels surrounding epithelial cells were less pronounced. The tubercles' smaller size, proximal location on the toes and shallow cells make them probably less prone to buckling and thus ideal for gripping curved surfaces. Each pad adheres by secreting a watery fluid, generating capillary forces resulting from the thin fluid layer between the pads and the surface. Previous studies have investigated the details of the attachment mechanisms and the attachment performance of various tree frog species –4. Althoet al. [et al. [Litoria caerulea) have shown that these structures barely come into contact with a flat surface [A recent study by Herrel et al.  tested t [et al.  studied  [et al. . However [et al. . To the  surface . We prop surface ,16 whichRhacophorus dennysi) climb and hold on to cylindrical objects by (i) measuring the ground reaction forces involved in climbing one fixed-sized cylindrical column, (ii) measuring the contact area of the adhesive pads and subarticular tubercles coming into contact when climbing differently-sized cylindrical tubes, and (iii) by comparing the maximum friction and adhesion forces generated by individual pads and tubercles in restrained animals. We ask the following questions: do tree frogs use clamping forces to climb cylindrical structures or do they rely solely on tangential friction forces to propel themselves upwards (similar to climbing a flat surface)? Is their clamping grip affected by surface roughness? How much do the subarticular tubercles come in contact with the surface when digits are wrapped around an object and do they aid the friction forces generated by the pads?In this study, we investigate how Chinese gliding frogs (2.http://dx.doi.org/10.5061/dryad.pd7vt) [Our (non-invasive) experiments adhered to the Animal Behaviour Society guidelines (United Kingdom) for the use of animals in research. All data are available from the Dryad repository (d.pd7vt) .(a)Rhacophorus dennysi) were obtained from a local supplier in China. This species was chosen for its large body size and large limb span, which would enable them to grasp around our larger diameter cylinders (see below). The animals were housed in simple terraria that contained broad-leaved indoor plants and dry branches for climbing and resting. Frogs were kept at room temperature (20–28°C) and were fed with water and crickets ad libitum.Six individuals of the Chinese gliding frog  was measured to the nearest millimetre by carefully stretching out the limbs along a ruler. Electronic supplementary material, table S1 lists the mass, the snout–vent length and the forelimb span for the individual frogs. For our experiments, we tried to use each frog equally often, when possible. The detailed number of repetitions per frog is given for each experiment in the data available electronically.(b)x-axis as the left–right axis of each force transducer which would resolve the lateral force component (Fx) of a climbing frog. The y-axis was defined along the direction of gravity and would resolve the fore–aft components of a climbing frog. The z-axis was defined as the normal component, perpendicular to the surface of each transducer (Fz) and would resolve the tensile and compressive forces.To measure ground reaction forces of frogs climbing a cylindrical column, 24 separate custom-built three-dimensional force transducers (similar to ) were arAs the hindlimbs of a vertically upwards climbing animal usually push into the surface  in order to compensate for the pivoting torque around the centre of gravity, these normal forces would mask the clamping forces; we therefore focused our force analysis on the forelimbs only. However, as the sensors were arranged around the sides of an octagon, i.e. in a 45° angle towards each other, positive normal force could also occur when a frog pulled on a sensor in an oblique way  inset. ‘In addition to the force measurements, climbing animals were filmed using three synchronized high-speed video cameras filming the position of the animal from two sides  and one top view . From the different camera perspectives, we extracted the positions of the limbs placed on the corresponding force sensors.To examine the effect of gripping force  versus attachment force , we used different ‘coatings’ on the force transducer platform tiles. We used the bare platforms as a smooth surface, and the platform segments covered with a rough sandpaper . Adhesive pads adhere well to smooth surfaces ,19 but a−1  frogs eventually slipped and detached. In only two out of 17 trials, did frogs manage to stay attached until the board reached a vertical position (90°). In all other cases, frogs detached on reaching an angle of 75 ± 6° (mean ± s.d.). This is in contrast to the attachment of the frogs to a flat smooth vertical surface, where frogs adhered without any problems.The anti-adhesive nature of the rough sandpaper substrate was tested by challenging the frogs to stay attached to a flat, platform covered with the sandpaper which could be tilted. By slowly rotating the platform from a horizontal into a vertical position at approx 16 ± 9 deg s(c)To measure the contact area of pads and subarticular tubercles in climbing frogs we used transparent, Perspex substrates. We allowed the frogs to climb a flat sheet and two cylindrical tubes −1). By moving the force transducer with a motorized stage controlled through a customized LabView program, we performed two sets of measurements: (i) a lateral movement to measure friction (travel of 6 mm in 10 s with a preload of 2 mN) in the proximal and distal direction of the pad to test for directionality and (ii) three detachment movements (2 mm in 10 s) to measure adhesion, namely after an initial attachment of the pad and after the two lateral movements.To measure the adhesion and friction forces on individual pads, we used a force transducer set-up similar to the one used before by . We restSynchronously with the force data acquisition, the contact area of the pad/tubercle was recorded using a stereo microscope (Leica M80) equipped with co-axial illumination in order to yield high-contrast images . The conFriction and adhesion were tested for each digit of the fore and hindlimbs on the distally located adhesive pads and most of the first subarticular tubercle, just proximal to the pad.(e)−1 and its fore and hindlimbs severed. After fixation of the tissue with 2.5% glutaraldehyde for 24 h, specimens were rinsed with 0.1 M phosphate-buffered sucrose followed by distilled water. The specimens were dehydrated with an alcohol series and critical point dried. Individual toes were mounted and sputter coated with gold before being viewed at 2000× and 15 000× using a Phillips SEM 500 scanning electron microscope.One frog was sacrificed via a lethal dose of benzocaine 250 mg l3.(a)p > 0.05 for all comparisons).After each frog was placed carefully in a ‘head-upwards’ orientation, they either rested  or lowered themselves down . The similarities between the forces from frogs holding on at rest and frogs climbing down were such that they were pooled for further analysis  and Fz the normal force perpendicular to the surface. Additionally, we plotted the normal forces which were greater than the lateral forces (zF > xF). Against the smooth platform surface, the average lateral force per forelimb was 199.7 ± 165.3 mN  and 144.1 ± 147.4 mN for the normal force showing that frogs stayed attached by mainly using friction forces to compensate for their tilt. The average y-force  per forelimb was 354.0 ± 153.1 mN, therefore compensating a little bit less than a quarter of the body weight (23% of the average body weight of 157 g).R = 1841, z = −2.24, p < 0.05), whereas compressive normal forces nearly doubled . This indicates a stronger clamping grip when frogs could not use their adhesion pads on the anti-adhesive rough surface. Along with a stronger grip, frogs also compensated a larger amount of their body weight with the forelimbs .When frogs were challenged to climb the rough surface, all force components increased significantly. The lateral force for a forelimb increased 1.3 times ; for the few cases , the compressive forces on the rough surface (310.4 ± 281.3 mN) were significantly larger than on the smooth surface .As frogs often pulled in an oblique way at the force transducers and thus creating a normal component which could mask potential adducting forces, we considered only the cases where (b)p > 0.05) and thus pooled the data for further analysis.Similar to the experiments on the force platform, frogs either rested  or climbed downwards . We did not find differences in the contact area  between resting frogs or climbing frogs . The pad area of the forelimbs was similar on the flat and 120 mm tube but lower on the smaller 44 mm tube.The subarticular tubercles did not differ in contact area between fore and hindlimbs and only made contact when frogs climbed curved surfaces. After having presumably reached the maximum contact area on the 120 mm tube, the area of tubercles did not increase further on the smaller tube.Frogs also increased the overall contact area of their limbs by using other parts on their feet, like parts of the palms/soles or the ventral digit skin without pads and tubercles. In particular, the hindlimbs significantly increased contact area by often involving such areas of the feet and legs (e.g. see the images in (c)a–c show the peak friction forces, contact area and the force per area (shear stress) during a proximal pull and distal push. Friction forces for pads and tubercles were similar between each other but showed higher forces in the pulling than in the pushing direction . Similarly, the contact area for both structures is increased during a pull. However, as tubercles were much smaller in size (b), they exhibited about 6.6 times higher frictional stresses than pads (c), supporting our idea about their possible role as frictional pads.We collected the data from all digits of fore and hindlimbs of one frog and pooled fore and hindlimbs for further analysis. Figure 4 in size b, they ehan pads c, suppord–f) were roughly an order of magnitude smaller compared with friction forces. Peak adhesion forces were dependent on the time of detachment and the structure being probed . For both structures (pads and tubercles) forces were significantly larger after a pull than before a drag or after a push. Adhesive forces were similar for pads and tubercles (detachment after a pull) but as pads were much larger in contact area (e), the adhesive stress was about 6.2 times lower than for the tubercles (f).Adhesive forces d–f were act area e, the adubercles f.(d)R. dennysi is typical of that of other rhacophorid  tree frogs [The fine structure of the toe pads of ee frogs ,23,24. SThe subarticular tubercles, situated more proximally on each digit, are considerably smaller than the toe pads (indicated in schematic of 4.