A Self-Scaffolding Model for G Protein Signaling

Activation of heterotrimeric G proteins is generally believed to induce dissociation of Gα and Gβγ subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Gαq subunits remain complexed with its major effector, phospholipase Cβ1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Gαi and eCFP-Gβγ remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cβ2 and eGFP-Gβγ is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gβγ subunits and changes in their fluorescence properties and accessibility upon activation and Gβγ binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors.     

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called ‘tricks-of-the-trade’ and include case studies of several mammalian transporters.     

Theoretical and experimental behaviour of the muscle viscosity coefficient during maximal concentric actions

The aim of this study was to calculate the theoretical variation of the nonlinear damping factor (B) as a function of the muscle shortening velocity, and then to compare the theoretical values with the experimental data obtained on both the elbow flexor and the ankle extensor muscles. The theoretical variation of the B factor was determined from a muscle model consisting of a contractile component in parallel with a viscous damper both in series with an elastic component, and by using, the charateristic equation of the force velocity curve. In this muscle model, the viscous element modelled the inability of the muscle to generate as big a contracting force (while shortening) as possible under isometric conditions. Eight volunteer subjects performed maximal concentric elbow flexions and ankle extensions on an isokinetic ergometer at angular velocities of 60, 120, 180, 240, 300 and 360°·s^–1, and held two maximal isometric actions at an elbow angle of 90° (0° corresponds to the full extension) and at an ankle angle of 0° (0° corresponds to the foot flexion of 90° relative to the leg axis). From these measurements, the force and the shortening velocity values of each muscle were determined by using a musculo-skeletal model of the joint. The results showed that the theoretical behaviour of the B factor would seem to be dependent on the shortening velocity and on the parameter which varies according to the muscle fibre type composition and affects the curvature of the force-velocity curve (a_f). For each muscle group, the experimental data of B fitted with the theoretical equation, and the best fit was obtained for an of of 0.28 for the ankle extensor and of 0.32 for the elbow flexor muscles. These results indicated that from the muscle model used in the present study it is possible to describe the mechanical behaviour of the muscle during maximal concentric action.     

Peak triceps surae muscle activity is not specific to knee flexion angles during MVIC

There is limited research on peak activity of the separate triceps surae muscles in select knee flexion (KF) positions during a maximum voluntary isometric contraction (MVIC) used to normalize EMG signals. The aim of this study was to determine how frequent peak activity occurred during an MVIC for soleus (SOL), gastrocnemius medialis (GM), and gastrocnemius lateralis (GL) in select KF positions, and if these peaks were recorded in similar KF positions. Forty-eight healthy individuals performed unilateral plantar-flexion MVIC in standing with 0°KF and 45°KF, and in sitting with 90°KF. Surface EMG of SOL, GM, and GL were collected and processed in 250 ms epochs to determine peak root-mean-square amplitude. Peak activity was most frequently captured in standing and rarely in sitting, with no position selective to SOL, GM or GL activity. Peak GM and GL activity was more frequent in 0°KF than 45°KF, and more often in similar KF positions than not. Peak SOL activity was just as likely in 45°KF as 0°KF, and more in positions similar to GM, but not GL. The EMG amplitudes were at least 20% greater in positions that captured peak activity over those that did not. The overall findings support performing an MVIC in more than one KF position to normalize triceps surae EMG. It is emphasized that no KF position is selective to SOL, GM, or GL alone.     

Impact of ankle foot orthosis stiffness on Achilles tendon and gastrocnemius function during unimpaired gait

Ankle foot orthoses (AFOs) are designed to improve gait for individuals with neuromuscular conditions and have also been used to reduce energy costs of walking for unimpaired individuals. AFOs influence joint motion and metabolic cost, but how they impact muscle function remains unclear. This study investigated the impact of different stiffness AFOs on medial gastrocnemius muscle (MG) and Achilles tendon (AT) function during two walking speeds. We performed gait analyses for eight unimpaired individuals. Each individual walked at slow and very slow speeds with a 3D printed AFO with no resistance (free hinge condition) and four levels of ankle dorsiflexion stiffness: 0.25 Nm/°, 1 Nm/°, 2 Nm/°, and 3.7 Nm/°. Motion capture, ultrasound, and musculoskeletal modeling were used to quantify MG and AT lengths with each AFO condition. Increasing AFO stiffness increased peak AFO dorsiflexion moment with decreased peak knee extension and peak ankle dorsiflexion angles. Overall musculotendon length and peak AT length decreased, while peak MG length increased with increasing AFO stiffness. Peak MG activity, length, and velocity significantly decreased with slower walking speed. This study provides experimental evidence of the impact of AFO stiffness and walking speed on joint kinematics and musculotendon function. These methods can provide insight to improve AFO designs and optimize musculotendon function for rehabilitation, performance, or other goals.     

