Simulations demonstrated that estimates of realized genetic gain from linear mixed models using regional trials are biased to some degree. Thus, we recommend multiple selected models to obtain a range of reasonable estimates.Glycine max (L.) Merr.] breeding programs were performed to evaluate linear mixed models to estimate the realized genetic gain (RGG) from annual multi-environment trials (MET). True breeding values were simulated under an infinitesimal model to represent the genetic contributions to soybean seed yield under various MET conditions. Estimators were evaluated using objective criteria of bias and linearity. Covariance modeling and direct versus indirect estimation-based models resulted in a substantial range of estimated values, all of which were biased to some degree. Although no models produced unbiased estimates, the three best-performing models resulted in an average bias of Genetic improvements of discrete characteristics are obvious and easy to demonstrate, while quantitative traits require reliable and accurate methods to disentangle the confounding genetic and non-genetic components. Stochastic simulations of soybean [The online version contains supplementary material available at 10.1007/s00122-023-04470-3. The purpose of plant breeding is to improve genetic contributions to plant characteristics. For many discrete characteristics such as fruit and seed color, pubescence, herbicide and disease resistance, etc., the genetic improvements are obvious and easy to demonstrate. However, for continuous characteristics, such as seed or grain yield, the genetic contributions are incremental and difficult to separate from management practices or variable and changing environments. Plant breeders have used various experimental and statistical methods to estimate incremental genetic improvements of quantitative traits  of traits evaluated on continuous scales are composed of discretely inherited polygenic effects (G), continuous environmental effects (E), and GE interaction effects (GEI) . For diploid species, additive effects refer to those associated with discrete alleles that can be inherited across generations, while non-additive genetic effects refer to genotypes that are not transmitted through inheritance. Thus, cycles of selection and reproduction affect genetic variability only through inherited alleles, also known as breeding values . The difference between the averaged phenotypic values representing two consecutive cycles of recurrent selection is known as the realized response to selection (R). Thus, under an additive (linear) model . Further, the years in which the trials are conducted will favor newer cultivars that are more likely to be adapted to the environments in which the era trial is conducted  used to evaluate genotypes during their development have been used to assess changes across time for common bean , the inclusion of either a positive or negative non-genetic trend linked to the calendar year , and two breeding schemes. The RGG was then estimated for a quantitative trait composed of additive effects at 1000 loci from simulated era and yield trials with different modeling strategies based on LMM. The study concluded that (i) the evaluated estimators were inaccurate, (ii) the error associated with the estimates was dependent on the breeding scheme, non-genetic trend, and heritability, and (iii) if the goal is restricted to determine if RGG is greater than zero, some indicators like the expected rate of genetic gain and the equivalent complete generations are useful  were randomly assigned to 1000 loci distributed across 20 chromosomes, according to a genetic linkage map (2145.5 cM) estimated from the Soybean Nested Association Mapping population  were simulated to produce a population of FPhenotypic values of Fmentclass2pt{minimTen percent of the BT1 experimental lines were selected for evaluation in BT2. For the remaining stages, a 20% selection intensity was applied. Yearly selections were carried out by ranking the predicted eBLUP values of experimental line means obtained only from the data of the current year. Experimental lines in the BT1 phase of development were genotyped with 2400 SNP markers, although the marker genotypes were not used for genomic selection. Rather, SNP information was used by some of the LMM to estimate RGG by providing information about covariance across cycles of development.Field trials within evaluation stages  included three to six check cultivars, where the actual number of checks was randomly determined for each simulation Fig. . In otheBLUP selection of breeding  lines was performed in BT3 with combined phenotypic data from BT1, BT2, and BT3 . Assuming that the creation of the experimental lines prior to evaluation in BT1 could be conducted at off-site continuous nurseries with 3 seasons per year, the time per cycle of line development would be five years. Explicitly, it will require two years to begin with the initial crosses, create FG) was accomplished using AlphaSimR as described in section “ijkth genotype (line) lth plot, L and Y represent the locations and years’ main effects, respectively. All model terms except L were considered random effects sampled from various distributions described below . Simulations labeled B2-M are a modification of B2 where the sampled variance values for GL, GY, and GLY were divided by an ad-hoc factor of 10 to minimize the contribution of GEI components. Lastly, simulations labeled B2-R represent B2 where a random sample of experimental and breeding lines were retained . Heterogeneous residual variances represented plot-to-plot variability within trials and were sampled from a Log-Logistic distribution  the most advanced set of experimental lines evaluated in the URT, (ii) data from experimental lines that are first evaluated in PYT and subsequently in the URT, (iii) data from BT3, PYT, and URT that provide information from three years of trials for the most advanced experimental lines as well as information from breeding lines , and (iv) data from all MET stages . The first and second data sets are typical of historical data available for most crop species. For our simulations, we also evaluated the third data set to determine if estimating RGG exclusively from breeding lines would provide more accurate estimates. The fourth data set was not considered in this study.ith experimental line is available. For public soybean empirical MET data, this occurs in the PYT. K is the number of years in the data set, ith experimental line, kth year, and Check cultivars were included in the models to provide estimates of non-genetic trends across years but were not considered to estimate RGG. The estimation models can be classified as providing either direct or indirect estimates of RGG, as they differ in the decomposition of the genotypic effects  is accounted for because Thus, Twenty-one models were applied to simulated data sets consisting of PYT and URT Table . The firGL and GY interaction effects. Models E0, E1, E2, E3, E4, E5, and E6, assumed independent effects with homogeneous variance, whereas their counterparts E0V, E1V, E2V, E3V, E4V, E5V, and E6V, involved correlated random effects with heterogeneous variances. We restricted investigations of diagonal and first-order factor-analytic models to avoid convergence issues across simulation runs. For Models E0G, E0GV, and E7G, we also investigated the RGG estimation with genomic estimated breeding values .” For example, in a MET dataset of 10 years, if R = year 5, the corrections for the year effects will be performed with a forward-backward regression algorithm among years, e.g., R = 1, only a forward process  an initial model was fit within years to obtain eBLUP values for experimental lines . The same procedure was applied to compare independent versus correlated genotypic effects, and direct versus indirect estimates of RGG.Models E0, E1, Because s) Model  is an esmentclass2pt{minimt-test  of the slope  quantitative trait and constant genetic variance, then a linear trend between years of line development and genotypic means for MET should be evident. For indirect models where marginal genotypic (line) effects are estimated/predicted, we formally tested for linearity with the sieve-bootstrap version of the Student’s entclass1pt{minimaModels that demonstrated the least bias in analyses of simulated data were used to estimate RGG from the empirical data. This data refers to historical soybean seed yield records of maturity groups II and III evaluated in PYT and URT between 1989 to 2019. The data set contains 39,006 data points, 4257 experimental genotypes derived from multiple public breeding programs, and 591 observed environments located in the United States and Canada. The dataset can be obtained from the R package SoyURT. Refer to Krause et al.  for moreIn total, Including covariance models for GEI significantly affected contrasts of estimated RGG values. Three contrasts were statistically significant when a random sample of breeding lines was used to create a new cycle of breeding (B2-R). The inclusion of the genomic relationship matrix  resulted in relative bias across simulations from els Fig. . Howeverp-values from the z-ratio statistic showed the linearity metric could be used to indicate there was RGG, except for Models E6, E6V, and E12, where results were similar to the simulation model B2-R . The point estimates of RGG ranged from 0.27 to 0.59 bu/acSince the beginning of selection for quantitative traits, the development of analytical methods to estimate RGG has been pursued by animal and plant breeders Eberhart . The motSimilar to breeding using recurrent population improvement methods Jenkins , cultivaDistinguishing RGG from genotypic and/or commercial gain will help avoid misleading interpretations. Genotypic gain, not genetic gain, is RGG plus the expression of non-additive genetic effects such as epistatic , then unbiased estimates of genetic effects could be obtained from the second-stage model.Another alternative for the benchmark Model EB was including a variable for environments (location–years combination). The idea underlying this approach is from Diers et al.  and MontGL, GY, and GLY from the benchmark model, where the estimated genotypic variance changed from 6.30 (bu/ac)GL, GY, and GLY variances associated with the experimental genotypes. Thus, while checks account for variability among environments of MET, the GEI effects from experimental genotypes are still confounded with the genotypic effects.While this second modeling strategy successfully captured RGG and GEI effects, it significantly inflated the estimates of genotypic variance, and consequently, the estimates of heritability . For example, estimates of genotypic variance and heritability using empirical soybean MET data changed, respectively, from 6.30 (bu/ac)The aforementioned inflation due to GEI applies to other mixed models used for analyzing data from MET. For example, the “EBV” model from Rutkoski  only accRobinson  and hencRobinson . Thus, aEvery model assessed to estimate RGG in the simulations demonstrated some degree of bias. For example, the average bias was similar between simulation models A1 and A2 (simple GEI effects), and in B1 and B2 (complex GEI effects), but were relatively different between the A and B simulations. Including a positive, cumulative, non-genetic gain did not result in major bias when comparing simulation models A1 versus A2, and B1 versus B2. Ideally, RGG should be estimated with models with small values for root mean squared error . Even though all evaluated estimation models produced biased results, note that for some models the bias was consistently in the same negative/positive direction relative to the “true” simulated RGG values, indicating that the estimated RGG values underestimate/overestimated on average the true RGG. Such directional bias can be seen as insurance that the estimated RGG from empirical datasets is likely under or overestimated, depending on the model of choice.The simulator was constructed to mimic public soybean breeding programs responsible for maturity zones II and III in the USA. These programs evaluate nearly homozygous experimental lines in replicated field trials for four to five years Fig. , but theBased on theory, larger samples will asymptotically produce more accurate and precise estimates. We focused on estimating RGG from 30 years of advanced MET and could not obtain an unbiased, robust estimator. Results considering only 10 years of data revealed a much larger bias and root mean squared error, as also observed by Rutkoski . The simWe used simulated data from two or three years of MET, but excluded the simulated data from local breeder trials. We excluded these because empirical data from local breeder trials historically have not been published nor recorded in accessible databases. However, it is well known that a pattern of missing data also can introduce bias in trend  least-squares yield numerically equivalent estimates. The first issue is that data from MET are rarely balanced within years, and largely unbalanced across years. Thus, RGG estimates from raw phenotypes are completely confounded with non-genetic effects. As emphasized by Hartung et al. , carefulper se performance. We did not test for this approach, but results from Rutkoski Below is the link to the electronic supplementary material.