(a)As tree frogs use adhesive pads to climb even flat surfaces, it was unclear to what extent clamping forces would be used on the force measurement array. Unfortunately, our force platform did not allow us to distinguish in all cases between ‘true’ adducting forces and normal forces resulting from an oblique pull. However, the cases where normal forces were greater than the lateral forces showed a difference between the two substrates tested. In addition, the overall normal forces  increased significantly when frogs struggled to attach to non-adhesive surfaces, whereas the lateral forces did not change which points towards a clamping grip.By clasping around the object and creating adducting forces, the friction between the limb skin surface and the substrate can increase. The clamping grip we describe here was caused by opposing limbs and not within a hand, foot or even tail as described for many other arboreal animals using prehensile grips . HoweverOn both surfaces (smooth and rough) frogs generated large shear forces by positioning their fore limbs around the structure. Against a flat platform, vertically climbing animals usually pull on the surface with their forelimbs  and push into the surface with their hind limbs  in order to compensate the pitching moment of their body  showed that two species were able to climb the (narrow) slippery stems of their host plant (Macaranga) by not only interlocking their fine claws with the waxy layer but by stretching their limbs further outwards and thus using adduction forces.For smaller insects such as ants, most curved surfaces (tree trunks or larger branches) appear to be virtually flat. Nevertheless, a comparative study by Federle & Brüning  on the c(b)When frogs climbed our transparent surfaces, in all cases, the adhesive pads came into contact with the substrate. Strikingly, on curved surfaces, the frogs additionally recruited subarticular tubercles for an increased contact area. Frogs increased the contact area to the substrate further by employing ventral skin from other parts of their body too. Not only did they often use the ventral skin on their toes, lying between the pads and tubercles but sometimes even large parts of the thighs and belly. However, it was evident from a few cases in our study and other earlier studies ,30, thatet al. [et al. [Phyllomedusa) managed to walk on even narrower (e.g. 1 mm and 4 mm diameters), horizontally placed tubes by wrapping individual digits around the substrate. The fact that the frog's centre of mass needs to be balanced above the substrate suggests that the skin on the digits has to compensate for potentially strong torques around a cylindrical substrate, and highlights the need for friction pads.The possible function of these tubercles as friction pads was mentioned by Noble & Jaeckle  and Ernset al. ; however [et al.  showed t(c)adhesive stresses, which was surprising given that the pads are usually the primary attachment devices. We believe that the pads still perform this role as main attachment devices as they are the only structures coming into contact when the frog is climbing a flat vertical surface. Individual contact area of tubercles are about five times smaller than the average pad area (measured during our pull-off experiments), but the sum of all tubercles in contact on curved surfaces is very close to the area of the pads (The force measurements on individual pads and tubercles in restrained frogs showed that tubercles exhibited higher frictional stresses than pads. This finding supports our idea that tubercles are used to enhance a friction grip. However, tubercles also showed larger the pads . We theret al. [Our results showed only weak evidence for directionality on the pads or tubercles for friction and a small but significant increase in adhesion after a pull. Previous research ,34 has set al.  showed tet al. ,37. We fThe chances of buckling and collapsing of the structures are less likely when the structures are subject to a pull and confirms the reorientating behaviour of frogs to climb up or down a vertical surface with their heads pointing upwards . When do5.Our study demonstrated how tree frogs using adhesive pads for climbing smooth surfaces can cope with curved and anti-adhesive substrates by applying clamping grips. Such grips use not just the adhesive pads found on the distal ends of each digit but also recruit additional adhesive structures located proximally to the adhesive pads. Compared with the adhesive pads, subarticular pads can withstand higher friction forces in potentially different directions and are thus ideal for climbing narrow substrates like twigs or branches.Understanding how animals with adhesive pads use gripping forces to aid their climbing will provide insights that will be beneficial for the development of climbing robots. The use of gripping forces on curved structures increases climbing efficiency and reduces the dependence on specialized adhesive structures. In addition, because adhesive structures often depend on shear and loading forces for effective function, gripping forces will actually aid the adhesive forces (e.g. in geckos  or artif


The purpose of this study was to retrospectively evaluate the contrast enhancement of the PVS.In textbooks, the perivascular space (PVS) is described as non-enhancing after the intravenous administration of gadolinium-based contrast agent (IV-GBCA). We noticed that the PVS sometimes has high signal intensity (SI) on heavily T2-FL images were obtained before and 4 h after a single dose of IV-GBCA. No subjects had renal insufficiency. On axial MRC at the level of the anterior commissure (AC)-posterior commissure (PC) line, 1 cm circular regions of interest (ROIs) were drawn centering on the PVS in the bilateral basal ganglia and thalami. Three-millimeter diameter ROIs were set in the cerebrospinal fluid (CSF) of the bilateral ambient cistern. The ROIs on MRC were copied onto the hT2-FL images and the SI was measured. The SI ratio (SIR) was defined as SIRPVS = SI of PVS/SI of the thalami, and SIRCSF = SI of CSF/SI of the thalami. The average of the bilateral values was used for the calculation. The SIRCSF, SIRPVS, and SI of the thalami were compared between before and 4 h after IV-GBCA. In 8 healthy subjects and 19 patients with suspected endolymphatic hydrops, magnetic resonance cisternography (MRC) and hTP < 0.01). The SI of the thalami showed no significant difference.The SIR was increased significantly from 1.02 ± 0.37 to 2.65 ± 0.82 in the CSF (P < 0.01) and from 1.20 ± 0.35 to 2.13 ± 1.23 in the PVS at 4 h after IV-GBCA  space and the PVS. This might be a first step in the imaging evaluation of the glymphatic system (waste clearance system) of the brain. The enhancement of the PVS, though, has been reported in a patient with renal insufficiency.11 The PVS represents an entrance point to the glymphatic system, a recently discovered macroscopic waste clearance system of the brain.13In textbooks, the perivascular space (PVS) is described as non-enhancing after the intravenous administration of gadolinium-based contrast agent (IV-GBCA).The purpose of this study was to retrospectively evaluate the contrast enhancement of the PVS both prior to and 4 h after a single dose of IV-GBCA in subjects who underwent MR imaging.2-FL images were obtained on a 3T scanner  prior to and 4 h after a single dose of IV-GBCA. No subjects had renal insufficiency. The 8 volunteers and 7 of the patients received IV-GBCA of 0.1 mmol/kg body weight or 0.2 ml/kg body weight using Gadoteridol  and the other 12 patients received the same amount of IV-GBCA using Gadodiamide . In this study, we defined renal insufficiency as the history of proteinuria within 3 months or the estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2.In 8 healthy men (ages: 29–53) and 19 patients with suspected endolymphatic hydrops , MR cisternography (MRC), and hT2-FL images. The voxel size was 0.5 × 0.5 × 1.0 mm3. All sequences utilize a frequency selective fat suppression pre-pulse. Further details of the MR parameters are listed in All MR scans were performed using 32-channel array head coil and GRAPPA (acceleration factor of 2). The slice thickness, field of view, matrix size, and slice position were identical in both the MRC and hT14The PVS was defined as “small, sharply delineated structures with CSF-like intensity on the MRC that followed the course of the perforating arteries.” If a CSF-like signal area of 3 mm or above was found, coronal reformatting was performed to exclude the CSF containing lacunes by checking the shape .14The circular 1 cm diameter ROI for the PVS in basal ganglia was placed according to the instructions given below.2-FL image and the SI of the ROI was measured  of the PVS was given as SER = SIR of the post-contrast administration/SIR of the pre-contrast administration.The SI ratio (SIR) was defined as: SIRCSF, SIRPVS, and SI of the thalami were compared prior to and 4 h post IV-GBCA using Student’s t-test. The SER of the volunteers and patients were compared using Mann-Whitney’s test. We set 5% as a significance level for the statistical test.The SIRFor the healthy volunteers’ scans, the medical ethics committee of our institution approved the study and written informed consent was obtained from all volunteers.For the patients’ retrospective study, the medical ethics committee of our institution approved this retrospective study with a waiver of written informed consent from the patients.CSF increased significantly from 1.02 ± 0.37 to 2.65 ± 0.82 after the IV-GBCA administration (P < 0.01) and the mean SIRPVS increased significantly from 1.20 ± 0.35 to 2.13 ± 1.23 after IV-GBCA (P < 0.01)  , 4.No significant difference was found between the SI of the thalami prior to and 4 h after IV-GBCA.CSF increased significantly from 0.91 ± 0.32 to 2.35 ± 0.72 after the IV-GBCA administration (P < 0.01) and the mean SIRPVS increased significantly from 1.00 ± 0.16 to 1.38 ± 0.14 after IV-GBCA (P < 0.01).When limited to the 8 volunteers and 7 patients who received IV-GBCA by Gadoteridol, the mean SIRCSF increased significantly from 1.15 ± 0.11 to 3.03 ± 0.22 after the IV-GBCA administration (P < 0.01) and the mean SIRPVS increased significantly from 1.46 ± 0.10 to 3.07 ± 0.37 after IV-GBCA (P < 0.01).When limited to the 12 patients who received IV-GBCA by Gadodiamide, the mean SIRThe SER of the PVS and the SER of the CSF showed no significant difference between the 8 volunteers and 19 patients. When limited to the Gadoteridol group, the SER of the PVS and the SER of the CSF showed no significant difference between the 8 volunteers and 7 patients. Among the patients who received IV-GBCA by Gadodiamide and that by Gadoteridol, there was no significant difference in the SER of the PVS or the SER of the CSF.2-FL image to a low concentration of GBCA in fluid allowed the detection of the slightest enhancement of the PVS in subjects without renal insufficiency.8 It has been reported that GBCA injected intrathecally enters into the PVS and brain parenchyma.15 In this study, it is speculated that the IV-GBCA entered into the PVS from the CSF. In the previous healthy volunteer study, the timing of enhancement is earlier in the optic nerve sheath, Meckel’s cave around trigeminal nerve, and the fundus of the internal auditory canal around the seventh and eighth cranial nerves than in the CSF of the ambient cistern.10 These findings suggested that GBCM permeated from the peripheral part of the cranial nerve or nerve sheath to CSF, even in the subjects with intact blood–brain barrier.In this study, the enhancement of the PVS and CSF was confirmed even in subjects without renal insufficiency. The high sensitivity of the hT12; however, one report is describing the PVS around the artery as both the entry and outlet.16 If the PVS around perforating artery acts also as an outlet, more intense enhancement of PVS than CSF seems to be reasonable. The MR scans at most time points should be performed in the future to reveal the precise kinetics of GBCA in the brain. The PVS in another region of the brain should also be investigated. The glymphatic system is reported to be active during sleep17; thus, the sleep condition should be controlled during the investigation of the glymphatic system.On the other hand, some of the PVS signals were visually higher than that of the CSF at 4 h after IV-GBCA . Simple 19 Intact gadolinium-chelates might penetrate the brain parenchyma through the CSF and PVS .The findings of this study suggest that the blood–CSF barrier and the CSF–interstitial fluid (IF) barrier might be leakier than the blood–brain barrier. The deposition of gadolinium in the brain parenchyma such as in the dentate nucleus and globus pallidus is a recent hot topic in the field of MR imaging.21 It has been demonstrated that arterial pulsation drives the subarachnoid CSF flow into the PVS, clearing soluble proteins such as amyloid beta (Aβ) from the brain.21 The wastes are washed out of brain from peri-venous spaces to CSF space. Dysfunction of the PVS pathways thus may lead to an enlarged PVS, increased Aβ deposition, and subsequent neuronal dysfunction and loss, which clearly would have profound implications for Alzheimer’s disease.21Recently, several reports have suggested that the PVS is a part of the brain “lymphatic” system (glymphatic system) through which the interstitial solutes are cleared from the brain20 The results of this study might open the door to an examination of the glymphatic system in humans by a clinically feasible method using MR imaging and IV-GBCA.In the animal study, an intrathecal injection of gadolinium was utilized to investigate glymphatic function by analyzing the CSF-ISF exchange across the brain.The enhancement of the PVS at 4 h after a single dose of IV-GBCA was confirmed in human subjects without renal insufficiency. This might represent the first steps using MR imaging to evaluate the glymphatic system in humans.