Kinematics of lower limbs during walking are emulated by springy walking model with a compliantly connected,off-centered curvy foot

The dynamics of the center of mass (CoM) during walking and running at various gait conditions are well described by the mechanics of a simple passive spring loaded inverted pendulum (SLIP). Due to its simplicity, however, the current form of the SLIP model is limited at providing any further information about multi-segmental lower limbs that generate oscillatory CoM behaviors and their corresponding ground reaction forces. Considering that the dynamics of the CoM are simply achieved by mass-spring mechanics, we wondered whether any of the multi-joint motions could be demonstrated by simple mechanics. In this study, we expand a SLIP model of human locomotion with an off-centered curvy foot connected to the leg by a springy segment that emulates the asymmetric kinematics and kinetics of the ankle joint. The passive dynamics of the proposed expansion of the SLIP model demonstrated the empirical data of ground reaction forces, center of mass trajectories, ankle joint kinematics and corresponding ankle joint torque at various gait speeds. From the mechanically simulated trajectories of the ankle joint and CoM, the motion of lower-limb segments, such as thigh and shank angles, could be estimated from inverse kinematics. The estimation of lower limb kinematics showed a qualitative match with empirical data of walking at various speeds. The representability of passive compliant mechanics for the kinetics of the CoM and ankle joint and lower limb joint kinematics implies that the coordination of multi-joint lower limbs during gait can be understood with a mechanical framework.     

不同内固定方式对后踝骨折固定的生物力学效果及临床疗效分析

目的：比较不同内固定方式对后踝关节骨折固定的生物力学效果及临床疗效。方法：选择2009年1月-2010年12月来我院就诊经X线检查确诊的后踝骨折患者28例为研究对象,根据所使用内固定方法不同,分为拉力螺钉组和微型钢板组,随访比较两组的手术时间、出血量、固定成功率、住院时间、后遗症及生物力学效果。结果：所有患者均随访至拆除内固定,拉力螺钉组和微型钢板组随访时间（t=-2．102）进行比较,P〉0．05两组差异无统计学意义。两组手术时间（t=4．293）、手术出血量（t=6．827）、固定成功率（2=,5．568）和住院时间（t=3．903）比较,P均〈0．05,差异有统计学意义。当骨折块波及胫骨远端关节面s25％和其〉25％时,两组内固定失效临界压力比较,P〈均0．05,两组内固定压力差异无统计学意义。结论：由于后踝关节的特殊结构和复杂的力学特点,拉力螺钉内固定在治疗效果优于微型钢板内固定法,但当骨折块波及胫骨远端关节面〉25％生物力学效果微型钢板内固定法更佳,因此根据患者个体骨折差异选择合适的内固定方法。     

Effect of alignment changes on socket reaction moments during gait in transfemoral and knee-disarticulation prostheses: Case series

The alignment of a lower-limb prosthesis is critical to the successful prosthetic fitting and utilization by the wearer. Loads generated by the socket applied to the residual limb while walking are thought to be different in transfemoral and knee-disarticulation prostheses. The aim of this case series was to compare the socket reaction moments between transfemoral and knee-disarticulation prostheses and to investigate the effect of alignment changes on them. Two amputees, one with a transfemoral prosthesis and another with a knee-disarticulation prosthesis, participated in this study. A Smart Pyramid™ was used to measure socket reaction moments while walking under 9 selected alignment conditions; including nominally aligned, angle malalignments of 6° (flexion, extension, abduction and adduction) and translation malalignments of 15 mm (anterior, posterior, medial and lateral) of the socket relative to the foot. This study found that the pattern of the socket reaction moments was similar between transfemoral and knee-disarticulation prostheses. An extension moment in the sagittal plane and a varus moment in the coronal plane were dominant during stance under the nominally aligned condition. This study also demonstrated that alignment changes might have consistent effects on the socket reaction moments in transfemoral and knee-disarticulation prostheses. Extension and posterior translation of the socket resulted in increases in an extension moment, while abduction and lateral translation of the socket resulted in increases in a varus moment. The socket reaction moments may potentially serve as useful biomechanical parameters to evaluate alignment in transfemoral and knee-disarticulation prostheses.     

Spotlight: Assembly of protein complexes by integrating graph clustering methods

As is generally assumed, clusters in protein–protein interaction (PPI) networks perform specific, crucial functions in biological systems. Various network community detection methods have been developed to exploit PPI networks in order to identify protein complexes and functional modules. Due to the potential role of various regulatory modes in biological networks, a single method may just apply a single graph property and neglect communities highlighted by other network properties.     

The Parkinson's disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of autosomal dominant Parkinson's disease (PD). LRRK2, a member of the ROCO protein family, contains both Ras GTPase-like (Roc) and kinase (MAPKKK) domains, as well as other functional motifs. Here, we have identified LRRK2 as the first mammalian ROCO protein that is an authentic and functional GTPase, defined by the ability to bind GTP and undergo intrinsic GTP hydrolysis. Furthermore, the Roc domain is sufficient for this native GTPase activity and binds and hydrolyzes GTP indistinguishably from the Ras-related small GTPase, Rac1. The PD-associated mutation, R1441C, located within the Roc domain, leads to an increase in LRRK2 kinase activity and a decrease in the rate of GTP hydrolysis, compared to the wild-type protein, in an in vitro assay. This finding suggests that the R1441C mutation may help stabilize an activated state of LRRK2. Additionally, LRRK2-mediated phosphorylation is stimulated upon binding of non-hydrolyzable GTP analogs, suggesting that LRRK2 is an MAPKKK-activated intramolecularly by its own GTPase. Since GTPases and MAPKKKs are upstream regulators of multiple signal transduction cascades, LRRK2 may play a central role in integrating pathways involved in neuronal cell signaling and the pathogenesis of PD.