Wearable assistive robotics has emerged as a promising technology to supplement or replace motor functions and to retrain people recovering from an injury or living with reduced mobility. We developed delayed output feedback control for a wearable hip-assistive robot, the EX1, to provide gait assistance. Our purpose in this study was to investigate the effects of long-term exercise with EX1 on gait, physical function, and cardiopulmonary metabolic energy efficiency in elderly people. This study used parallel experimental (exercise with EX1) and control groups (exercise without EX1). A total of 60 community-dwelling elderly persons participated in 18 exercise intervention sessions during 6 weeks, and all participants were assessed at 5 time points: before exercise, after 9 exercise sessions, after 18 sessions, and 1 month and 3 months after the last session. The spatiotemporal gait parameters, kinematics, kinetics, and muscle strength of the trunk and lower extremities improved more after exercise with EX1 than in that without EX1. Furthermore, the effort of muscles over the trunk and lower extremities throughout the total gait cycle (100%) significantly decreased after exercise with EX1. The net metabolic energy costs during walking significantly improved, and functional assessment scores improved more in the experimental group than in the control group. Our findings provide evidence supporting the application of EX1 in physical activity and gait exercise is effective to improve age-related declines in gait, physical function, and cardiopulmonary metabolic efficiency among older adults. Increase in step variability and the metabolic cost of walking and decrease in preferred gait speed, step length, and range of motion of the ankle, knee, and hip joints have been reported as typical gait pattern changes associated with increased age5. In many cases, declines in physical conditioning and gait function with age ultimately lead to a sedentary lifestyle, which is strongly correlated with a variety of cardiovascular and metabolic diseases6. As life expectancy continues to increase worldwide, two major questions have arisen: (a) Will the increase in life span include several years of healthy life? and (b) How do we promote health-related quality of life into old age?7. The concepts of “aging well,” including “active-”, “successful-”, and “healthy aging”, have emerged in recent years to promote mental and physical health throughout aging9.Life expectancy is increasing in most countries, and rapid population aging is being reported worldwide. Because the prevalence of gait disorders increases with age, the expected demographic changes will result in increased numbers of people affected in the coming decades. Elderly adults normally experience decreases in their physical capabilities due to deterioration of the neuromusculoskeletal system, which causes them to walk with a changed gait pattern and to become more cautious10. The risk of fall increases with age for many reasons. The most common causes of falls by elderly people are trips and slips, and the ability to rapidly move the entire leg is a potentially important aspect of preventing such falls11. Older adults generally redistribute joint moment and force across their lower extremities to maintain a gait performance similar to those of young people. Specifically, older adults tend to transfer the mechanical demands of walking on the lower extremity from distal to proximal, producing significantly more net positive work at the hip to compensate for less net positive work at the ankle12. The hip joint is vital for generating movement throughout the whole leg and is important for fall prevention11.The risk of falls increases with age, and fall prevention among older adults is one of the most important public health issues in today’s aging society13. Participation in physical activity and exercise can help to maintain or improve health, physical function, and quality of life and reduce falls among elderly people in general and in those with morbidities in particular14. Improvements in mental health; psychological, emotional, and social well-being; and cognitive function are also associated with regular physical activity and exercise15. However, the reduced bodily functions of many older adults are a major barrier to regular execution of a standard exercise program18. Maintaining physical activity and exercise usually requires substantial support and supervision. Even then, many seniors fail to maintain such activity due to difficulties negotiating everyday participation, such as schedule conflicts and competing sedentary activities or health issues19. In addition, mobility limitations can be a challenge for many older adults trying to maintain an exercise program20. Therefore, alternative exercise programs providing regular physical activity to the elderly should be suggested.Regular physical activity and exercise are recommended as a primary countermeasure to the natural decline of physical function with age21. Robotic assistance has the potential to assist locomotion in a variety of areas, including recreation, clinical rehabilitation and assistance, and workplaces23. The use of wearable assistive devices has become one of the most promising ways to assist individuals with gait disorders25. Robot-assisted rehabilitation can reduce the heavy physical burden otherwise placed on therapists and can provide long training sessions with good consistency27. Several studies have reported that robot-assisted gait training is effective for improvement of gait and functional capability in people with mobility impairment32. In a study using hybrid Assistive Limb (HAL), HAL-assisted gait training significantly improved gait ability after intervention and the effect was maintained for 3 months after the training in chronic stroke patients28. Ogino et al. suggested that gait training using Gait Exercise Assist Robot is are effective for improving gait ability than treadmill gait training among subjects with chronic stroke29, and a study using Honda Walking Assistive device (HWA) reported that HWA-assisted gait training was safe and feasible, and could be effective for the elderly improvement of gait ability, hip function, and gait pattern after total hip arthroplasty30. Furthermore, a study using Alter G Bionic Leg Orthosis demonstrated that home-based robot-assisted gait training program could elicit improvements in clinical functional outcomes in patients with stroke and the effect was maintain for 22-week assessment32.Wearable assistive robotics has emerged as a promising technology to supplement or replace motor functions and to retrain people recovering from an injury or living with reduced mobility33. They can train the wearer’s muscles and assist movement by providing controllable assistive force/torque at the hip joint. They can also improve strength and endurance1. Compared with other human joints, the hip joint imposes higher metabolic costs to generate similar mechanical joint power due to differences in muscle characteristics34. Therefore, a robotic hip exoskeleton is a promising strategy for increasing the efficiency of human walking because the human hip joint produces large torque during the activities of daily living35.Robotic hip exoskeletons are human–robot cooperation systems that integrate robot power with human intelligence37. To further our investigation into the effectiveness of EX1, we conducted this study to demonstrate the long-term effect of EX1 use. Our goal was to determine whether task-specific physical activity and functional gait exercise with EX1 would be effective in improving spatiotemporal parameters, kinematics, kinetics, muscle effort, metabolic demand, and physical function in older adults compared to exercise without EX1.We developed a robotic hip exoskeleton, the EX1 Fig.  online tThe specific values for spatiotemporal gait parameters, joint kinematics, and kinetics at the pre-exercise (Pre), post-exercise (Post), and 1-month and 3-month follow up (1 m FU and 3 m FU) time points after exercise are presented in Table Figure The changed values for net metabolic energy cost (mL/kg/min) are shown in Fig. Figure Repeated measures ANOVA revealed significant group × time interactions in the 10MWT-SSV and -FV, FSST, and TUG (P < 0.05). The experimental group showed greater improvement than the control group in the 10MWT-SSV and -FV and FSST at Mid, Post, 1 m FU, and 3 m FU (P < 0.05) and in the TUG at Post, 1 m FU, and 3 m FU (P < 0.05).Our findings in this study suggest that task-specific physical activity and functional gait exercise with EX1 have several key advantages for gait, physical function, and metabolic energy efficiency over exercise without EX1. Spatiotemporal gait