The association of troponin-I levels and outcome in medical-surgical ICU patients has been studied before in populations with low to moderate prevalence of cardiovascular risk factors. The objective in this article is to examine the association of troponin-I levels with hospital mortality in patients with high prevalence of cardiovascular risk factors who were admitted with medical-surgical indications to a non-cardiac intensive care unit.Group I < 0.03, Group II = 0.03–0.3, Group III = 0.3–3 and Group IV > 3 ng/mL). Hospital mortality was the primary outcome. To study the association between elevated troponin-I and hospital mortality, we carried out multivariate logistic regression analyses with Group I as a reference group.This was a retrospective study of adult patients admitted to a tertiary medical-surgical ICU between July 2001 and November 2011. Data were extracted from prospectively collected ICU and clinical laboratory databases. Patients were stratified based on the highest troponin-I level in the first 72 h of admission into four groups  were diabetic and 1356 (40.2%) were chronically hypertensive. Among the study population, 2719 (81%) had elevated troponin-I levels (0.03 ng/mL and higher). Hospital mortality increased steadily as the troponin-I levels increased. Hospital mortality was 23.4% for Group I, 33.2% for Group II (adjusted odds ratio (aOR) 1.08, 95% confidence interval (CI) 0.84, 1.38), 49.6% for Group III , and 57.4% for Group IV . The association of increased mortality with increased troponin level was observed whether patients had underlying advanced heart failure or not. Subgroup analysis showed an increased mortality in patients aged < 50 years, non-diabetics and not on vasopressors.In a population with high prevalence of diabetes and hypertension, elevated troponin-I was frequently observed in medical-surgical critically ill patients, and showed a level-dependent association with hospital mortality.The online version of this article (10.1186/s12871-018-0515-7) contains supplementary material, which is available to authorized users. Troponin is a regulatory protein that controls calcium-mediated interaction of myosin and actin in the cardiac cells. Troponin-I elevation is an indicator of myocardial cell injury  and is cIn patients with acute coronary syndromes, elevated troponin-I levels have been shown to be associated with a proportionately increasing risk of mortality, therefore troponin-I has been used by clinicians for the prognostication of such patients –8. TropoThe prevalence of cardiovascular risk factors varies considerably around the globe. The incidence of diabetes in Saudi Arabia is very high (18.7%) vs. other Western countries 5–11%) . Accordi% . AccorTherefore, the aim of this study was to determine the association between elevated troponin-I and hospital mortality of medical-surgical critically ill patients in a population with high prevalence of cardiovascular risk factors and identify subgroups of critically ill patients with a particularly increased risk.This was a retrospective cohort study of critically ill patients admitted between July 2001 and November 2011 in the general medical-surgical (non-cardiac) ICU at King Abdulaziz Medical City, Riyadh, Saudi Arabia. The study was approved by the Institutional Review Board of the Ministry of National Guard Health Affairs, Riyadh, Saudi Arabia. All patients above 18 years of age admitted to the ICU with troponin-I levels measured at least once during the first 72 h were included in the study. Patients with burns and brain death were excluded from the study. For patients who were admitted to the ICU more than once during the same hospitalization, we included the first admission only. Patients admitted under cardiology or cardiac surgery such as those with an acute ST-elevation myocardial infraction  and post-cardiac surgery were admitted to cardiac ICUs and were not included in the study. Troponin-I test ordering was based on the discretion of the treating ICU team.Troponin-I was measured using Abbott ARCHITECT STAT Troponin-I Test, Abbot, Abbot Park, IL, USA. This troponin-I assay has a 10% coefficient of variation of 0.032 ng/mL (0.032 μg/L) according to the manufacturer. Data were extracted from prospectively collected ICU and clinical laboratory databases. The ICU has a database of all consecutive admissions with data being collected prospectively by a full-time data collector. The following variables were collected: age, gender, height, weight, acute physiology and chronic health evaluation (APACHE) II score , historyI < 0.03, Group II = 0.03–0.3, Group III = 0.3–3 and Group IV > 3 ng/mL [Patients were divided into four groups depending on the troponin-I level in the first 72 h Group  3 ng/mL . This caThe primary outcome was hospital mortality. The secondary outcomes were ICU and hospital length of stay (LOS), mechanical ventilation duration, need for renal replacement therapy (RRT).Statistical analysis was performed using the Statistical Analysis Software . Baseline characteristics, interventions and outcomes were reported as numbers and percentages for categorical variables and as means and standard deviations for continuous variables. They were compared among groups using Chi-square test and ANOVA, respectively.To determine if troponin-I category was an independent predictor for hospital mortality, stepwise multivariate logistic regression analysis was used with Group I as the reference, where variables included in the model were those showing statistical significance or those known to be clinically relevant . Results were presented as aOR and 95% confidence intervals. Furthermore, we analyzed the data based on troponin-I measured in the first 24 h.p-value of ≤.05 was considered statistically significant.Lastly, we carried out subgroup analyses with stratification by the following variables: age, gender, sepsis, diabetes, vasopressor use, operative admission category, chronic cardiac, respiratory and liver disease, chronic immunosuppression, acute kidney injury, hypertension, and cardiac arrest, adjusting for the same clinically relevant covariates mentioned above. Tests of interactions to assess whether these variables are effect modifiers of the association between troponin-I and hospital mortality. A p value< 0.0001). Similarly, APACHE II scores differed among the four groups , being lowest in Group I (20 ± 8) and highest in Group IV (29 ± 9). The prevalence of diabetes and hypertension differed between the groups with the lowest being in Group I (27% diabetic and 30% hypertensive) and the highest in Group IV (52% diabetic and 51% hypertensive) . Similar differences were seen with vasopressor use, the presence of sepsis, advanced heart failure, chronic renal disease, lactic acid, creatinine, and INR  were diabetic and 1356 (40.2%) were chronically hypertensive. Among the study population, 2719 (81%) had elevated troponin-I (≥0.03 ng/mL). Of those, 1572 (47%) patients had troponin-I level between 0.03 and 0.3 (Group II), 750 (22%) between 0.3–3.0 (Group III) and 397 (12%) had troponin-I level > 3 ng/mL (Group IV). At baseline, the four groups had stepwise differences in several variables. Patients in Group I were the youngest (47 ± 21 years) and Group IV the oldest  . Multivariate analysis  showed a level-dependent association between troponin-I levels and hospital mortality (adjusted OR (aOR) = 1.08, 95% CI = 0.84,1.38 for Group II, aOR = 1.64, 95% CI = 1.24, 2.17 for Group III and aOR = 1.80, 95% CI = 1.30, 2.49 for Group IV p-value for interaction = 0.0004 for Group IV). There was an incremental association of troponin-I levels with hospital mortality in non-diabetics but not in diabetics  . There was an association of troponin-I elevation with hospital mortality in patients who were on vasopressors but not those who were not on vasopressors (V) Table .Table 3SWhen these analyses were repeated with using troponin-I measurements in the first 24 h (instead of 72 h), we found similar results, which are presented in Additional file In this study, we assessed the association between troponin-I levels and hospital mortality in medical-surgical critically ill patients with high prevalence of cardiovascular risk factors who were admitted to non-cardiac ICU. We found that 81% of these patients with troponin-I measurements had an elevated level. We found that troponin-I levels were independently associated with hospital mortality.P < 0.001) and had an increased ICU stay by three days  [In a systematic review of 23 studies, Lim et al. assessed the frequency and association of elevated troponin-I with ICU mortality and length of stay and found a prevalence of troponin-I elevation of 43% (interquartile range 21–59%) . The sys = 0.04) . Most stOur findings are in accordance with the previously published systematic review . HoweverOne marked difference between our study and the studies included in the systematic review was the proportion of patients found to have elevated troponin-I. Our study population had a higher proportion of patients with elevated troponin-I, 81% compared to a mean of 43% in the articles studied in the systematic review . This diAnother difference is that Lim et al. systematic review suggested an association between troponin elevation and length of ICU stay, while our study did not demonstrate such an association .Our study indicated a level-dependent association of troponin-I elevation with hospital mortality in patients who were less than 50 years of age, those not on vasopressors, and who were non-diabetics. These findings are surprising and may, at first, appear to be counterintuitive. Interestingly, these findings are consistent with studies that showed no association between troponin-I and mortality in patients admitted to ICU with sepsis and in elderly ill patients presenting to the Emergency Department , 25. TheThe strengths of our study include the large sample size and the use of a prospectively collected ICU database. The limitations includes the retrospective design, being a single center study, utilizing troponin-I measurement instead of highly sensitive troponin (which was not available during the study period), unavailability of data on other risk factors (i.e. hyperlipidemia and smoking) because the data were obtained from an ICU database and unavailability of data regarding the specific causes of death. Although, troponin-I was ordered on a relatively large