Dendritic Integration of Sensory Evidence in Perceptual Decision-Making.

Perceptual decisions require the accumulation of sensory information to a response criterion. Most accounts of how the brain performs this process of temporal integration have focused on evolving patterns of spiking activity. We report that subthreshold changes in membrane voltage can represent accumulating evidence before a choice. αß core Kenyon cells (αßc KCs) in the mushroom bodies of fruit flies integrate odor-evoked synaptic inputs to action potential threshold at timescales matching the speed of olfactory discrimination. The forkhead box P transcription factor (FoxP) sets neuronal integration and behavioral decision times by controlling the abundance of the voltage-gated potassium channel Shal (KV4) in αßc KC dendrites. αßc KCs thus tailor, through a particular constellation of biophysical properties, the generic process of synaptic integration to the demands of sequential sampling.

A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity.

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.

SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics.

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

Energy storing and return prosthetic feet improve step length symmetry while preserving margins of stability in persons with transtibial amputation.

BACKGROUND: Energy storing and return (ESAR) feet are generally preferred over solid ankle cushioned heel (SACH) feet by people with a lower limb amputation. While ESAR feet have been shown to have only limited effect on gait economy, other functional benefits should account for this preference. A simple biomechanical model suggests that enhanced gait stability and gait symmetry could prove to explain part of the difference in the subjective preference between both feet. AIM: To investigate whether increased push-off power with ESAR feet increases center of mass velocity at push off and enhance intact step length and step length symmetry while preserving the margin of stability during walking in people with a transtibial prosthesis. METHODS: Fifteen people with a unilateral transtibial amputation walked with their prescribed ESAR foot and a SACH foot at a fixed walking speed (1.2 m/s) over a level walkway while kinematic and kinetic data were collected. Push-off work generated by the foot, center of mass velocity, step length, step length symmetry and backward margin of stability were assessed and compared between feet. RESULTS: Push-off work was significantly higher when using the ESAR foot compared to the SACH foot. Simultaneously, center of mass velocity at toe-off was higher with ESAR compared to SACH, and intact step length and step length symmetry increased without reducing the backward margin of stability. CONCLUSION: Compared to the SACH foot, the ESAR foot allowed an improvement of step length symmetry while preserving the backward margin of stability at community ambulation speed. These benefits may possibly contribute to the subjective preference for ESAR feet in people with a lower limb amputation.

Walking in an unstable environment: strategies used by transtibial amputees to prevent falling during gait.

OBJECTIVE: To investigate which strategies transtibial amputees use to cope with challenges of gait stability and gait adaptability, and how these strategies differ from strategies used by able-bodied controls. DESIGN: Cross-sectional study. SETTING: An instrumented treadmill mounted onto a 6°-of-freedom motion platform in combination with a virtual environment. PARTICIPANTS: Transtibial amputees (n=10) and able-bodied controls (n=9). INTERVENTIONS: Mediolateral (ML) translations of the walking surface were imposed to manipulate gait stability. To provoke an adaptive gait pattern, a gait adaptability task was used in which subjects had to hit virtual targets with markers guided by their knees. MAIN OUTCOME MEASURES: Walking speed, step length, step frequency, step width, and selected measures of gait stability (short-term Lyapunov exponents and backward and ML margins of stability [MoS]). RESULTS: Amputees walked slower than able-bodied people, with a lower step frequency and wider steps. This resulted in a larger ML MoS but a smaller backward MoS for amputees. In response to the balance perturbation, both groups decreased step length and increased step frequency and step width. Walking speed did not change significantly in response to the perturbation. These adaptations induced an increase in ML and backward MoS. To perform the gait adaptability task, both groups decreased step length and increased step width, but did not change step frequency and walking speed. ML and backward MoS were maintained in both groups. CONCLUSIONS: Transtibial amputees have the capacity to use the same strategies to deal with challenges of gait stability and adaptability, to the same extent as able-bodied people.

Cdk5 and GSK3ß inhibit fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

The concept of margins of stability can be used to better understand a change in obstacle crossing strategy with an increase in age.