parameters, kinematics, and kinetics improved significantly after EX1 exercise in the experimental group, and changes in spatiotemporal gait parameters  and hip joint ROM remained significant up to 3 m FU. In addition, the experimental group showed significant improvements in the MVC of the RA, LES, hip flexor, RF, TA, and GCM after EX1 exercise, and the MVC changes of RA, hip flexor, RF, TA, and GCM remained significant up to 1 m FU. The decreases of muscle effort in RA, RF, and BF were maintained to the post test, decreases of muscle effort in hip flexor and TA were maintained to the 1 m test, and decreases of muscle effort in LES and GCM were maintained to the 3 m FU. Furthermore, the experimental group showed significant change in net metabolic energy cost and the scores in 10MWT-SSV, 10MWT-FV, BBS, FSST, TUG, FRT, and SPPB from Pre to all other time points.40. Mobility and physical function impairments, often operationalized as spatiotemporal gait parameters, are strongly associated with concerns about falling. A decrease in gait speed is frequently used as a predictor of fall risk and disability and a marker of frailty because it correlates with functional loss and imminent death42. Therefore, spatiotemporal gait parameters are a major factor influencing mobility and physical function, and they are meaningful biomechanical assessments for monitoring the effects of exercise programs in older adults. According to our results, gait speed, cadence, and stride length improved significantly after EX1 exercise in the experimental group, and those improvements were maintained at the 1 m and 3 m FU time points after cessation of exercise. In contrast, the control group showed significant improvements in the spatiotemporal parameters after exercise, but those improvements were not maintained at the 1 m or 3 m FU assessment. These results lead us to postulate that task-specific physical activity and functional gait exercise with EX1 can improve spatiotemporal gait parameters and offer long-term health benefits. Several studies have shown that changes in certain spatiotemporal gait parameters such as velocity or stride length increase fall risk45, and aging is associated with an increased risk of falls46. Improvements in spatiotemporal parameters maintained in the FU assessments after EX1 exercise support that EX1 exercise has the ability to reduce the risk of fall. Furthermore, by providing appropriate levels of assistance, EX1 can provide extensive, repetitive, task-specific gait exercise that can facilitate durable improvement in spatiotemporal parameters. Our results demonstrate the superiority of repetitive EX1 exercise over that without EX1 in terms of the durability of improvements in spatiotemporal gait parameters.Within the natural processes of aging, decreases in gait speed, stride length, cadence, and relative duration of the swing phase as well as increases in gait speed variability and single- and double-support time are common and expected49. It is especially important to identify changes in joint movement angles when assessing the gait performance of elderly people. Biomechanical analyses of lower extremity activity in elderly people have shown decreased ROM of the hip, knee, and ankle joint in the sagittal plane during walking tasks52. That decrease is associated with changes observed in kinetic data for those joints. In particular, elderly people showed reduced hip extension moment at the loading phase of gait53 and plantar flexion peak moment of the ankle joint in the late stance phase compared with young persons54. Elderly individuals also demonstrate limited capacity to advance the limb during the push-off period due to decreased plantar flexor moments and force55. In our study, hip joint and ankle joint ROM significantly improved in the experimental group after EX1 exercise, and changes in hip joint ROM remained significant up to 3 m FU. The peak hip extension and ankle plantarflexion moment and the second peak of vertical ground reaction force also changed significantly from Pre to Post in the experimental group. In contrast, the control group showed no significant change in joint ROM, peak moment, or vertical ground reaction force. The occurrence of falls in older adults is related to various factors, such as balance disorders, unstable lower limbs, and biomechanical and neuromuscular changes56. These results indicated that EX1 exercise could increase lower-limb joint ROM, joint moment, and force. In other words, EX1 exercise can be recommended to help prevent falls by reducing age-related changes in biomechanical gait characteristics.Age-related changes in biomechanical gait characteristics are associated with limited lower-limb joint ROM and significant reductions in joint moment and force as a result of physiological and neuromuscular changes57. During the late stance phase, decreased push-off power could affect trunk stabilization and trunk progression, which cause a shorter step length in elderly people58. As a potential compensation for reduction of ankle push-off power, older adults rely more on the hip musculature for power generation than young adults, a phenomenon known as distal-to-proximal redistribution59. This redistribution could explain, at least in part, why elderly people pay greater metabolic energy costs for walking than younger people and might be considered maladaptive60. The functional consequences of higher metabolic energy costs include reduced physical activity and independence, gait inefficiency, and lower quality of life61. In our study, the experimental group showed significant improvements in muscle strength of the trunk  and lower extremities  after EX1 exercise, and the changes in muscle strength of RA, hip flexor, RF, TA, and GCM remained significant up to 1 m FU. Thus, EX1, which provides assistance torque only to the hip joint, affects not only the strength of hip joint muscles, but also the strength of trunk and ankle muscles. Older adults have been reported to rely less on ankle strategies for balance, resulting in greater use of hip strategies62. We assume that EX1 exercise is effective in improving both ankle and hip strategies, along with strength of the muscles around these joints in older adults. Furthermore, the experimental group showed significant changes in the muscle effort of the trunk  and lower extremities  during the total gait cycle (100%) after EX1 exercise. Decreases of muscle effort in hip flexor and TA were maintained to the 1 m test, and decreases of muscle effort in LES and GCM were maintained to the 3 m FU. The reduced muscle effort during walking demonstrated that EX1 exercise is effective. The experimental group demonstrated a greater than 20% net metabolic decrease when walking. As such, decreased walking energy cost is predicted to significantly improve health, quality of life, and life expectancy in older adults6. Our results indicated that EX1 exercise could increase muscle strength and help older adults to walk more efficiently by reducing the muscle effort required by their gait, which could lead to a reduction in cardiopulmonary metabolic costs. In other words, the gait inefficiency common in older adults could be improved through EX1 exercise.Older adults generally have plantar flexor muscle weakness, which could cause a weak push-off power63. Both dynamic and static balance are key components in many activities of daily living, from simple activities such as quiet standing to more complex activities such as walking while talking or changing direction. As people age, their ability to control their balance and gait function deteriorates due to alterations in the visual, vestibular, somatosensory, musculoskeletal, and central nervous systems65. Therefore, specific exercise programs to improve age-related changes in balance and gait function should be suggested for older adults. In this study, we conducted functional assessments of gait and balance to examine how EX1 exercise affected elderly people. In addition, participants completed the GDS-SF to monitor changes in depression after exercise. We found greater improvements in all the functional assessments after exercise with EX1 than without EX1. Previous studies have shown that better physical function is generally related to a lower incidence of depressive symptoms among older adults67. It is also associated with better mental health, well-being, and quality of life68. In this study, only the experimental group, which showed great improvement in functional assessments after exercise with EX1, showed a significant change in the GDS-SF. Improved physical functioning after EX1 exercise might have a positive effect on depression in elderly people.Gait and balance disorders are common causes of falls by