proportion of patients (36%), it was not obtained from the others, because routine troponin-I levels are not considered standard of care, and therefore the prevalence of high troponin-I cannot be generalized to all ICU patients. Nevertheless, this reflects real-life practice. Additionally, data on medications that may be cardioprotective were also not available. This latter effect was recently studied by Poe et al. who found that statins, β-blockers and aspirin have the potential to modify the mortality in patients with elevated cardiac troponin-I . NotablyUnlike patients with STEMI, who myocardial infarction due to an atherosclerotic plaque,  troponinOur study indicates that troponin-I elevation demonstrates a significant level-dependent association with hospital mortality in critically ill patients. As such, it may be used in conjunction with other measures as well clinical expertise to help clinicians prognosticating critically ill patients, as is currently done for troponin elevation in acute coronary syndromes . FurtherAdditional file 1:Table S1. Categories of main reasons of ICU admission. Table S2. Baseline characteristics of the four troponin-I groups based on troponin-I measured in the first 24 h. Table S3. Multivariate analysis of the four troponin-I groups and different clinical outcomes based on troponin-I measured in the first 24-h. Table S4. Subgroup analysis for the association between different levels of troponin-I measured in the first 24 h  and hospital mortality. The following variables were used as covariates in the model: age, APACHE II, sex, admission diagnosis, diabetes, chronic liver disease, chronic respiratory disease, chronic renal diseases, chronic immunosuppression, vasopressor use, sepsis, cardiac arrest, acute kidney injury, Glasgow Coma Scale, platelet, INR, bilirubin and lactic acid levels) Interaction test was performed for each subgroup. (DOCX 45 kb)


Upper-extremity exoskeletons have demonstrated potential as augmentative, assistive, and rehabilitative devices. Typical control of upper-extremity exoskeletons have relied on switches, force/torque sensors, and surface electromyography (sEMG), but these systems are usually reactionary, and/or rely on entirely hand-tuned parameters. sEMG-based systems may be able to provide anticipatory control, since they interface directly with muscle signals, but typically require expert placement of sensors on muscle bodies. We present an implementation of an adaptive sEMG-based exoskeleton controller that learns a mapping between muscle activation and the desired system state during interaction with a user, generating a personalized sEMG feature classifier to allow for anticipatory control. This system is robust to novice placement of sEMG sensors, as well as subdermal muscle shifts. We validate this method with 18 subjects using a thumb exoskeleton to complete a book-placement task. This learning-from-demonstration system for exoskeleton control allows for very short training times, as well as the potential for improvement in intent recognition over time, and adaptation to physiological changes in the user, such as those due to fatigue. Upper-extremity exoskeletons have demonstrated the potential to augment the capabilities of human users ,2, to asControl of upper-extremity devices is typically more difficult than control of lower-extremity exoskeletons, since the latter can rely on archetypical gait patterns, which do not have an upper-extremity analog. The degrees of freedom involved in common upper-extremity movements are also greater than those involved in most lower-extremity movements. Control of upper-extremity exoskeleton devices with sEMG will thus often use classification systems to reduce the complexity of the state space. sEMG classification systems have been demonstrated for in-air gesture recognition for teleoperation, sometimes in conjunction with inertial measurement (IMU) units. Wolf et al.  used thiWe aim to build on the work of these in-air systems to implement personalized sEMG control for upper-extremity exoskeletons, and take advantage of the sEMG signals preceding limb movement in order to improve fluency of human-exoskeleton movement. Beckers et al.  showed tIn contrast to sEMG inputs provided by in-air gestures, there is a particular paradox inherent in using sEMG inputs to control exoskeletons that raises a unique challenge. For the most fluent movement, sEMG inputs for exoskeletons should be collected from the muscles of the limb that are coupled to the exoskeleton. However, if movement of a limb is constrained by an exoskeleton, the muscles of that limb may not be able to express the range of sEMG signals that are necessary for the desired movement, or the motor strategy itself may be adapted to the constrained movement. This situation means that a constrained limb cannot appropriately train an sEMG-based exoskeleton if it cannot move the exoskeleton, but the exoskeleton cannot move appropriately if it has not been trained to understand sEMG inputs.One way to address this paradox was developed by Hamaya et al. ,18, who Kiguchi et al.  present Robot learning from demonstration (LfD) is a related body of work that can inform another solution. In this framework, appropriate robot actions are learned from examples provided by a human teacher, typically in a form that is very similar to the robot action . For exaThis paper builds on the sEMG-based anticipatory signal classification work of Siu et al.  to applyThere are several challenges faced by such a system. First, we use sEMG signals in a physically coupled human-robot system, where exoskeleton contact can change the signal. Second, since the system uses human-in-the-loop machine learning, care must be taken to ensure that the system acts appropriately even when it is being trained to avoid discomfort or injury. We implement novice (non-specific) placement of the sEMG sensors, since this is likely to occur when donning an exoskeleton in an operational setting as the muscles of the forearm can be difficult for a non-expert to palpate. In learning appropriate exoskeleton reactions, the system must also account for shifts in the muscles under the skin, which would occur even with expert sensor placement. In contrast to the work done by Hamaya et al. ,18, our We present an exoskeleton control method that learns user intentions from interaction pressure between the user and the exoskeleton, along with and surface electromyography (sEMG) signals collected while the exoskeleton is in use. For this proof-of-concept, a single-degree-of-freedom thumb exoskeleton, the Interaction-based Learning Exoskeleton (ILEXOS Thumb) was used with the goal of assisting a user’s grasp and release motions . The sysSensing was accomplished by two Flexiforce force-sensitive resistors  mounted on either side of the thumb, as well as a band comprised of six Delsys Bagnoli double-differential sEMG sensors located on the forearm . The sEMG sensors were uniformly distributed along the band, and placed at the widest point of the forearm, without specific azimuthal placement of sensors on muscles. The force-sensitive resistors (FSRs) and sEMG sensors were connected to a single National Instruments USB-6351 Multifunction I/O device  which read inputs at 1000 Hz. The raw sEMG signals were processed with a 6th-order Butterworth filter (10–450 Hz), followed by a 60 Hz notch filter to remove noise from surrounding electrical equipment.sEMG data were divided into 125 ms epochs for feature extraction, and successive epochs have 100 ms of overlap. This epoch size was chosen because it was between the 50 and 500 ms epochs used in literature ,29,30,31Exoskeleton operation was segmented into a training phase and a test phase. During the training phase, a pressure-based limit switch controller was used. The test phase used one of three controllers: the pressure-based limit switch (LS), a non-adaptive EMG controller (NA), or an adaptive EMG controller (AC).The pressure-based limit switch controller defaults the exoskeleton to an open grip, and closes the grip if sufficient pressure is applied to the interior FSR. The threshold for grip closure is adjusted on a person-by-person basis due to variations in thumb size and the way the exoskeleton is worn. sEMG data was collected along with pressure data during the phase, but was not used as a control input.The NA controller combines the pressure and sEMG data collected during the training phase to allow the exoskeleton to learn a user-specific correspondence between user intent and sEMG features via a supervised K-nearest-neighbors (KNN) classifier on fourteen signal features for each of the six sEMG sensors. Labeled data are generated by the user during a training period when the LS controller is used, with intent labels from the FSR pressure, and features from the sEMG. The collected sEMG features are standardized via mean-subtraction and divided by the feature values’ standard deviation, forming the model used for classification.We use a supervised KNN for online learning of exoskeleton control because there is no model training time after data are collected, and it works well on small datasets. Both of these considerations are important for human-in-the-loop machine learning and control, where training time is limited by the patience and comfort of the user. The supervised KNN classifier is not the same as the more commonly used unsupervised KNN clustering algorithm, since the latter does not used labeled data .t is applied to the features from epoch t − 1, so that labels apply to the 125 ms time window that ended 25 ms before the original label time. The last epoch in a trial is assumed to have the same label as the epoch before it. The optimal amount of epoch shift depends on the length of each epoch and amount of time between successive epochs, and the single-epoch shift used here was chosen after preliminary experiments using zero to six epochs of shifting.To encourage the model to learn a classification that anticipates the motion of the user rather than simply replicating the reactive behavior of the limit switch controller, the labels for all features are shifted backwards in time by a single epoch. Thus, the label for epoch Unlabeled data during the test phase were classified by comparing new data points to its K nearest neighbors. We set When this controller is in use, pressure data continued to be collected for post-hoc analysis, though