The purpose of the current study was to use the margins of stability (MoS) to investigate how older adults choose between minimizing the risk of a forward fall when crossing an obstacle and the ease of maintaining forward progression during the steps taken behind the obstacle. In the current study 143 community-dwelling older adults aged between 55 and 83 years old, were divided into three age groups based on tertials of age. All participants were asked to complete five trials of obstacle walking and five trials of normal walking. For the trials of normal walking, the main difference between groups was that MoS at initial contact was lower in the older age groups. For the trials of obstacle crossing the MoS at the instants of obstacle crossing with both the leading and trailing limb became smaller with an increase in age. This result might imply that older people choose to use a strategy during obstacle crossing that results in smaller chance of falling forward if an obstacle was struck. A negative consequence of this more conservative strategy was a smaller MoS at the instants of initial contact after crossing the obstacle, thus a larger chance of a backward fall. These findings provide more insight into the regulation of stability during obstacle crossing and specifically in the differences in strategy between younger and older people, and therefore these results might be used for further research to investigate whether obstacle crossing strategies are trainable in older adults, which could be used as advisory programs aimed at fall prevention and/or engagement in an active lifestyle.

Publisher Correction: FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis.

In the version of this Letter originally published, the name of co-author Safa Lucken-Ardjomande Häsler was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

FBP17 and CIP4 recruit SHIP2 and lamellipodin to prime the plasma membrane for fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells1, but several clathrin-independent endocytic routes exist in parallel2,3. One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the ß1 adrenergic receptor4. FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin4,5. However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5'-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5-10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation.

Regulation of step frequency in transtibial amputee endurance athletes using a running-specific prosthesis.

Running specific prostheses (RSP) are designed to replicate the spring-like behaviour of the human leg during running, by incorporating a real physical spring in the prosthesis. Leg stiffness is an important parameter in running as it is strongly related to step frequency and running economy. To be able to select a prosthesis that contributes to the required leg stiffness of the athlete, it needs to be known to what extent the behaviour of the prosthetic leg during running is dominated by the stiffness of the prosthesis or whether it can be regulated by adaptations of the residual joints. The aim of this study was to investigate whether and how athletes with an RSP could regulate leg stiffness during distance running at different step frequencies. Seven endurance runners with an unilateral transtibial amputation performed five running trials on a treadmill at a fixed speed, while different step frequencies were imposed (preferred step frequency (PSF) and -15%, -7.5%, +7.5% and +15% of PSF). Among others, step time, ground contact time, flight time, leg stiffness and joint kinetics were measured for both legs. In the intact leg, increasing step frequency was accompanied by a decrease in both contact and flight time, while in the prosthetic leg contact time remained constant and only flight time decreased. In accordance, leg stiffness increased in the intact leg, but not in the prosthetic leg. Although a substantial contribution of the residual leg to total leg stiffness was observed, this contribution did not change considerably with changing step frequency. Amputee athletes do not seem to be able to alter prosthetic leg stiffness to regulate step frequency during running. This invariant behaviour indicates that RSP stiffness has a large effect on total leg stiffness and therefore can have an important influence on running performance. Nevertheless, since prosthetic leg stiffness was considerably lower than stiffness of the RSP, compliance of the residual leg should not be ignored when selecting RSP stiffness.

Quality of life after staged revision for infected total hip arthroplasty: a systematic review.

INTRODUCTION: The aim of this review was to assess (health-related) quality of life ([HR]Qol) after 1-stage or 2-stage revision for prosthetic joint infection of the hip (PJI). Additionally, we compared (HR)QoL scores with normative population scores to assess whether QoL is reduced after revision for PJI. METHODS: A systematic search was performed in Embase, Cochrane and Pubmed. We included articles that reported (HR)QoL questionnaires after staged revision for hip PJI with a minimum follow-up of 24 months. Methodological quality was assessed using the MINORS score. RESULTS: The search produced 11,195 results. We selected 12 papers describing 2-stage revisions. The mean MINORS score was 9.8. Average WOMAC scores of 185 patients with a mean follow-up of 69.8 months were 73.Average Short Form 36 (SF-36) scores of 159 patients with a mean follow-up of 40.7 months were 40.4 for the physical component score (PCS) and 51.6 for the mental component score (MCS). Average Short Form 12 (SF-12) scores of 154 patients with a mean follow-up of 73.4 months were: a PCS of 35.4 and a MCS of 49.1. The WOMAC, SF-12 (PCS) and SF-36 (PCS) were respectively 12%, 26.7% and 14.8% lower, compared to normative values. DISCUSSIONS: Patients who underwent 2-stage revision for hip PJI had substantially lower (physical component) (HR)QoL scores, but mental scores were comparable to the general population.