elderly people, and falls often lead to injuries, disability, loss of independence, and low quality of life60. A study using an exoskeletal Active Pelvis Orthosis (APO)16 investigated its feasibility for cardiopulmonary gait training in the elderly and showed a decrease in Metabolic Cost of Transport (− 26.6 ± 16.1%) during treadmill walking after a 4-week APO-assisted training program, similar to our results. Our prior study with stroke subjects using an earlier version of the EX124 demonstrated improvement in spatiotemporal gait parameters and muscle efforts and decrease in net cardiopulmonary metabolic energy cost (− 14.71%) after the training intervention. Furthermore, a study using a soft robotics suit69 reported the long-term rehabilitation effectiveness of the robotic suit in elderly persons and showed a improved gait characteristics and an increased walk ratio with an average of 9.8% compared with the initial state. While the exercise program of these studies consisted only of treadmill and overground walking, the intervention session in the present study consisted of long-term task-specific physical activity and functional gait exercise that elderly people can perform in their daily lives, including obstacle avoidance and navigation, multidirectional stepping, walking on varied surfaces, stair climbing, and community mobility. Therefore, our findings support the adoption of EX1 not only for training and rehabilitation to facilitate active aging in the elderly, but also for physical activity and gait exercises in daily life for community dwelling elderly. In addition, the maintained effect of EX1 exercise could be suggested the EX1-assisted exercise can promote sustained improvement in gait and functional abilities and can be recommended as a community exercise program.Globally, several robot-assisted gait devices have been developed, and the research into rehabilitation robotics has increased. Pre-clinical studies with robotic hip exoskeletons have demonstrated improvements in gait and physical function, muscle effort, and cardiopulmonary metabolic efficiency in elderly individuals and individuals with strokeThis study has limitations related to inadequate control of everyday activity level of the participants beyond the experimental session. During the long-term intervention and follow-up period, other factors that might have affected the study outcome, such as an individual’s overall amount of physical activity, were not controlled or monitored. All participants were allowed to perform their usual daily activities and completed the study without drop-outs or intervention- or fall-related injuries during the study period. We believe that this limitation should not significantly affect our results. An additional limitation is related to sample size. The statistical power of this study was low because of the small number of participants; therefore, our results cannot be generalized to the entire elderly population. Nevertheless, this study demonstrated that use of EX1 in addition to task-specific physical activity and functional gait exercise produced better gait, physical function, and gait efficiency outcomes than similar exercise without EX1.This study is the first randomized controlled trial to consider gait, physical function, and gait efficiency in elderly people after long-term, task-specific, and functional gait exercise with a wearable robot. Our findings provide evidence supporting the application of EX1 to regular physical activity and exercise to improve age-related declines in gait and physical functioning. All participants in this study completed protocols with EX1 without any specific adverse events, indicating that EX1 exercise is safe and does not pose risks to apply for older adults. Nonetheless, several participants suggested improvements to the weight, noise, and design of EX1. Future works will examine the usability of and satisfaction with EX1.A total of 60 subjects was included in this study, and their characteristics are shown in Table This study used a repeated-measure, single-blinded , randomized, controlled, and parallel design. All participants attended 23 experimental visits: 5 testing visits  and 18 exercise visits.70. The exercise intervention was conducted at a perceived exertion between 12 and 16 on Borg’s Rating of Perceived Exertion (RPE) scale (somewhat hard ~ hard)71 for 6 weeks, with 3 exercise sessions per week . The duration of each exercise session was 60 min: 5 min of warm-up, 50 min of exercise with 5 min of rest, and 5 min of cool-down. Exercise intensity was assessed with verbal questions twice during the intervention session based on the Borg RPE scale and was controlled by giving the subject a rest period and adjusting assistive torque of EX1 applied to the subject. For subject safety, a physical therapist supervised the intervention. If a participant missed an exercise session, an additional session was offered at another time of the week or at the end of the intervention period.All participants performed task-specific physical activity and functional gait exercise, including obstacle avoidance and navigation, multidirectional stepping, walking on varied surfaces, stair climbing, and community mobility  with EX1  or without EX1 (control group)Participants were assessed at 5 time points: Pre, Mid, Post, 1 m FU, and 3 m FU after the last exercise session. Each assessment evaluated spatiotemporal gait parameters, kinematics, kinetics, muscle activity, cardiopulmonary metabolic energy efficiency, and functional outcomes (performance-based and subject-reported measures) except the Mid time point, at which only the functional outcomes were measured. The participants were asked to walk at a comfortable, self-selected speed along a 10-m walkway, and spatiotemporal, kinematic, kinetic, and muscle activity data were collected simultaneously. Cardiopulmonary metabolic energy efficiency was measured before placement of the sensors for participant comfort during 6 min of treadmill walking. All outcome measures were collected when subjects were not wearing the EX1 device.72. The motion capture system allowed us to define each marker during collection, so marker position was recorded in real time. A standing calibration was used to obtain a rotation matrix for each limb segment and align the local  reference frame for the thigh, shank, and foot to the global (laboratory) reference frame. Movement data were automatically converted to 3-dimensional coordinates with CORTEX version 6.2 motion capture software . Spatiotemporal, kinematic, and kinetic gait parameters were calculated for each gait cycle using Ortho Track 6.6.4 software .Spatiotemporal and kinematic data were measured using a 3D motion capture system with 8 infrared cameras , and kinetic data were obtained using 2 force plates  embedded midway along the walkway. We collected the trajectories of 19 markers placed on anatomical landmarks using the Helen Hayes marker model73 for reliable sensor placement. The skin was prepared for sEMG collection by shaving, abrading, and cleansing with alcohol to reduce surface impedance. In addition, footswitches  were placed on the right toe and heel to identify the timing of the stance and swing gait phases while walking.To obtain surface electromyography (sEMG) signals , bipolar surface electrodes (Ag/AgCl) were positioned on the right RA, LES, G Max, RF, BF, TA, and GCM, and the electrodes for the hip flexor were placed in the femoral triangle inferior to the inguinal ligament just medial to the rectus femoris. sEMG sensor placement was performed for all four assessments by the same professional therapist, following the recommendations of the Non-Invasive Assessment of Muscles Project (SENIAM)2) and carbon dioxide production (VCO2). The participants wore the system on the upper body with a face mask that prevented exposure to outside air for the breath analysis.Cardiopulmonary metabolic energy efficiency was measured using a validated and reliable portable telemetric gas analyzer system . The COSMED K5 portable cardiopulmonary metabolic system uses combined breath-by-breath technology to measure oxygen consumption . SPPB consists of three performance subtasks  that measure balance, lower limb muscle strength, and mobility. The score of each of the three tests ranges from 0 to 4, where 4 indicates the best result and 0 the worst result, and a summary score ranges from a minimum of 0 points (worst performers) to a maximum of 12 points (best performers)75. BBS is a valid, qualitative, and efficient measure of balance measured through performance of functional activities that incorporate most components of postural control in older individuals. The tool consists of 14 items scored on an ordinal scale of 0 to 