only the sEMG data were used as control inputs within the test phase. The sEMG-to-actuation mapping used in this controller remained constant for all trials.The AC controller was initialized in the same way as the NA controller. This initialization means that the first test trial using either the AC or the NA controllers is equivalent (though different mappings may be learned depending on the associated training). This controller diverges from the NA controller after the first test trial.Between test trials, the AC controller performs an update to the sEMG mapping via the KNN classifier. First, epochs from the preceding test trial were labeled. A “close” label was applied if the pressure on the interior FSR exceeded that of the exterior FSR by the threshold set for LS control. An “open” label was applied if the pressure differential exceeded the threshold in the opposite direction. If neither threshold is exceeded, the open/close actuation command previously given for the epoch is assumed correct and the corresponding label is applied.Next, a “full KNN model” is created using all the data collected during the previous AC trials, along with the training trial. Since KNN runtime scales linearly with the number of data points, using the full model is prohibitively expensive for real-time exoskeleton operation. We thus construct a reduced model with fewer data points that is as close to the full model as possible, as measured by the L2 norm between the models. A reduced model consisting of 1500 randomly sampled points from the previous trial is built, and the L2 norm between this and the full KNN model was calculated.Since calculating the L2 norm via integration is infeasible on the 84-dimensional feature space, we use a Monte Carlo integration of the norm with importance sampling  to emphaReduced models are constructed and evaluated via random resampling of new sets System fit. The user dons the sEMG armband and the exoskeleton, and adjustments are made to the thumb holder and actuator placement to ensure comfort and an aligned center of rotation.sEMG calibration. The user holds his or her arm steady, laterally in his or her lap, while the armband records five seconds of sEMG data, in order to establish a nominal resting mean and standard deviation for each sensor.Range-of-motion calibration. The user moves the unpowered exoskeleton to acceptable “close” and “open” positions for the desired task, which are then recorded by the exoskeleton.Limit switch deadband calibration. A deadband for the limit switch of the pressure-controlled case is set for the individual.Training. The LS mode is used initially to allow the user to familiarize themselves with using the exoskeleton, and then the desired task is performed.Testing. If the NA or AC mode is being used, the system is switched to an sEMG-controlled mode, which uses a classification model based on previous data to move the exoskeleton according to the inferred user intent. If the LS mode is being used, then the controller remains the same as it was in training.Updates. If the AC mode is being used, the system periodically updates its sEMG mapping. For the experiment (described below), these updates occur during breaks in the task.A typical concept of operations for training and using the sEMG control mode is described as follows:The efficacy of the learned sEMG control system was evaluated using a tabletop book-shelving task. Twenty people were consented for the study, but two subjects’ data were unusable due to technical issues with the exoskeleton in one case, and unforeseen distractions for the subject in another. For one participant, data from two out of the twelve total test trials was unusable as the exoskeleton had a wire that became disconnected and data were not captured. The remaining trials for this subject were appropriately captured and are included in the analysis. The 18 participants that completed the study and were included in data analysis consisted of eleven males and seven females, all of whom were right-handed, had 20/20 or corrected to 20/20 vision, and had no self-reported disabilities or injuries affecting the use of their right arms. The experiment protocol was approved by the MIT Committee on the Use of Humans as Experimental Subjects (protocol 1454402), and all participants provided written informed consent. All participants received up to $20 as compensation. Subject age ranges and collected anthropometry data are shown in During the experiment, participants were seated in front of a tabletop bookshelf with six shelves (70 cm wide by 41.5 cm high by 15 cm deep), and asked to move a book between the tabletop and shelves on the bookshelf using their right hands while wearing the exoskeleton. Participants were instructed to move the book in a palm-down position, using their thumbs to support the bottom of the book . Two corA single iteration of the task consisted of grasping a book on the tabletop, placing it in one of six locations on the bookshelf, releasing the book, touching the table where the book was originally placed, and then grasping and moving the book back to its original location .Steps 1 through 4 of the concept of operations were followed at the beginning of the experiment to adjust the system for each user. The experiment itself consisted of three blocks, one to test each controller. After the system adjustment steps at the beginning of the experiment, each block began with a single-trial training phase consisting of six iterations of the task, once per location available on the bookshelf. The training period was always performed with the limit switch controller (step 5 of the concept of operations). This training period was not set at a fixed time duration, but rather ended when a user finished one set of six motions, utilizing all the spots on the shelf. There is thus some variability in training time, as well as the amount of data and the proportion of the data that is labeled as grasp or release.During the subsequent test phase, subjects performed four trials, each consisting of twenty-four iterations of the task, where each of the six locations on the bookshelf was used four times . These test phases were performed with the LS, NA, or AC controllers, depending on the block (step 6), with the subjects unaware of the nature of the controllers they were using. Trials were separated by a two-minute break and blocks were separated by five-minute breaks to allow the subject to rest. Subjects took Likert scale and NASA Task Load Index  surveys For the purpose of these comparisons, a grasping action (thumb close) is considered a positive result , and a release (thumb open) is a negative result . Ground truth was determined by the pressure on the FSR sensors, which should be high when a subject is holding the book, regardless of the controller used, due to the nature of the grasp being performed. Thus, a true positive occurred when the controller actuates to a close position when the user has a high pressure signal on the palmar thumb FSR (beyond the pre-set activation threshold for the LS controller). A false positive occurred when the controller actuated to a close position when the user did not have a high palmar thumb FSR signal, and continues to not have a high signal for the duration of the close actuation. Note that this scheme for obtaining ground truth means that false positives cannot occur with the limit switch controller by definition, since it uses the pressure signal directly for actuation. This setup also allows us to use the limit switch controller as a baseline against which to compare the other controllers. Data analysis for this paper focuses on examining three aspects of the exoskeleton behavior that influence system usability: (1) the training time of the system; (2) the precision  of the EMG intent classification; and (3) whether or not the EMG controllers anticipated the human.p < 0.05), so a one-way analysis of variance (ANOVA) was used to find differences in the times between controllers.The training times were calculated as the time between the first exoskeleton actuation command to either the last actuation command or the end of the trial (whichever came first). The times were normally distributed according to a Shapiro-Wilk normality test (not to grasp. The duration of these positives is important because it acts as a measure of how useful a true positive was (if it lasted the duration of an intended grasp) or how detrimental a false positive was (how long the user was stopped from doing what he or she intended). Both the number and duration of positive responses were determined to be non-normal using the Shapiro-Wilk test (p > 0.05), so nonparametric Kruskal-Wallis tests were used. The precision of the sEMG-based prediction was evaluated in two ways—(1) the number of true and false positives across trials and (2) the duration of the positive responses. Recall, a positive response is defined to be when the system is in a closed state. We evaluate precision  because ground truth information on negative predictions  is harder to infer. The grasping pressure provides a much clearer signal than the equivalent pressure signal when the intent is p > 0.05), thus trial data were pooled to examine the other independent variables. As the Kruskal-Wallis test does not include interaction effects in the omnibus test, an independent variable was defined to enable examination of the individual treatments. For the number of positive responses and action duration, the independent variable defined included controller and response type , with trials pooled as previously described. Omnibus Kruskal-Wallis tests were then performed for both number and duration, and Dwass-Steel-Critchlow-Fligner post-hoc pairwise comparisons were performed if the corresponding omnibus test was significant, enabling underlying interactions to be examined.An initial Kruskal-Wallis test was performed for each combination of controller and response type, with an independent variable of trial . All tests for trial were non-significant for both number and duration of grasps  between crossings . These ianticipation time for a true positive grasp as the time between the start of an EMG-triggered grasping motion and the time when the pressure threshold is crossed , and the mean time across all cases is 51 ± 14 s. Thus, users have similar exposure to the system within each training period.The mean training time for the three