Stride frequency and length adjustment in post-stroke individuals: influence on the margins of stability.

OBJECTIVE: To investigate whether post-stroke participants can walk at different combinations of stride frequency and stride length and how these adaptations affect the backward and medio-lateral margins of stability. SETTING: Computer Assisted Rehabilitation Environment (CAREN). PARTICIPANTS: Ten post-stroke individuals. INTERVENTION: Six trials of 2 min walking on a treadmill at different combinations of stride frequency and stride length. Treadmill speed was set at the corresponding speed, and subjects received visual feedback about the required and actual stride length. OUTCOME MEASURES: Mean stride length and frequency and backward and medio-lateral margins of stability for each trial. RESULTS AND CONCLUSION: Stroke patients were able to adjust step length when required, but had difficulty adjusting step frequency. When a stride frequency higher than self-selected stride frequency was imposed patients additionally needed to increase stride length in order to match the imposed treadmill speed. For trials at a high stride frequency, in particular, the increase in the backward and medio-lateral margins of stability was limited. In conclusion, training post-stroke individuals to increase stride frequency during walking might give them more opportunities to increase the margins of stability and consequently reduce fall risk.

Stepping asymmetry among individuals with unilateral transtibial limb loss might be functional in terms of gait stability.

BACKGROUND: The asymmetry in step length in prosthetic gait is often seen as a detrimental effect of the impairment; however, this asymmetry also might be a functional compensation. An advantage of a smaller step length of the nonprosthetic leg, and specifically foot forward placement (FFP), might be that it will bring the center of mass closer to the base of support of the leading foot and thus increase the backward margin of stability (BW MoS). OBJECTIVE: The purpose of this study was to characterize differences in step length, FFP, and the concomitant difference in BW MoS between steps of the prosthetic and nonprosthetic legs (referred to as prosthetic and nonprosthetic steps, respectively) of people after transtibial amputation. DESIGN: This was an observational and cross-sectional study. METHODS: Ten people after transtibial amputation walked for 4 minutes on a self-paced treadmill. Step length and FFP were calculated at initial contact. The size of the BW MoS was calculated for the moment of initial contact and at the end of the double-support phase of gait. RESULTS: Step length (5.4%) and FFP (7.9%) were shorter for the nonprosthetic step than for the prosthetic step. The BW MoS at initial contact was larger for the nonprosthetic step, but because of a significant leg × gait event interaction effect, BW MoS did not differ significantly at the end of the double-support phase. LIMITATIONS: All participants were relatively good walkers (score of E on the Special Interest Group in Amputee Medicine [SIGAM] scale). CONCLUSIONS: The smaller step length and FFP of the nonprosthetic step help to create a larger BW MoS at initial contact for the nonprosthetic step compared with the prosthetic step. Hence, step length asymmetry in people after transtibial amputation might be seen as a functional compensation to preserve BW MoS during the double-support phase to cope with the limited push-off power of the prosthetic ankle.

Steps to take to enhance gait stability: the effect of stride frequency, stride length, and walking speed on local dynamic stability and margins of stability.

The purpose of the current study was to investigate whether adaptations of stride length, stride frequency, and walking speed, independently influence local dynamic stability and the size of the medio-lateral and backward margins of stability during walking. Nine healthy subjects walked 25 trials on a treadmill at different combinations of stride frequency, stride length, and consequently at different walking speeds. Visual feedback about the required and the actual combination of stride frequency and stride length was given during the trials. Generalized Estimating Equations were used to investigate the independent contribution of stride length, stride frequency, and walking speed on the measures of gait stability. Increasing stride frequency was found to enhance medio-lateral margins of stability. Backward margins of stability became larger as stride length decreased or walking speed increased. For local dynamic stability no significant effects of stride frequency, stride length or walking speed were found. We conclude that adaptations in stride frequency, stride length and/or walking speed can result in an increase of the medio-lateral and backward margins of stability, while these adaptations do not seem to affect local dynamic stability. Gait training focusing on the observed stepping strategies to enhance margins of stability might be a useful contribution to programs aimed at fall prevention.