4, with 0 being the worst and 4 being the best performance of independent tasks for a total of 56 points76. For TUG measurement, subjects sit on a chair placed against the wall. They are instructed to stand up from a chair, walk at their normal pace on a 3 m pathway, turn around, walk back, and sit down on the chair again. The stopwatch is started on the word “go” and stopped when the subject sits down again77. FRT assesses subject stability by measuring the maximum distance (cm) an individual can reach forward while standing in a fixed base of support78, and GDS-SF consisting of 15 questions is a useful screening tool for monitoring mood status and evaluating depression79. All assessments were conducted by an experienced physical therapist blinded to intervention assignments.For the 10MWT-SSV and -FV measurements, subjects were asked to walk a total of 15 m at a comfortable and maximum speed, respectively80, and the latest version of the EX1 has been further reduced in weight by improving the structure of the wearable frame81. The device is worn around the user’s waist and thighs and assists with hip joint flexion and extension. The EX1 consists of snap-together components weighing 4.7 lb (2.1 kg) in total. The waist part houses 2 actuator modules, a Bluetooth module, and a control pack. Each actuator module houses a motor, an embedded angular position sensor, and a controller. The control pack contains a central processor, an inertial measurement unit sensor, a rechargeable battery pack, and a power switch. The torque assistance is transmitted to the user’s thighs via thigh frames. The trained physical therapist who operates the device can change the assist settings through software that runs on a mobile device.EX1, which was developed at Samsung Electronics Co., Ltd. , is a lightweight, robotic exoskeleton designed to assist in the ambulatory function of elderly people. EX1 is designed based on a slim and backdrivable actuator82. While the user is sitting on a chair, the hips need to be bent up to 100°. Therefore, the flexion angle of the device is mechanically limited to 110°, and the extension angle is set to 45°. However, a flexion angle of 100° and extension angle of 40° are set with the software to ensure the safety of both the users and the device. The device is torqued off when the user’s leg angle exceeds the range. Furthermore, the device alerts the physical therapist by sending a warning message to the mobile application when the joint angle reaches the limit.EX1 defines the angle of the hip joint to be 0° when the user is wearing the device and standing upright. The hip flexion angle is increased up to 70° during stair climbingThe maximum assistance torque of the device is about 12 Nm. Furthermore, maintaining torque tracking performance is essential to deliver sustained hip power to the wearer during walking. The hip angular velocity measured by the device is 4–5 rad/s when the user is walking at 3–5 km/h. The device can provide torque assistance up to a maximum hip speed of 6 rad/s to accommodate faster swing speeds. For example, when the hip swing velocity needs to be increased to avoid obstacles, the torque tracking performance is ensured up to 6 rad/s . Since the EX1 can walk for 2 h continuously at 3 km/h, it can perform walking training for more than 1 h.83.We developed the DOFC, a time-delayed, self-feedback controller for gait assistance with EX1. By assigning the appropriate time-delay and self-feedback gain, we can generate stable assistive torque through interactions between the wearer and the exoskeleton. This control algorithm is simple but powerful because of its generalizability to changes in the user’s walking patterns. The assistance method changes the assistive torque according to gait pattern changes without requiring changes to the control parameters. Its implementation was first illustrated in87. They have also focused on immediate effects rather than training effects. There are no tests that take into account gait training for more than 30 min in large numbers of older adults, as was done in this study. In elderly subjects with low muscle strength, torque greater than 10 Nm can cause gait instability, and some complained of discomfort due to the strong assistive force. Further research is needed to easily find the optimal torque level for elderly subjects with low muscle strength, and this is one of our future projects.Most of the existing studies have focused on minimizing gait energy and strength loads and have been conducted with young and healthy subjectsMotion capture data were filtered with a Butterworth filter algorithm with a cutoff frequency of 6 Hz, 2-pass, fourth-order, and zero-phase shift filter. Hip, knee, and ankle moments were first calculated using inverse dynamics, kinematics, ground reaction forces applied to the foot, and the distance between the application of force and the center of mass of the segment. Moment data were scaled by body weight, and values for peak ankle plantarflexion moments and peak flexion and extension moments at the knee and hip were averaged across trials for each subject. The calculation of joint moments was accomplished using Ortho Track 6.6.4 software. Hip and knee joint ROM were calculated by subtracting the minimum joint angle during the gait cycle from the maximum joint angle achieved during the swing phase. Ankle joint ROM was calculated by subtracting the minimum ankle joint angle at the toe off point from the maximum ankle joint angle during the gait cycle. The vertical ground reaction force data were scaled by body weight. The values for the first peak, developed at the instant of loading response, and the second peak, developed at the time of push-off (onset of forward propulsion), were averaged across trials for each subject.74. The MVC test is the normalizing method proposed by the SENIAM and Kinesiology guidelines and is the most widely used normalization method88. To normalize the sEMG signal amplitude, three 5-s maximal muscle contraction measurement trials were performed with each muscle to determine each participant’s MVC. To reduce movement artifacts, a sampling frequency of 1000 Hz was used, and raw sEMG signals were bandpass filtered between 10 and 350 Hz and full-wave-rectified with Noraxon software (MyoResearch XP Master Edition). In addition, the root mean squared values of the signals were calculated using a sliding 100-ms window for analysis. The data were passed through a sixth-order Butterworth low-pass filter with a 6-Hz cutoff frequency to create a linear envelope and normalized to the MVC data obtained prior to the tasks. The average normalized sEMG activity was processed within the selected phases of the gait cycle using MATLAB software 89. Individual gait cycles were determined using the footswitch data, where each stride was considered the period between successive heel-strikes by the same leg. The sEMG patterns for each stride were time normalized and expressed as a percentage of the total gait cycle (0–100%).We applied sEMG muscle effort, defined as the percentage of maximum voluntary contraction (%MVC) averaged across all participants. %MVC was calculated using the following equation: % MVC = [(EMGtask − EMGresting)/(EMGmax − EMGresting)] × 100 (%)Baseline cardiopulmonary metabolic energy efficiency was measured in a comfortable standing position for 5 min. Then, the metabolic energy cost was recorded during 6 min of treadmill walking at each participant’s most comfortable gait speed. Participants identified their most comfortable gait speeds by walking at their own pace for 3 min without using handrails. The metabolic energy cost test at all time points was performed on the same treadmill at the same speed and using the same measurement method. The net cardiopulmonary metabolic cost (mL/kg/min) was calculated by subtracting the standing oxygen demand (baseline) from the average oxygen uptake during the last 2 min.2 tests for categorical variables to compare participants' baseline characteristics between the experimental and control groups. Repeated measures ANOVA was used to examine the main effects of exercise over time in both groups  and time points . Post-hoc tests determined whether changes from baseline were significant at Mid, Post, 1 m FU, and 3 m FU in each group, and the significance levels of those tests were adjusted using Bonferroni correction.Statistical analyses were performed with SPSS version 22.0 , and the significance level was set at 0.05. Descriptive statistics are expressed as mean . To determine the appropriate statistical tests, we checked the normal distribution of the data, based on which we applied parametric tests. We used independent t tests for continuous variables and χSupplementary Information.


