Transfemoral limb loss modestly increases the metabolic cost of optimal control simulations of walking.

Background: In transtibial limb loss, computer simulations suggest that the maintenance of muscle strength between pre- and post-limb loss can maintain the pre-limb loss metabolic cost. These results are consistent with comparable costs found experimentally in select cases of high functioning military service members with transtibial limb loss. It is unlikely that similar results would be found with transfemoral limb loss, although the theoretical limits are not known. Here we performed optimal control simulations of walking with and without an above-knee prosthesis to determine if transfemoral limb loss per se increases the metabolic cost of walking. Methods: OpenSim Moco was used to generate optimal control simulations of walking in 15 virtual "subjects" that minimized the weighted sum of (i) deviations from average able-bodied gait mechanics and (ii) the gross metabolic cost of walking, pre-limb loss in models with two intact biological limbs, and post-limb loss with one of the limbs replaced by a prosthetic knee and foot. No other changes were made to the model. Metabolic cost was compared between pre- and post-limb loss simulations in paired t-tests. Results: Metabolic cost post-limb loss increased by 0.7-9.3% (p < 0.01) depending on whether cost was scaled by total body mass or biological body mass and on whether the prosthetic knee was passive or non-passive. Conclusions: Given that the post-limb loss model had numerous features that predisposed it to low metabolic cost, these results suggest transfemoral limb loss per se increases the metabolic cost of walking. However, the large differences above able-bodied peers of ∼20-45% in most gait analysis experiments may be avoidable, even when minimizing deviations from able-bodied gait mechanics. Portions of this text were previously published as part of a preprint (https://www.biorxiv.org/content/10.1101/2023.06.26.546515v2.full.pdf).

Modulation of Vertical Ground Reaction Impulse With Real-Time Biofeedback: A Feasibility Study.

Given its apparent representation of cumulative (vs peak) loads, this feasibility study investigates vertical ground reaction impulse (vGRI) as a real-time biofeedback variable for gait training aimed at reducing lower limb loading. Fifteen uninjured participants (mean age = 27 y) completed 12 2-min trials, 1 at each combination of 4 walking speeds (1.0, 1.2, 1.4, and 1.6 m/s) and 3 targeted reductions in vGRI (5, 10, and 15%) of the assigned ("target") limb, with the latter specified relative to an initial baseline (no feedback) condition at each speed. The ability to achieve targeted reductions was assessed using step-by-step errors between measured and targeted vGRI. Mean (SD) errors were 5.2% (3.7%); these were larger with faster walking speeds but consistent across reduction targets. Secondarily, we evaluated the strategy used to modulate reductions (ie, stance time or peak vertical ground reaction force [vGRF]) and the resultant influences on knee joint loading (external knee adduction moment [EKAM]). On the targeted limb, stance times decreased (P < .001) with increasing reduction target; first and second peaks in vGRF were similar (P > .104) across all target conditions. While these alterations did not significantly reduce EKAM on the target limb, future work in patients with knee pathologies is warranted.

Functional Outcomes of Service Members With Bilateral Transfemoral and Knee Disarticulation Amputations Resulting From Trauma.

As longitudinal studies for those with bilateral transfemoral amputation (BTFA) or knee disarticulation (KD) are lacking, it is important to quantify performance measures during rehabilitation in an effort to determine reasonable expectations and trends that may influence the rehabilitation process. At initial evaluation (date of first independent ambulation) and follow up (median 135 [range = 47-300] days later), 10 participants with BTFA/KD completed 6 minute walk testing and Activity Specific Balance Confidence and Lower Extremity Functional Scale questionnaires. Of these, six participants also completed stair ambulation; ascent time and stair assessment index (SAI) scores were calculated. Patients utilized their prescribed prostheses at each visit. Participants were able to cover a significantly greater distance (135.3 [70.1] m) in 6 minutes at the follow-up visit (*p = 0.005). The change in SAI scores for stair ascent and descent was not statistically significant (p = 0.247). Stair ambulation confidence scores were significantly greater at the final visit (*p = 0.034). Stair negotiation appears to plateau early; however, confidence builds despite absence of functional gains over time. Service members with BTFAs/KDs are able to achieve functional community ambulation skills. Thus, this investigation suggests that clinicians can realign rehabilitation paradigms to shift focus towards community distance ambulation once safe stair ascent and descent is achieved.

Performance of conventional and X2® prosthetic knees during slope descent.