Stepping strategies for regulating gait adaptability and stability.

Besides a stable gait pattern, gait in daily life requires the capability to adapt this pattern in response to environmental conditions. The purpose of this study was to elucidate the anticipatory strategies used by able-bodied people to attain an adaptive gait pattern, and how these strategies interact with strategies used to maintain gait stability. Ten healthy subjects walked in a Computer Assisted Rehabilitation ENvironment (CAREN). To provoke an adaptive gait pattern, subjects had to hit virtual targets, with markers guided by their knees, while walking on a self-paced treadmill. The effects of walking with and without this task on walking speed, step length, step frequency, step width and the margins of stability (MoS) were assessed. Furthermore, these trials were performed with and without additional continuous ML platform translations. When an adaptive gait pattern was required, subjects decreased step length (p<0.01), tended to increase step width (p=0.074), and decreased walking speed while maintaining similar step frequency compared to unconstrained walking. These adaptations resulted in the preservation of equal MoS between trials, despite the disturbing influence of the gait adaptability task. When the gait adaptability task was combined with the balance perturbation subjects further decreased step length, as evidenced by a significant interaction between both manipulations (p=0.012). In conclusion, able-bodied people reduce step length and increase step width during walking conditions requiring a high level of both stability and adaptability. Although an increase in step frequency has previously been found to enhance stability, a faster movement, which would coincide with a higher step frequency, hampers accuracy and may consequently limit gait adaptability.

Stepping strategies used by post-stroke individuals to maintain margins of stability during walking.

BACKGROUND: People recovering from a stroke are less stable during walking compared to able-bodied controls. The purpose of this study was to examine whether and how post-stroke individuals adapt their steady-state gait pattern to maintain or increase their margins of stability during walking, and to examine how these strategies differ from strategies employed by able-bodied people. METHODS: Ten post-stroke individuals and 9 age-matched able-bodied individuals walked on the Computer Assisted Rehabilitation Environment. Medio-lateral translations of the walking surface were imposed to manipulate gait stability. To provoke gait adaptations, a gait adaptability task was used, in which subjects occasionally had to hit a virtual target with their knees. We measured medio-lateral and backward margins of stability, and the associated gait parameters walking speed, step length, step frequency, and step width. FINDINGS: Post-stroke participants showed similar medio-lateral margins of stability as able-bodied people in all conditions. This was accomplished by a larger step width and a relatively high step frequency. Post-stroke participants walked overall slower and decreased walking speed and step length even further in response to both manipulations compared to able-bodied participants, resulting in a tendency towards an overall smaller backward margins of stability, and a significantly smaller backward margin of stability during the gait adaptability task. INTERPRETATION: Post-stroke individuals have more difficulties regulating their walking speed, and the underlying parameters step frequency and step length, compared to able-bodied controls. These quantities are important in regulating the size of the backward margin of stability when walking in complex environments.

Speeding up or slowing down?: Gait adaptations to preserve gait stability in response to balance perturbations.

It has frequently been proposed that lowering walking speed is a strategy to enhance gait stability and to decrease the probability of falling. However, previous studies have not been able to establish a clear relation between walking speed and gait stability. We investigated whether people do indeed lower walking speed when gait stability is challenged, and whether this reduces the probability of falling. Nine healthy subjects walked on the Computer Assisted Rehabilitation ENvironment (CAREN) system, while quasi-random medio-lateral translations of the walking surface were imposed at four different intensities. A self-paced treadmill setting allowed subjects to regulate their walking speed throughout the trials. Walking speed, step length, step frequency, step width, local dynamic stability (LDS), and margins of stability (MoS) were measured. Subjects did not change walking speed in response to the balance perturbations (p=0.118), but made shorter, faster, and wider steps (p<0.01) with increasing perturbation intensity. Subjects became locally less stable in response to the perturbations (p<0.01), but increased their MoS in medio-lateral (p<0.01) and backward (p<0.01) direction. In conclusion, not a lower walking speed, but a combination of decreased step length and increased step frequency and step width seems to be the strategy of choice to cope with medio-lateral balance perturbations, which increases MoS and thus decreases the risk of falling.