A standardized protocol for electroencephalography (EEG) under standing sedation for the investigation of epilepsy in foals is needed.To evaluate a modified standardized EEG protocol under standing sedation using sublingual detomidine hydrochloride in Egyptian Arabian foals.Nineteen foals .Descriptive clinical study. Foals were classified as controls or epileptic based on history or witnessed seizures and neurological examination. Foals were sedated using sublingual detomidine hydrochloride at a dosage of 0.08 mg/kg to avoid stress associated with injectable sedation. Once foals appeared sedated with their heads low to the ground and with wide base stance (30 minutes), topical lidocaine hydrochloride was applied at the determined locations of EEG electrodes. Fifteen minutes were allowed for absorption and electrodes were placed, protected, and EEG recording performed.Level of sedation was considered excellent with no need of redosing. The EEG recording lasted from 27 to 51 minutes and provided interpretable data. Epileptic discharges (ED) were noted predominantly in the central‐parietal region in 9 of 10 epileptic foals. Photic stimulation triggered ED in 7 of 10 epileptic foals and in none of the controls. Foals were not oversedated and recovered uneventfully.Sublingual detomidine hydrochloride is a safe, painless, simple, and effective method of sedation for EEG recording in foals. Sublingual sedation allowed the investigation of cerebral electrical activity during states of sleep and arousal, and during photic stimulation for the investigation of epilepsy in foals. ECGelectrocardiogramEDepileptic dischargesEEGelectroencephalogramJIEjuvenile idiopathic epilepsyPDphotic drivingSWSslow wave sleep1Seizures and epilepsy in horses are reported infrequently in veterinary medicine compared to other species possibly reflecting lower occurrence in equids.Juvenile idiopathic epilepsy (JIE) is a well‐characterized epileptic disorder based on clinical, neurologic, EEG, and histopathological examination in Egyptian Arabian foals.Electroencephalography is an essential diagnostic modality to define, classify, and advance understanding of pathophysiology, and to improve treatment of epileptic disorders in veterinary medicine as well as in human medicine.22.1The study included 19 foals of Egyptian Arabian breed between the ages of 1.5 and 12 months, 9 colts and 10 fillies. Foals were classified as healthy or epileptic based on history of presence (witnessed or recorded using video surveillance) or absence of seizures, and physical and neurological examination findings. Neurological examination was performed before each EEG study by board‐certified large animal internal medicine specialists. The foals under study were housed alone or with their dams in a padded stall and the study was performed at the Equine Veterinary Medical Center (EVMC) in Doha, Qatar in 2022. The protocol was approved by the Institutional Animal Care and Use Committee of the EVMC (#2021‐1179).2.2Foals were sedated using sublingual detomidine hydrochloride  at a dosage of 0.08 mg/kg. Adequate level of sedation was determined to be when foals adopted a head position with ears below the level of the withers, wide‐based stance, and apparent somnolent state with minimal reactivity to external stimuli. Once foals appeared sedated within 30 minutes, the specific location for the placement of EEG electrodes was determined by using a flexible ruler and marked with a surgical skin pen . Local anesthetic cream  was applied and massaged into the skin using a glove, and absorption was allowed for 15 minutes before electrode placement in the identified locations. Forty‐five minutes elapsed from the time of sedative administration to the placement of SC needle electrodes and beginning of EEG recording.2.3A Nicolet NicOne digital EEG system  equipped with integrated photostimulator and video was used for the recording of cerebrocortical activity. Electrode nomenclature and placement were based on a modified 10‐20 system used in humans and described in horses previously.The electrodes were placed SC in the scalp located equidistant to each other at approximately 2 cm in cranial to caudal and transverse directions. A soft ruler was used to determine the location of the electrodes, which were marked with a washable pen Figure . The froOnce all of the electrodes were placed, a tubular mesh bandage was placed to cover the foal's head and protect the electrodes and prevent dislodgement of the electrodes by sudden head movements. Holes were cut in the mesh for the ears. Cotton was inserted in the ears and disposable face masks  were used to cover the eyes and minimize environmental sound and visual stimulation while recording the EEG Figure . The amp2.3.1Activation procedures were performed during the wake recording including 5 minutes of intermittent photic stimulation and 5 minutes of noise stimulation. Intermittent photic stimulation consisted of 10 second epochs of photic stimulation at different frequencies including 1, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 50, and 60 Hz. The foal's head was raised and positioned at a distance of at least 30 cm from the photic stimulator during the intermittent photic stimulation  and 5 fillies (1.5‐9 months old). Six of 10 foals with epilepsy had generalized onset seizures, 1 had focal onset, and 3 had unknown onset with recorded postictal signs such as blindness, obtundation, tongue paresis, and ataxia a few days to a few weeks before the EEG study. Seizures were characterized as violent generalized tonic‐clonic with facial motor component, trismus, and loss of consciousness. Focal seizures consisted of facial motor alterations including trismus, chewing, tongue paresis, and progression to generalized tonic‐clonic seizures. Postictal signs included disorientation, obtundation, cortical blindness, decreased palpebral reflex, dysphagia, tongue protrusion, proprioceptive deficits of all limbs, and ataxia. Duration of postictal signs varied from a few minutes to days to 1 week . Otherwise, foals were neurologically normal during the interictal period. Common complications included corneal ulceration, head trauma that in 1 case that resulted in multiple comminuted skull fractures, and dysphagia.3.2Oral sedation was considered excellent for the purpose of the study and did not require redosing. A time of 45 minutes was needed for the level of sedation required  for placement of the SC needle electrodes and recording.3.3Historically, 9 of 10 foals were receiving antiseizure medication as single  or multidrug therapy . Antiseizure medication was discontinued in the 3 oldest foals  days to weeks before the EEG study. Therefore, at the time of the EEG study, only 6 of 10 foals were receiving antiseizure medication. Timing of the EEG from the last seizure ranged from 6 days to 6 months . The data obtained was thoroughly examined from EEG tracings and simultaneous video recording for each foal under study. Total duration of EEG recording ranged from 27 to 51 and 40 to 47 minutes in controls and epileptic foals, respectively, and included all phases of vigilance with sleep at the beginning (because of sedation) and wakefulness at the end of the recording.3.3.1In all controls as well as in most affected foals (9 of 10), the background during wakefulness was normal and characterized by medium amplitude mixed alpha (8‐9 Hz) and theta (6‐7 Hz) frequencies, seen predominantly in the parasagittal regions Figures . During 3.3.2Epileptiform discharges (ED) were seen in most affected foals (9 of 10) in the form of either focal, multifocal, or generalized spike and wave discharges Figure . Focal E3.3.3Auditory stimulation via a loud clap did not result in any changes on the EEG tracing in either control or affected foals. Intermittent photic stimulation resulted in either no response (1 of 10), normal photic driving (2 of 10), abnormal photoparoxysmal responses in the form of increased ED during stimulation (6 of 10), and mixed ED and photic driving (1 of 10). Photoparoxysmal discharges were not associated with clinical manifestations while recording in foals with JIE. Photic driving was seen in 7 of 9 controls. Examples of photic responses are shown on Figure 4We found that sublingual detomidine hydrochloride provided an excellent plane of sedation for the duration required for foal preparation and performance of video EEG with additional activation procedures. This protocol proved to be simple, safe, and likely less stressful for the foals than when using IV sedation. Furthermore, sublingual detomidine induced SWS, which facilitated observation of ED in foals with JIE and helped confirmation and classification of foals as epileptic. This sedation protocol proved to be simple, apparently painless, and provided interpretable EEG data in foals, essential for proper phenotyping for future genetic studies.Our study describes a noninvasive, easy, practical, low cost, and efficient standardized protocol for sedation and EEG for the investigation of epilepsy in foals. Also, this protocol allowed us to minimize the amount of personnel required for foal restraint. It is essential for the recording and interpretation of EEG to avoid movement artifacts associated with struggling. Placement of multiple SC needle electrodes for the recording of EEG can result in struggling. Therefore, an effective sedation protocol was imperative. Detomidine hydrochloride, an α2‐adrenergic agonist, is known for its cardiovascular and respiratory effects such as decreased heart rate, increased occurrence of atrioventricular block, decreased cardiac output, increased or decreased mean arterial pressure in a dose dependent manner, and decreased respiratory rate.Detomidine hydrochloride induced SWS in all 19 foals as described in adult horses.None of the foals with JIE in our study had obvious preictal signs. Sudden violent generalized tonic‐clonic seizures with loss of consciousness were observed in 6 of 10 foals, and 1 foal had focal facial, oral, and motor seizures that affected the tongue. Three other foals were not observed to have seizures but were found disoriented, obtunded, blind and with tongue protrusion and dysphagia. The postictal phase in foals with observed seizures consisted of disorientation, obtundation, cortical blindness, dysphagia, proprioceptive deficits, and ataxia. The semiology of seizures in these foals with JIE was similar to that previously described for foals with JIE from the United States.Activation procedures included photic and sound stimulation. Sound stimulation did not trigger ED. Photic stimulation did not result in ED in control foals, whereas ED were triggered in 7 of 10 epileptic foals. Photic stimulation is widely used in routine EEG to aid in the diagnosis of epilepsy in humans.Limitations of our study were the low numbers of foals for each group, particularly the control group. Absence of interictal EEG abnormalities does not rule out epilepsy and cases could have been misclassified. Longer duration or repeated EEG recordings in those cases would have been helpful.5We described an easy, low cost, safe, less stressful, and efficient mode to sedate foals sublingually with detomidine hydrochloride for EEG investigation. Sublingual detomidine provided an excellent plane of sedation and sufficient duration of time for EEG recording of interpretable data and performance of activation procedures. Our protocol provided a novel practical standardized process for EEG recording and interpretation which is an essential component for accurate phenotyping in the investigation of epilepsy for future genetic studies. Furthermore, EEG is paramount to classify foals as nonepileptic versus epileptic. Photic stimulation must be included as a routine activation procedure during EEG recording for the investigation of epilepsy because it may trigger ED not detected on routine EEG.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.The protocol was approved by the IACUC of the Equine Veterinary Medical Center (#2021‐1179), Doha, Qatar.Authors declare human ethics approval was not needed for this study.