controllers is not significantly different  = 1.92, The number of true and false positives across trials for each controller is shown in p < 0.01), and the response time . For the number of responses, the median number of true positives for LS was significantly greater than both NA (p < 0.01) and AC (p < 0.01). There was no significant difference in the median number of true positive responses between NA and AC. The median number of false positives was significantly greater for AC than NA (p = 0.045). The median number of true positives was significantly different from false positives for NA (p < 0.001), although was not significantly different for AC (p > 0.05). For the median duration, there was no significant difference between the median true positive durations for LS and NA (p > 0.05) or LS and AC (p > 0.05). The duration of true positives was found to be greater than the duration of false positives for all cases (p < 0.05). The Kruskal-Wallis tests with an independent variable of controller crossed with response found significant effects for both the number of responses , actuation occurs as a reactive measure when the pressure of the user’s thumb crosses a given threshold. Part of the purpose of ILEXOS was to reduce the movement delay and the need for constant pressure by the user with an anticipatory sEMG control scheme. p < 0.01 for NA; W = 72, p < 0.01 for AC). The Wilcoxon Rank-Sum test supported a significant difference between the anticipation times for the two controllers .The results show that personalized sEMG-based exoskeleton control is possible with machine learning for this grasping task on a 1-DOF exoskeleton with short training periods. The learning from demonstration scheme implemented in ILEXOS allowed us to use a traditional limit switch controller to learn sEMG inputs, and then improve on that performance by anticipating the user by time-shifting the intent labels collected during the limit switch phase. The learned mappings are robust to both novice sensor placement and to subdermal muscle shifts. We specifically assessed three elements of the experiment: the training time of the system, the precision of the EMG intent classification, and the system’s ability to anticipate the user’s movements.The training time of our system was based on completing twelve placement motions on six shelf locations, and took approximately one minute for a novice user to complete. During this training time the exoskeleton moved in response to the user, but was controlled via a pressure-based limit-switch. This training time is on par with times used in other upper-extremity sEMG classification experiments, such as the two minutes used by Chan and Englehart  in theirComparing training times between studies is not straightforward, since many elements of these studies  are highly variable. However, shorter training time is certainly operationally desirable, as is robustness to imprecise sensor placement. In an industrial setting, for example, the ability to quickly don and begin using an augmentative system could translate to higher rates of adoption and lower user frustration in comparison to systems requiring significant training time for the classifier (to understand intent) and/or for the user (to precisely place sensors).It is promising to see that the duration of responses between true and false positives were significantly different in each case but that true positive durations in both sEMG-controlled cases were not different from the LS case . These rnumber of false positives. On the other hand, although the duration results show that the ILEXOS Thumb is usable under sEMG-controlled conditions, one potential issue is the relatively large While the tapping motion did remove this confusion from the system, it introduced a signal that was similar to the sEMG signal of a grasp if the subject was tapping hard. This was observed in many cases with both sEMG controllers, where tapping the table induced a short actuation by the exoskeleton, likely resulting in many of the 0.1 and 0.2-s false positives seen in Such false positives might be removed with additional training time or different AC controller updates assumptions (such as removing short-duration close labels from updates). However, the short duration of these motions means that they are largely operationally insignificant, though the presence of such unintended motions may affect the user’s trust in the system. On the other hand, such false positives could present a proprioceptive feedback to help the user learn the system’s behavior and adjust their own inputs as a kind of error augmentation, though such false positives would have to be limited to prevent user frustration or injury. We also considered the occurrence of multi-grasps—multiple grasps as indicated by pressure within a single period of exoskeleton close action. These occurrences had elements of true positive, false positive, and false negative (not opening when it should) responses, and were considered separately. In some cases, these were simply the result of the participant acting very quickly in letting go of and re-grasping the book when it was placed on the table, which may be an indication of a change in grasping strategy. These particular multi-grasps would likely have been reduced or eliminated with more explicit instructions to the participants. Although these grasps were not observed to be affecting the performance of the task.and that lack of pressure meant that the label obtained from the sEMG classifier was correct—drove the classifier to be heavily biased towards a close label. It is unclear why this occurred with these two subjects, but not with others. That such a phenomenon could happen, at least with two of the subjects and specifically with the AC controller, points to the need for additional refinements of the AC updates in order to prevent this particular failure mode , ,30, 18,3Although we present an instance of the ILEXOS system for a thumb exoskeleton that used a supervised KNN classifier, the interaction-based learning framework of ILEXOS is agnostic to the nature of the exoskeleton actuation, as well as to the machine learning method used. To that effect, there are several generalizable advantages of the ILEXOS framework. First, the exoskeleton is usable while it is training as long as a simple intent mapping is obtained . In contrast to systems that require the machine learning algorithms to explore the feature space by random perturbation ,18, our Second, the system allows novice sensor placement. Non-specific placement of sensors is likely to be the norm with operational systems, and our system is shown to be robust to such placement, in addition to subdermal muscle shifts that are typical of a pick and place task. Allowing novice sensor placement is an important difference to note in comparison to the sEMG-controlled systems developed by Hamaya ,18 and KThird, the system has the potential to improve over time, as additional data are collected from both the pressure and the sEMG sensors during the sEMG-controlled phase. The aforementioned modifications to the AC update assumptions may be necessary to ensure that times when the exoskeleton and the user are moving together fluently are labeled correctly, and we are able to avoid creating the strong bias towards a particular label that was observed in two of the subjects. The effect of different assumptions and timings for these updates should be considered in future work.Since we tested ILEXOS using a discrete classifier with two states, our results are limited to considering operation in those states for the case of the book-placing task. The interactive learning-from-demonstration framework could be extended to a continuous control system, but that is not evaluated in the present study.The system is generally applicable to different numbers of sensors and features. This study used six sEMG sensors, and fourteen sEMG features, but it does not specifically depend on any of these. Preliminary experiments have shown that intent recognition accuracy does not seem to suffer when decreasing the number of sensors and features, so further work could use a smaller set of sensors and features to reduce cost and computation time. However, it should be noted that as the number of sensors decreases, the precision with which the sensors must be placed would need to increase, since less of the forearm will be covered by sensors, presenting a trade-off that must be considered.We also note that while supervised KNN is a fairly simple algorithm for nonlinear feature inputs like the present sEMG features, the algorithm has a linear time and space complexity of n  n  a) aO(nd) We motivated how the robust and anticipatory nature of our method has potential to improve the fluency for human-exoskeleton interaction in the augmentative case. The effects of the different controllers on human-exoskeleton fluency measures using motion capture and survey data collected during this experiment is ongoing work. The desire for improved fluency in the assistive case, where the exoskeleton aids in the movement of a non-healthy individual but is not doing so in a rehabilitative context is also of interest. For assistive exoskeletons, the robustness of our method to sensor placement may mean greater ease of use for at-home use cases. However, additional complexities arise when considering the assistive and rehabilitative use case. Progressive robot-assisted therapy, such as those using the wearable MyoPro exoskeleton  or the MThis paper presented a learning from demonstration exoskeleton system that transitions from a reactive pressure-based limit switch controller that is common in the current state of the art to an anticipatory sEMG-based controller. We show that this sEMG-based method is robust to novice sensor placement and subdermal muscle shifts during movement. The exoskeleton controllers show similar performance to a limit switch controller in terms of true positive grasp counts and times, but are able to move in time with the users’ thumbs by anticipating their actions approximately 0.2 to 0.3 s ahead of the pressure trigger. This learning from demonstration method has broader applicability to increasing the fluency of exoskeleton control by creating personalized sEMG mappings for individuals, and may also be applied to more complex multi-DOF exoskeletons without the need for perturbation-based system identification.