BACKGROUND: Individuals with transfemoral amputation often have difficulty descending sloped surfaces due to increased lower extremity range of motion and torque requirements. The X2®, a new microprocessor-controlled prosthetic knee, claims to improve gait over sloped terrain. The aim of this study was to evaluate how experienced prosthesis users descended a sloped surface using the X2®, compared to a conventional knee, either mechanical (MECH) or microprocessor (MP). METHODS: Descent technique and biomechanics were assessed in 21 service members with unilateral transfemoral amputation as they descended an instrumented 10° slope at a self-selected walking velocity. FINDINGS: Use of the X2® in the MECH group resulted in greater hill assessment scores (8.5 to 11.0, P=0.026), due primarily to decreased reliance on handrail use. The use of the X2® in the MP group increased prosthetic knee flexion to a median of 6.4° at initial contact (P=0.002) and 73.7° in swing (P=0.005), contributing to longer prosthetic limb steps (P=0.024) and increased self-selected velocity (P=0.041). Additionally, the use of the X2® in the MP group increased prosthetic limb impact peaks (11.6N/kg, P=0.004), improving impact peak symmetry to -1.3% (P=0.004). INTERPRETATION: Decreased reliance on handrail use as MECH users descended in the X2® indicate improved function and perhaps greater confidence in the device. Additional biomechanical improvements for existing MP users suggest potential longer-term benefits with regard to intact limb health and overuse injuries.

Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries.

Leaves and flowers begin life as outgrowths from the edges of shoot apical meristems. Stem cell divisions in the meristem center replenish cells that are incorporated into organ primordia at the meristem periphery and leave the meristem. Organ boundaries, regions of limited growth that separate forming organs from the meristem, serve to isolate these two domains and are critical for coordination of organogenesis and meristem maintenance. Boundary formation and maintenance are poorly understood processes, despite the identification of a number of boundary-specific transcription factors. Here we provide genetic and biochemical evidence that the Arabidopsis thaliana transcription factor lateral organ boundaries (LOB) negatively regulates accumulation of the plant steroid hormone brassinosteroid (BR) in organ boundaries. We found that ectopic expression of LOB results in reduced BR responses. We identified BAS1, which encodes a BR-inactivating enzyme, as a direct target of LOB transcriptional activation. Loss-of-function lob mutants exhibit organ fusions, and this phenotype is suppressed by expression of BAS1 under the LOB promoter, indicating that BR hyperaccumulation contributes to the lob mutant phenotype. In addition, LOB expression is BR regulated; therefore, LOB and BR form a feedback loop to modulate local BR accumulation in organ boundaries to limit growth in the boundary domain.

Misregulation of the LOB domain gene DDA1 suggests possible functions in auxin signalling and photomorphogenesis.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene family encodes plant-specific transcription factors. In this report, the LBD gene DOWN IN DARK AND AUXIN1 (DDA1), which is closely related to LATERAL ORGAN BOUNDARIES (LOB) and ASYMMETRIC LEAVES2 (AS2), was characterized. DDA1 is expressed primarily in vascular tissues and its transcript levels were reduced by exposure to exogenous indole-3-acetic acid (IAA or auxin) and in response to dark exposure. Analysis of a T-DNA insertion line, dda1-1, in which the insertion resulted in misregulation of DDA1 transcripts in the presence of IAA and in the dark revealed possible functions in auxin response and photomorphogenesis. dda1-1 plants exhibited reduced sensitivity to auxin, produced fewer lateral roots, and displayed aberrant hypocotyl elongation in the dark. Phenotypes resulting from fusion of a transcriptional repression domain to DDA1 suggest that DDA1 may act as both a transcriptional activator and a transcriptional repressor depending on the context. These results indicate that DDA1 may function in both the auxin signalling and photomorphogenesis pathways.

LATERAL ORGAN BOUNDARIES defines a new family of DNA-binding transcription factors and can interact with specific bHLH proteins.

Conserved in a variety of evolutionarily divergent plant species, LOB DOMAIN (LBD) genes define a large, plant-specific family of largely unknown function. LBD genes have been implicated in a variety of developmental processes in plants, although to date, relatively few members have been assigned functions. LBD proteins have previously been predicted to be transcription factors, however supporting evidence has only been circumstantial. To address the biochemical function of LBD proteins, we identified a 6-bp consensus motif recognized by a wide cross-section of LBD proteins, and showed that LATERAL ORGAN BOUNDARIES (LOB), the founding member of the family, is a transcriptional activator in yeast. Thus, the LBD genes encode a novel class of DNA-binding transcription factors. Post-translational regulation of transcription factors is often crucial for control of gene expression. In our study, we demonstrate that members of the basic helix-loop-helix (bHLH) family of transcription factors are capable of interacting with LOB. The expression patterns of bHLH048 and LOB overlap at lateral organ boundaries. Interestingly, the interaction of bHLH048 with LOB results in reduced affinity of LOB for the consensus DNA motif. Thus, our studies suggest that bHLH048 post-translationally regulates the function of LOB at lateral organ boundaries.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen.

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.