The type of traumatic peripheral nerve injury is a key factor for determining optimal treatment. Proper assessment of peripheral nerve injury facilitates appropriate treatment, significantly affects prognosis, and reduces disabilities. This study evaluated ultrasonography (US) to assess upper limb traumatic nerve injuries and compared the US with electrodiagnostic studies as the gold standard.Participants were 69 adults  with a total of 96 peripheral nerve injuries (duration of 1 month–3 years). High-frequency US examinations and electro-physiologic studies confirmed upper limb peripheral nerve injury.Nerve discontinuation was diagnosed in 15 (15.6%) nerves; the cross-sectional area was increased in 33 (34.4%) nerves. Of 96 injuries, 54 (56.3%) were median, 24 (25%) were ulnar, and 18 (18.8%) were radial nerves. No statistically significant difference was found between US and electro-physiologic studies for nerve injury diagnosis (p = 0.054).No significant differences were found between US and electro-physiologic studies for diagnosis of nerve injuries; however, US was valuable to assess surrounding tissue and supplied muscles. The capabilities to detect nerve injury and associated distal muscular, vascular, and other regional structures position the US as a complementary diagnostic tool. Peripheral nervous system trauma is rare, but this injury can have a significant negative influence on a patient's health and functional capacities. The overall incidence is 2–3% of patients who visit major trauma centers worldwide, or 13–23 per 100,000 people annually. The most frequent cause of peripheral nerve injuries (PNIs) during calm times is motor vehicle collisions, whereas less prevalent causes are penetrating trauma, falls and industrial accidents [Specific mechanisms of trauma increase the risk of specific nerve injury. To determine which nerves may be impacted, knowledge of the precise trauma mechanism is necessary. This knowledge should take precedence over the conventional neurologic methodology when localizing the peripheral nervous system disease during workup . Most PNDespite the fact that localization of PNIs is clinically possible accurate localization of the injury is challenging, especially when many nerves are involved. Electrophysiological studies are useful to detect the injury severity and location. However, because the most accurate diagnostic information from these studies is only accessible after 2 weeks when Wallerian degeneration is fully developed, electro-diagnostic techniques are not well suited for the acute phase and are inherently painful. In addition, in individuals with a total loss of nerve conduction, electro-diagnostic testing cannot reveal the precise location of the lesion site. Furthermore, these studies cannot display the morphologic changes of the injured nerve , 4.Compared with electro-diagnostic tests, ultrasonography (US) can accurately and painlessly provide this information. Within the whole clinical workup for PNI, the US has a complementary and expanding role. It not only offers a quick and affordable method to probe extensive stretches of peripheral nerves, but it also has unique benefits based on its dynamic, real-time nature and its small number of clinical limitations and contraindications. Thanks to developments in high-frequency transducers and post-processing methods, the function of the US is expanding .When using the US, it is essential to determine the following factors: whether the nerve is still continuous; the size and position of any gaps in the nerve course; the presence of any focal neuromas or extra nerve damage sites, such as tandem lesions; the presence of any foreign bodies; the degree of neighboring scar tissue; and the condition of surrounding tissues and structures, such as tendons, arteries, and bones . As an aOur study will evaluate the value of the US in the assessment of traumatic nerve injuries of the upper limb compared with the role of electro-diagnostic studies as the current gold standard diagnostic modality.From December 2021 to June 2022, adult patients with a history of PNIs were recruited from the clinical neurophysiology unit at our university after undergoing physical examination by an orthopedic surgeon subspecialized with 10 years of experience. The study participants were 69 patients with 96 nerve injuries, including 57 (83%) men and 12 (17%) women, with a mean age of 36.3 ± 13.5 years. All of the study participants gave informed written consent to the study.Inclusion criteria were a clinical picture of PNI in the upper limb, with a duration of 1 month up to 3 years; and surgical exploration of the PNI. Exclusion criteria were diabetes mellitus, polyneuropathies, renal or hepatic disorders, or radiculopathy ; and refusal to participate in the study. All patients underwent thorough history-taking and clinical examination. These patient history data were collected: date of injury date; onset of neurologic manifestations, such as weakness, numbness, and loss of sensation; and history of surgical intervention for the injury.The following clinical examinations were performed for all patients.The electrophysiological research was performed using the Neuropak MEB-9200G/K EP/EMG measurement device . Standard motor nerve conduction studies (NCS) were performed to record the nerve damage for these sites and data: abductor pollicis brevis; abductor digiti minimi; wrist; below and above the elbow stimulation for the ulnar nerve, extensor indices properties; and forearm and elbow stimulation for the radial nerve. The second digit (antidromic stimulation at the wrist for the median nerve), the fifth digit (antidromic stimulation at the wrist for the ulnar nerve), and the anatomical snuff box were recorded as part of routine sensory NCS .To determine the level of injury needle electromyography (EMG) was performed for the muscles supplied by the injured nerve using concentric needles first distally and then proximally. To rule out numerous sites of nerve damage an EMG investigation was also performed for neighboring muscles.Conventional high-resolution US in the brightness mode was performed for all patients using Aplio 500  with a linear probe of 714 MHz frequency. The US examinations were performed by a radiologist with 15 years of experience and who was blinded to the results of the electrodiagnostic tests and clinical Examinations. Depending on the patient's overall condition, patients were either examined while lying flat on their back or comfortably sitting facing the radiologist. The elbow was supported and flexed, and the forearm was extended when the affected nerve was examined at the forearm. To examine the hand, the wrist was held steady and positioned slightly hyperextended. A generous amount of gel was used to improve contact between the probe and the skin's surface; in cases of painful neuromas, little pressure was used. Using sterile gel and taking proper antiseptic precautions, such as using disposable non-sterile gloves and cleaning the US machine and transducer before and after scanning is advised for susceptible patients or while scanning in wound areas.To compare the healthy and injured nerves, a US examination was also performed on the healthy unaffected side. Using a recognized anatomic landmark, the nerves were identified sonographically. Once identified, the nerve was tracked proximally and distally while remaining in the center of the US image along its short axis. The area of interest was then positioned properly focused during US inspection using transverse and longitudinal scanning planes.Identification of the injured nerve lesion site by elicitation of the Tinel sign during US examination because it can help identify the nerve lesion site while using sonopalpation, or the compression of the US probe.Testing for nerve continuity, which includes determining whether the nerve's fascicle is swollen and hypoechoic, whether it lacks a typical fascicular pattern, and whether a neuronal stump is present.Sunderland grade 1: No pathologic findings on the US are visualized or only mild swelling of the nerve with an intact fascicular pattern.Sunderland grade 2: Enlarged cross-sectional area (CSA) of affected fascicles and nerves at the lesion site because of axonal swelling and edema are evident.Sunderland grades 3 and 4: Lesion shows a loss of the normal nerve architecture and echotexture with a disruption of the fascicular pattern and often sizable hypoechogenic enlargement of the nerve.Sunderland grade 5: Lesion shows transection of the nerve with a loss of nerve continuity.Degree of nerve injury, determined to be partial or complete based on the Sunderland classification of PNI , as follAmount of nerve retraction and the gap after complete nerve transaction.Nerve thickening, by measuring the CSA of the injured nerve within the hyperechoic epineurial rim of the nerve and the transducer perpendicular to the nerve.Bony fragments pressing against the nerve near the site of the bone fracture.Proximal terminal neuroma, characterized by uniform texture, hypoechoic echogenicity, and concentric swelling at the terminal end of a transected nerve, with measurement of its size by placing several diameter markers proximal to, at, and caudal to the lesion site.Neuroma in-continuity, by determining if it is intact and manifests as a broadening of the nerve's contour in a nodular shape.Perineural fibrosis, vascular injury, and retained foreign bodies, by evaluating for their presence.Muscular injury, recognized in this study as an increase in echogenicity of the supplied muscle and/or reduced girth (atrophic changes).Doppler brightness mode with color or power Doppler to quantify blood flow for both intraneural and epineural compartments.To interpret the images, the following steps were taken and factors were assessed:p < 0.05 was considered significant.Statistical analysis was conducted using SPSS . Quantitative variables were presented as mean ± standard deviation and range, and qualitative variables were presented as frequency and percentages. Paired comparison of diagnostic tests was conducted using the McNamara test, comparison between proportions was conducted using the chi-squared test. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPP), and overall diagnostic accuracy were calculated using a 2 × 2 contingency table. Any value of A total of 69 patients with 96 nerve injuries were eligible for inclusion in the final analysis, with 57 (83%) men and 12 (17%) women. The mean age was 36.3 ± 13.5 years. Some patients had more than one injured nerve.The mode of trauma was crush injury 27 cases), cut wounds (27 cases), compression (9 cases), and traction 6 cases). Of the 96 injured nerves, 27 were on the right side and 69 71.9%) were on the left side. The injury level was at the arm in 39 nerves, followed by the forearm (33 nerves), and 24 injuries were at the hand. Injured nerves were mainly median nerve in 54 (56.3%) cases, ulnar in 24 (25%) cases, and radial nerve in 18 (18.8%) cases. Table .9% were 7 cases,  cases. OIncreased echogenicity of supplied muscle was observed in 81 84.4%) nerve injuries. In addition, 69 (71.9%) injured nerves had a reduced muscle girth, as shown in Table .4% nerveThe 27 cases with intact nerves shown in the US were diagnosed by electrophysiological studies with mild to moderate nerve injuries. These studies showed that the severity of nerve injury was mild in 18 (18.8%) nerves, moderate in 45 (46.9%) nerves, and severe in 21 (21.9%) nerves, with complete injury in 9 nerves (9.4%), whereas 3 nerves were intact. The axonal injury was reported by NCS in 96 (100%) nerves. These data are presented in Table p = 0.054; sensitivity 100%; specificity 95.8%; NNP 100%; PPV 95.8%; overall diagnostic accuracy 97.8%). Tables No statistically significant difference was found between US and NCS in terms of the diagnosis of nerve injuries  men and 12 (17%) women, with a mean age of 36.3 ± 13.5 years old. These findings are consistent with the evidence in the literature from Ciaramitaroi  and BöscKamble  reportedA study by Martins  showed tKoenig  divided In the present study, no statistically significant difference was revealed between US and NCS for diagnosis of PNI . These findings have high agreement with those by Assy , who assThe limitations of this study are the relatively small number of patients and the limited availability of postoperative findings in all patients.Results showed that the US can be used efficiently as a complementary diagnostic tool in the assessment of PNIs based on its capability not only to detect the type of injury and morphological abnormalities of affected nerves, but because it also allows proper assessment of the surrounding tissue, including swelling, scar tissue, and neuroma formation, thus altering management decisions as needed.