Metabolic profiling of cerebrospinal fluid (CSF) is a promising technique for studying brain diseases. Measurements should reflect the in vivo situation, so ex vivo metabolism should be avoided.To investigate the effects of temperature (room temperature vs. 4 °C), centrifugation and ethanol, as anti-enzymatic additive during CSF sampling on concentrations of glutamic acid, glutamine and other endogenous amines.CSF samples from 21 individuals were processed using five different protocols. Isotopically-labeled alanine, isoleucine, glutamine, glutamic acid and dopamine were added prior to sampling to trace any degradation. Metabolomics analysis of endogenous amines, isotopically-labeled compounds and degradation products was performed with a validated LC–MS method.O-phosphoethanolamine concentrations were also lower when ethanol was added. Degradation of isotopically-labeled compounds (e.g. glutamine to glutamic acid) was minor with no differences between protocols.Thirty-six endogenous amines were quantified. There were no statistically significant differences between sampling protocols for 31 out of 36 amines. For GABA there was primarily an effect of temperature (higher concentrations at room temperature than at 4 °C) and a small effect of ethanol (lower concentrations if added) due to possible degradation. Most amines can be considered stable during sampling, provided that samples are cooled immediately to 4 °C, centrifuged, and stored at − 80 °C within 2 h. The effect of ethanol addition for more unstable metabolites needs further investigation. This was the first time that labeled compounds were used to monitor ex vivo metabolism during sampling. This is a useful strategy to study the stability of other metabolites of interest.The online version of this article (10.1007/s11306-018-1333-0) contains supplementary material, which is available to authorized users. Novel high-throughput biochemical analysis technologies used in metabolomics and proteomics allow for large-scale profiling of metabolites and proteins in body fluids  on the stability of the metabolic profile of primary and secondary endogenous amines. To this end we compared five different sample handling protocols for CSF. In addition, we quantified the degradation of allegedly unstable metabolites such as glutamine by adding isotopically-labeled versions of these metabolites to sampling tubes before CSF sampling.We obtained human CSF samples from 21 individuals  as part of a research program on migraine pathophysiology. The study was approved by the Medical Ethical Committee of Leiden University Medical Centre (LUMC). All subjects gave written informed consent prior to collection.CSF sampling was performed before 12.00 am via lumbar puncture. All subjects were overnight fasted and only allowed water in the 8 h preceding lumbar puncture. Local skin was disinfected with Chlorhexidine (5 g/L)/denatured ethanol 70% . CSF was sampled between the L3/L4, L4/L5 or L5–S1 interspace with an atraumatic Sprotte® needle . For routine CSF diagnostics we collected 3 mL CSF followed by 12 mL for migraine research purposes.For this study we additionally collected five times 1 mL CSF in five separate 15-mL polypropylene falcon tubes  that already contained a 100-µL mix of isotopically-labeled compounds . Tubes were inverted at least ten times to mix the CSF and ILC-mix. For each of the five tubes there was a unique sample handling protocol to study the effects of temperature, centrifugation and the addition of ethanol. The five protocols are depicted in Fig. For protocol 1, CSF collection and centrifugation were performed at room temperature; for the additional four protocols sampling tubes were prepared on ice before CSF was added and further sample handling was also done on ice. For protocol 2, the sampling tube also contained 2 mL cold ethanol  besides the ILC-mix. For protocol 3, centrifugation was performed and ethanol was later added during aliquoting. For protocol 4, we performed no additional sample handling steps. For protocol 5, the samples were centrifuged.All centrifugation steps  were performed for 5 min  directly after sampling. Centrifugation in protocol 1 was performed at 21 °C and for protocols 3 and 5 at 4 °C. After centrifugation, the supernatant was transferred to another 15-mL polypropylene falcon tube, and divided in aliquots. Samples from protocols 1, 4 and 5 were divided in 0.25-mL aliquots [into 0.5-mL cryotubes ], samples from protocols 2 and 3 in 0.75-mL aliquots [into 1.5-mL cryotubes (Eppendorf)] because of the additional ethanol. All aliquots were immediately placed on dry ice after processing, within 30–60 min from sampling, and transferred to − 80 °C for storage within 120 min from sampling. All samples remained at − 80 °C until sample preparation, no extra freeze–thaw cycles were allowed.13C2-alanine  and 13C15N-isoleucine  , U13C–U15N–UD-glutamic acid , and 1,2,3,4,5,6-13C6-dopamine . Isotopically-labeled compounds were selected to minimize overlap of their isotopic envelopes. Concentrations of isotopically-labeled compounds were chosen such that they approximate physiological concentrations after addition of CSF. The ILC-mix was prepared in one batch, then divided into aliquots, and stored at − 80 °C until the day of sampling. Maximal 1 h before sampling the ILC-mix was defrosted at room temperature. For each subject, we prepared the five 15-mL polypropylene collection tubes for sampling by adding 100 µL of the ILC-mix.To assess whether potential differences between protocols were due to metabolic degradation, and not due to other factors, we added ILC-mix containing two stable and three potentially unstable compounds to the collection tubes. Stable isotopically-labeled compounds were: 2,3-13C–U15N-labeled amino acid mix (Cambridge isotope laboratories), D4-β-alanine, D6-ornithine, D4-histamine, D6-2-aminobutyric acid, D3-3-methyl-histidine, D3-3-methoxytyrosine and D4-3-methoxytyramine (CDN Isotopes) were used as internal standards. Acquired data were evaluated using MassHunter software  by integration of assigned SRM peaks and normalization using proper internal standards (ISTDs). For analysis of amino acids, their U–13C–U15N-labeled analogues were used as ISTDs. For other amines—the closest-eluting ISTD was used. Relative concentrations of endogenous compounds were reported as response ratios to their respective ISTDs. Data quality was monitored and compensated for shifts in sensitivity of the mass spectrometer over time using additionally measured quality control (QC) samples as described before . The fact that both endogenous (from CSF) and isotopically-labeled metabolites (from ILC-mix) were increased indicated that there was less ethanol in samples from protocol 2 than anticipated. This systematic error could result from evaporation of ethanol before, during and after sampling, or might be caused by inconsistencies in pipetting of cooled volatile ethanol.l-alanine and l-isoleucine . l-alanine and l-isoleucine are compounds with hardly any degradation ex-vivo . See Supplemental Methods for full descriptions and examples of the applied corrections.In order to test for differences in endogenous metabolite levels between all five protocols, without the influence of the systematic error described above, we standardized concentrations of all endogenous metabolites to the concentrations of endogenous t-test comparisons were applied with Bonferroni’s correction. The same ANOVA strategy was used to compare degradation percentages of 13C2-glutamine to 13C2-glutamic acid (volume correction and log-transformation were not necessary for these data). Repeated measures ANOVA was performed with R software (package “ez” version 4.2-2).Relative concentrations of endogenous compounds and absolute concentrations of stability markers were log-transformed prior to statistical analysis . Routine CSF diagnostics were performed and were all within reference limits. Median erythrocyte count was 2.5 cells/3 µL (range 0–200); mean leucocyte count 3.50 ± 2.50 cells/3 µL; mean total protein 0.33 ± 0.11 g/L; and mean glucose levels 3.24 ± 0.19 mmol/L. With LC–MS we identified and quantified 36 amino acids and 7 isotopically-labeled compounds.O-phosphoethanolamine (PE), gamma-aminobutyric acid (GABA), l-glutamic acid, l-methionine sulfoxide and l-aspartic acid  but only compared to protocol 3 .There were no significant concentration differences between the sampling protocols for 31 out of 36 (86.1%) amines. Five amines showed significant differences: id Table a. GABA cid Table b. Howeveid Table b. Concen13C2-alanine, and 13C15N-isoleucine) and the potentially unstable isotopically-labeled markers  .There were no significant differences between protocols in concentrations of the stable  on GABA, and effects of ethanol on PE and partly on GABA. In addition, our labeling strategy with isotopically-labeled compounds allowed us to successfully monitor degradation of l-glutamine to l-glutamic acid and showed that the amount of degradation was extremely minimal, with no difference between protocols.When performing metabolomics in CSF we want our measurements to reflect the in vivo situation as close as possible. It is therefore important to avoid ex vivo metabolism. The aim of this study was to investigate the effect of three major factors  on the stability of primary and secondary endogenous amines. To this end we compared five different CSF sample handling protocols. In addition, we aimed to quantify the degradation of allegedly unstable metabolites such as Our observation that most endogenous amines are stable under controlled sampling conditions with minimal delay in sample storage is in line with other large scale stability studies which have measured multiple amino acids in CSF  and no observable decrease at − 80 °C  degradation of l-glutamine and l-glutamic acid is therefore the most important factor leading to the alleged instability of these compounds  the impact will be similar for all protocols.The labeling strategy applied in this study allowed the selective monitoring of the conversion of l-glutamic acid and l-methionine sulfoxide in samples at room temperature (protocol 1) compared to cooled samples containing ethanol (protocol 3). Taken together, the effect of temperature seems limited for most of the metabolites provided that the CSF samples are processed quickly, cooled to − 20 °C within 1 h and stored at − 80 °C within 2 h. In conclusion, it is advisable to quickly cool CSF samples and perform sample processing at 4 °C to reduce enzymatic activity, which seems especially relevant for GABA.The rate of chemical and enzymatic activity is highly dependent on the sample temperature, which combined with a delayed storage determines the actual degree of change


What are the perspectives of adolescents conceived using surrogacy, egg or sperm donation regarding their conception and the third party involved?The majority of adolescents described feeling indifferent about their conception, and yet simultaneously reported an interest in the third party involved, or were in contact with them.There is an assumption that children conceived through reproductive donation will feel negatively about their origins in adolescence. However, little is known about the views of adolescents who have been conceived through different types of reproductive donation.Forty-four adolescents, all of whom had been told about their conception in childhood, participated in a semi-structured interview as part of the sixth phase of a longitudinal, multi-method, multi-informant study of assisted reproduction families in the UK.n = 22), egg donation (n = 13) or sperm donation (n = 9) to heterosexual couples, and varied in terms of their information about, and contact with, the third party involved in their conception. Semi-structured interviews were conducted in participants’ homes. Interviews were analysed qualitatively to determine adolescents’ perceptions of their conception, and their thoughts and feelings about the surrogate or donor involved.All adolescents were aged 14 years, had been conceived using surrogacy (n = 7), indifferent (n = 32) or ambivalent (n = 5) about their conception. Amongst adolescents not in contact with the surrogate or donor, most were interested (n = 16) in the surrogate or donor, and others were ambivalent (n = 4), or not interested (n = 6) in them. Adolescents in contact with the surrogate or donor expressed positive (n = 14), ambivalent (n = 1) or negative (n = 1) feelings about them.Adolescents were found to feel positive (Of 56 adolescents invited to take part in the study, 47 consented to take part, giving a response rate of 84%. It was not possible to obtain information from adolescents who do not know about their conception.The findings contradict the assumption that children conceived through reproductive donation will feel negatively about their origins in adolescence and suggest that it may be helpful to draw a distinction between adolescents’ feelings about their conception in general, and their feelings about the surrogate or donor in particular.This study was funded by the Wellcome Trust [097857/Z/11/Z]. The authors have no conflicts of interest to declare. Reproductive donation is a form of assisted reproduction in which a third party assists in the conception of a child, either through donated gametes (sperm or egg) or by hosting the pregnancy, either using their own egg  or the commissioning mother’s  .The body of empirical evidence on donor-conceived offspring’s perspectives is growing, but it is often limited to non-representative samples of adults conceived by sperm donation  or lesbiThe transition from childhood to adolescence has been described as a crucial time for identity formation and the development of personal autonomy , adolescRecent research has shown that the rate of parental disclosure is increasing both amongst parents who have used donors who may be identified by offspring, and those who have used anonymous donors . The preAdolescents’ views of their donor conception have largely been obtained through research using questionnaire methods. These surveys have recruited adolescents via an online forum for those interested in making connections with the donor or those conceived using the same donor  or singlThis study is the first to have asked adolescents conceived through different types of reproductive donation  directly for their views. It reports the thoughts and feelings of a systematic sample of adolescents who have been raised in heterosexual two-parent families that were initially recruited to the study when the adolescents were infants through fertility clinics, the UK Office for National Statistics, and Childlessness Overcome Through Surrogacy (COTS), the only UK surrogacy organization at the time. The study sought to ascertain whether or not children feel distressed about the circumstances of their conception or birth when they reach adolescence, and what they think and feel about the surrogate or donor involved.The data analysed in this article are from the sixth phase of the UK Longitudinal Study of Assisted Reproduction Families that has examined the impact of reproductive donation on children’s psychological wellbeing and parent–child relationships from infancy to adolescence , 13 through egg donation, and 9 through sperm donation). The remaining three adolescents, who responded to the initial question of ‘Can you tell me more about how you were made?’ with ‘don’t know’, or otherwise diverted the interview to another topic, were asked no further questions, and their interviews were subsequently excluded from the analyses.All participants came from different families, and all had been told about their conception in childhood. In total, 28 (64%) were females and 16 (36%) were males. All participants were born in the year 2000, before the removal of donor anonymity in the UK in 2005. Overall, 14 (64%) of the adolescents conceived through surrogacy had contact with their surrogate, and 2 (15%) of the adolescents conceived through egg donation had contact with their egg donor , indifferent (n = 32) and ambivalent (n = 5) (Table Adolescents’ feelings were found to correspond to three distinct categories: positive (5) Table .‘I was really confused [when first told] but then afterwards I felt quite special.’ ‘I felt really special [when first told] because no one else was like that… I think I still feel like that. It’s different but it doesn’t really make me different.’ Some adolescents expressed positive feelings about their conception, describing it as ‘cool’, ‘interesting’ and a ‘special’ fact about themselves:‘Sometimes my friends ask me about it, they’re just like “oh, it’s really cool how you were made”…It’s a really cool process, and I’m always like, “oh, yeah, I was lucky I was made this way”.’ (Egg donation)‘It’s cool, I think it’s cool [laughs]. I quite like talking about it because it’s an interesting fact about me… My friends thought it was cool as well [laughs].’ Some of these adolescents also highlighted that their peers had responded positively:‘I don’t think I really minded [when first told] to be honest…I still don’t really care. It doesn’t make any difference.’ (Sperm donation)‘[I] don’t really mind. It doesn’t really affect my daily life.’ ‘I don’t really mind. Yeah, I don’t really mind.’ (Egg donation)‘I didn’t really care to be honest. I still don’t really care.’ The vast majority of adolescents expressed feeling indifferent about their conception. Adolescents in this category had been conceived through each of the reproductive techniques:‘Um, it didn’t really bother me. Mum is still my mum. Dad is still my dad.’ ‘I don’t think it really affects anything. I consider that my dad is still my dad, so.’ (Sperm donation)Some adolescents who described feeling indifferent also stated that their conception did not change the nature of their relationship with their parents:‘Um, [I’m] not like bothered about it but I think it’s quite interesting to know how I was made.’ (Egg donation)‘I don’t get like emotional or anything, it’s just like talking about anything else.’ (Egg donation)Others explained that they were indifferent at the same time as being interested in the process, or that they were comfortable discussing it with others:‘My friends ask questions that I don’t know the answer to or I don’t really want to know the answer to. Like “Where did your Mum get like the thing?”, “How much money did it cost?”, “How did you get like the sperm for it and stuff?”. I don’t really want to answer that.’ (Egg donation)However, others described sometimes finding such social encounters difficult:‘When I was little, I used to get called fostered and it sort of got to me “I’m not fostered! You don’t understand because you’re dumb!”…Frustrating when they say like “Oh you’re adopted, you’re fostered…” Surrogacy was described as particularly difficult to explain to peers:‘It makes you feel like you weren’t a mistake…but I sort of feel like I’ve got that part missing.’ (Sperm donation)‘Sometimes I can go, ‘Is it natural? Is it normal? Is it…a normal thing to happen?’ but kind of assure myself that it is fine.’ Some adolescents were ambivalent about their conception, and described a combination of different feelings:n = 28) had no contact with their surrogate or donor. These adolescents were found to be interested (n = 16), ambivalent (n = 4) or not interested (n = 6) in the surrogate or donor The majority of adolescents expressed a desire to either know who the surrogate or donor was, or to meet them. A list of these adolescents’ responses to the question about what they would like to ask the surrogate or donor is provided in Table ‘I would like to know who he is…quite a lot…Recently a lot more than I used to.’ (Sperm donation)‘It’s more important to me now….Um, and I’m just always thinking about what she looks like.’ (Egg donation)While most adolescents said that they infrequently thought about the donor, a minority stated that they thought about their donor frequently, and increasingly since entering adolescence:‘[I think about] who my real mum is…I just really want to know like, who she is and meet her maybe.’ ‘Yeah probably like [to know] who he is, and maybe meet him because he is my dad, but I’m not too bothered.’ (Sperm donation)Others who wished to know the identity of, or to meet the surrogate or donor described them as a ‘real’ parent:‘I would like to know who he is…quite a lot. And I told my mum that and I don’t want to tell my dad that because I don’t know how he would feel about that, so…’ (Sperm donation)‘I think mum might be a bit upset but she’d understand about it but…I’d only do it when I was 50 or 60…I don’t feel the need to now.’ (Egg donation)Yet others expressed concern that their interest in the donor might impact upon their non-genetic parent:‘Sometimes I think about it…wanting to meet my birth mum. [To] say thanks for being my birth mum [laughs]…It sounds really funny. Yeah, I don’t know, just thanks.’ One adolescent stated that they would like to meet the surrogate to express their gratitude:‘I’ve gone so long without knowing about her, it’s just easier…I think I’d want to meet to see if I had anything in common.’ (Egg donation)‘I heard it’s when you’re 18 or something you can try to find out who it was, so I would possibly consider doing that, but I don’t know really.’ (Egg donation)Four adolescents explained that they were either unsure, or expressed both wanting and not wanting to know more about, and/or to meet, the surrogate or donor:n = 6) in the surrogate or donor answered questions about whether they would like to know anything about, or to meet, the surrogate or donor with simply ‘no’, or ‘not really, no’.Those adolescents identified as ‘not interested’ (n = 16) were found to feel positive, ambivalent or negative about them (Table Those adolescents who had contact with their surrogate or egg donor (em Table . Most adn = 14) of adolescents described their relationship with the surrogate or egg donor positively. Many participants referred to them as a family friend, an aunt or a godparent, and reported having a close relationship:‘[Our relationship is] a good one. But I don’t talk to her like she’s my mum and she doesn’t talk to me like I’m her daughter.’ ‘She’s like family to me but we don’t see her that often because she lives quite far away.’ The large majority (Other adolescents emphasized that they didn’t really know the surrogate or donor well enough to say much about them. One adolescent was ambivalent about the surrogate, and in another case, the relationship between the surrogate and the adolescent and their family had broken down, with negative feelings reported as a result.This study sheds light on how adolescents make meaning of their conception during a developmental stage that is characterized by increased cognitive capabilities and marked identity development . The majFeelings of indifference towards their conception were found amongst adolescents conceived through each of the four types of reproductive donation under study: traditional surrogacy, gestational surrogacy, egg donation and sperm donation. Interestingly, adolescents’ lack of concern about their method of conception is consistent with their high levels of psychological wellbeing and the quality of relationships with their mothers at this age (In describing their experiences, several adolescents referred to the actual or anticipated responses of others—namely, parents and peers—to their feelings. It is noteworthy that a minority of adolescents described feeling concerned that their interest in their conception might upset the parent to whom they have no genetic connection, a finding that echoes earlier research (In terms of adolescents’ thoughts and feelings about the surrogate or donor, as in previous research on donor-conceived samples (Adolescents who were interested in the surrogate or donor mostly wanted to know more about why they had donated or acted as a surrogate, and some had questions about the surrogate or donor’s family, or children conceived using the same surrogate or donor. Previous studies of adolescents born through reproductive donation who use the internet to try to connect with donor relations have shown that adolescents desire to learn more about the donor and other children who share their genetic material in order to better understand themselves (While most of the adolescents who had contact with their surrogate or donor continued to have positive relationships with them (The findings of this study are based on a sample of adolescents who are all aware of their conception and have been raised in heterosexual two-parent families, and therefore tell us little about the experiences of adolescents in other family circumstances, or those who remain unaware of the circumstances of their conception or birth. Indeed, many parents in the overall sample of this longitudinal study have not told their children about the circumstances of their conception (Although there has been much concern about how children conceived using reproductive donation would feel about their origins as they grow older, the adolescents in this study mainly reported being unconcerned about their conception. The fact that none of the adolescents conceived through any of the types of reproductive donation were found to feel distressed about their conception is of considerable importance given such longstanding concerns. The findings also indicate that it is important to differentiate between adolescents’ feelings about their conception, and their feelings about the surrogate or donor. How these feelings may change as the adolescents enter adulthood remains to be seen.