Pseudomonas aeruginosa. We found that bacterial adhesion in the absence of SDS was dependent on the cation concentration rather than the total ionic strength and that combined treatment with several millimolar NaCl and SDS can increase bacterial adhesion. The addition of low concentrations of SDS (2 mM) to tens to hundreds millimolar concentrations of NaCl, typical of systems that suffer seawater incursion, reduced bacterial adhesion dramatically. Combined treatment with Ca+2  and SDS produced a small increase in total adhesion but a dramatic increase in the strength of adhesion. We conclude that the type and concentration of salts in water can have a considerable effect on the efficacy of soap in reducing bacterial adhesion and should be taken under consideration in critical applications.Sodium dodecyl sulfate (SDS) is a common surfactant used in various hygienic products. Its interactions with bacteria were studied previously, but the three-way interaction between surfactants, bacteria, and dissolved salts in the context of bacterial adhesion has not been studied. Here, we examined the combined effects of SDS  and salts, sodium chloride, and calcium chloride  on the adhesion behavior of the common opportunistic pathogen IMPORTANCE Surface-adhering bacteria are a reoccurring problem in many settings, including households, municipal water systems, food production facilities, and hospitals. Surfactants, and specifically sodium dodecyl sulfate , are commonly used to remove bacterial contamination, but data regarding the interaction of SDS with bacteria and especially the effects of water-dissolved salts on this interaction are lacking. Here, we show that calcium and sodium ions can dramatically affect the efficacy of SDS on bacterial adhesion behavior and conclude that salt concentrations and ion species in the water supply should be considered in SDS applications. Surfactants are a cornerstone of modern hygiene, facilitating the removal of bacteria from surfaces , 2. ManyBoth bacteria and surfactants interact with dissolved salts, affecting their behavior. The critical micelle concentration (CMC), the threshold concentration of surfactant required for the formation of micelles, is inversely correlated with the ionic strength of the solution . Higher 6–While surfactants facilitate the removal of bacteria from surfaces 2 at 8 mM SDS  ions were mostly concentrated at the surface and thus not interacting with the bacteria in the bulk liquid, while the counter sodium ions were able to disperse throughout the volume and interact with a far larger portion of the bacterial population. The difference in adhesion at these SDS concentrations versus the comparable NaCl concentrations  in concentrations typical of those in drinking water, thus increasing adhesion. At higher concentrations, the Na+ and Ca+2 cations can form layers on both the surface and the bacterial surface, reestablishing electrostatic repulsion and reducing adhesion. Indeed, the correlation between adhesion and the cation concentration, rather than ionic strength, supports this hypothesis and suggests that the DLVO model, which uses ionic strength as a predictor of adhesion . This treatment also caused a reduction in adhesion strength. Higher SDS concentrations (8 and 16 mM) combined with 1 and 2 mM NaCl reduced adhesion and adhesive strength. In municipal water systems, these effects may translate into different adhesion patterns, depending on the salt content of the water. In developed countries, NaCl concentrations in drinking water are generally limited to 4 mM, as higher concentrations affect the flavor of the water . Under these conditions, dilution of SDS products below the CMC is likely to cause some excess adhesion. In contrast, in tap water in regions suffering from seawater incursion into costal aquifers, NaCl concentrations can reach as high as 140 mM , corresponding to 2.2 mM Ca+2 , cation species in water should be considered and adjusted, if needed. In water with a low sodium content—below 8 mM—the use of SDS at sub-CMC concentrations is likely to increase bacterial adhesion, making diluted soap solutions counterproductive, as the decrease in bacterial adhesion will occur only at higher SDS concentrations. This scenario is likely in developed countries and inland regions. In areas suffering from seawater incursion into costal aquifers, bacterial adhesion is likely to be fast and strong, but the addition of low SDS concentrations (~0.6 g/L) should reduce it dramatically. In karstic regions, where high dissolved CaPseudomonas aeruginosa PAO1 wild type (WT) was obtained from the Manoil laboratory at Washington University  supplemented with 200 μg/mL carbenicillin (Sigma). The bacteria were cultivated in 6 mL medium in 18-mm glass tubes placed in a water bath shaker set to 37°C and 120 rpm for 18 h. The bacteria were collected by centrifugation , washed once, and resuspended in deionized water.iversity . Cells w protein , 27 and 2 (2H2O), 64 mM SDS , and 100 mg/mL Allura red AC.All chemicals were purchased dry from Sigma and dissolved in sterile deionized water to make the following stock solutions: 32 mM NaCl, 16 mM CaClThe microtiter plates were clear, flat-bottom, 96-well plates made of sterile TC-treated polystyrene .600) of 0.1 in deionized water  supplemented with 1.6 mg/mL Allura red AC was added to each well, diluting the bacterial culture density to an OD600 value of 0.05, dye to 0.8 mg/mL, and salt/SDS to the designated concentrations. Bacteria were added using a multichannel pipettor, taking less than 30 s from the first to last column of the plate. In all cases, the plates were loaded immediately into the plate reader  and read for bottom fluorescence  once a minute for 30 min. After the last kinetic point was measured, the liquid was removed, along with sedimented, nonadhering, and weakly adhering bacteria, using a multichannel pipettor, and the wells were filled with 100 μL of 0.8 mg/mL Allura red AC solution in deionized water. The wells were read once more for bottom fluorescence, measuring the fraction of bacteria tightly bound to the well bottom. Eight replicates were performed for each experiment unless otherwise noted.The effect of salts and SDS on bacterial adhesion kinetics was studied using the method described in reference The intensity of fluorescent signals is highly dependent on the specific fluorometer used. Thus, the results are reported in relative fluorescent units (RFU).post hoc test in all cases.Statistical analyses were performed using SPSS statistics version 24.0.0 ; Tukey’s honestly significant difference (HSD) test was used as a All data are presented in the text; the raw data are available by request from the corresponding author.