Mediolateral damping of an overhead body weight support system assists stability during treadmill walking.

BACKGROUND: Body weight support systems with three or more degrees of freedom (3-DoF) are permissive and safe environments that provide unloading and allow unrestricted movement in any direction. This enables training of walking and balance control at an early stage in rehabilitation. Transparent systems generate a support force vector that is near vertical at all positions in the workspace to only minimally interfere with natural movement patterns. Patients with impaired balance, however, may benefit from additional mediolateral support that can be adjusted according to their capacity. An elegant solution for providing balance support might be by rendering viscous damping along the mediolateral axis via the software controller. Before use with patients, we evaluated if control-rendered mediolateral damping evokes the desired stability enhancement in able-bodied individuals. METHODS: A transparent, cable-driven robotic body weight support system (FLOAT) was used to provide transparent body weight support with and without mediolateral damping to 21 able-bodied volunteers while walking at preferred gait velocity on a treadmill. Stability metrics reflecting resistance to small and large perturbations were derived from walking kinematics and compared between conditions and to free walking. RESULTS: Compared to free walking, the application of body weight support per-se resulted in gait alterations typically associated with body weight support, namely increased step length and swing phase. Frontal plane dynamic stability, measured by kinematic variability and nonlinear dynamics of the center of mass, was increased under body weight support, indicating reduced balance requirements in both damped and undamped support conditions. Adding damping to the body weight support resulted in a greater increase of frontal plane stability. CONCLUSION: Adding mediolateral damping to 3-DoF body weight support systems is an effective method of increasing frontal plane stability during walking in able-bodied participants. Building on these results, adjustable mediolateral damping could enable therapists to select combinations of unloading and stability specifically for each patient and to adapt this in a task specific manner. This could extend the impact of transparent 3-DoF body weight support systems, enabling training of gait and active balance from an early time point onwards in the rehabilitation process for a wide range of mobility activities of daily life.

Impact of Surgical Margins in Breast Cancer After Preoperative Systemic Chemotherapy on Local Recurrence and Survival.

BACKGROUND: While "no tumour on ink" is an accepted margin width for R0 resection in primary surgery, it's unclear if it's oncologically safe after neoadjuvant chemotherapy (NAC). Only limited data demonstrate that surgery within new margins in cases of a pathological complete response (pCR) is safe. We therefore investigated the influence of different margins and pCR on local recurrence and survival rates after NAC. METHODS: We retrospectively analysed data of 406 women with invasive breast cancer, treated with NAC and breast-conserving therapy between 1994 and 2014 in two certified Austrian breast health centres. We compared R ≤ 1 mm, R > 1 mm and RX (pCR) for local recurrence-free survival (LRFS), disease-free survival (DFS) and overall survival (OS). RESULTS: After a median follow-up of 84.3 months, the 5-year LRFS (R ≤ 1 mm: 94.2%, R > 1 mm: 90.6%, RX: 95.0%; p = 0.940), the 5-year DFS (R ≤ 1 mm: 71.9%, R > 1 mm: 74.1%, RX: 87.2%; p = 0.245) and the 5-year OS (R ≤ 1 mm: 85.1%, R > 1 mm: 88.0%, RX: 96.4%; p = 0.236) did not differ significantly between narrow, wide, nor RX resections. Regarding DFS and OS, a negative nodal status reduced the hazard ratio significantly. CONCLUSION: There is no significant difference in LRFS, DFS and OS comparing close, wide or unknown margins after pCR. We suggest that resection in new margins after NAC is safe according to "no tumour on ink". Resection of the clipped area in cases of pCR is emphasized.

Robotic body weight support enables safe stair negotiation in compliance with basic locomotor principles.

BACKGROUND: After a neurological injury, mobility focused rehabilitation programs intensively train walking on treadmills or overground. However, after discharge, quite a few patients are not able to independently negotiate stairs, a real-world task with high physical and psychological demands and a high injury risk. To decrease fall risk and improve patients' capacity to navigate typical environments, early stair negotiation training can help restore competence and confidence in safe stair negotiation. One way to enable early training in a safe and permissive environment is to unload the patient with a body weight support system. We here investigated if unloaded stair negotiation complies with basic locomotor principles, in terms of enabling performance of a physiological movement pattern with minimal compensation. METHODS: Seventeen able-bodied participants were unloaded with 0-50% bodyweight during self-paced ascent and descent of a 4-tread staircase. Spatio-temporal parameters, joint ranges of motion, ground reaction forces and myoelectric activity in the main lower limb muscles of participants were compared between unloading levels. Likelihood ratio tests of separated linear mixed models of the investigated outcomes assessed if unloading affects the parameters in general. Subsequent post-hoc testing revealed which levels of unloading differed from unsupported stair negotiation. RESULTS: Unloading affected walking velocity, joint ranges of motion, vertical ground reaction force parameters and myoelectric activity in all investigated muscles for stair ascent and descent while step width and single support duration were only affected during ascent. A reduction with increasing levels of body weight support was seen in walking velocity (0.07-0.12 m/s), ranges of motion of the knee and hip (2-10°), vertical ground reaction force peaks (10-70%) and myoelectric activity (17-70%). An increase with unloading was only seen during ascent for ankle range of motion and tibialis anterior activity at substantial unloading. CONCLUSIONS: Body weight support facilitates stair negotiation by providing safety and support against gravity. Although unloading effects are present in most parameters, up to 30% body weight support these changes are small, and no dysfunctional patterns are introduced. Body weight support therefore fulfills all the necessary requirements for early stair negotiation training.

Experiences with the standardized classification of surgical complications (Clavien-Dindo) in general surgery patients.

BACKGROUND: The standardized Clavien-Dindo classification of surgical complications is applied as a simple and widely used tool to assess and report postoperative complications in general surgery. However, most documentation uses this classification to report surgery-related morbidity and mortality in a single field of surgery or even particular intervention. The aim of the present study was to present experiences with the Clavien-Dindo classification when applied to all patients on the general surgery ward of a tertiary referral care center. METHODS: We analyzed a period of 6 months of care on a ward with a broad range of general and visceral surgery. Discharge reports and patient charts were analyzed retrospectively and reported complications rated according to the most recent Clavien-Dindo classification version. The complexity of operations was assessed with the Austrian Chamber of Physicians accounting system. RESULTS: The study included 517 patients with 817 admissions, of whom 463 had been operated upon. Complications emerged in 12.5%, of which 19% were rated as Clavien I, 20.7% as Clavien II, 13.8% as Clavien IIIa, 27.6% as Clavien IIIb, 8.6% as Clavien IVa, and 10.3% as Clavien V. No Clavien grade IVb complication occurred within the investigation. Patients having undergone more complex surgery or with higher scores experienced significantly longer lengths of hospital stay. CONCLUSION: The Clavien-Dindo classification can easily be used to document complication rates in general surgery, even though this collective was not included in the original validation studies of Clavien et al. and consisted of more heavily impaired patients.

Preserved gait kinematics during controlled body unloading.

BACKGROUND: Body weight supported locomotor training was shown to improve walking function in neurological patients and is often performed on a treadmill. However, walking on a treadmill does not mimic natural walking for several reasons: absent self-initiation, less active retraction of leg required and altered afferent input. The superiority of overground training has been suggested in humans and was shown in rats demonstrating greater plasticity especially in descending pathways compared to treadmill training. We therefore developed a body weight support system allowing unrestricted overground walking with minimal interfering forces to train neurological patients. The present study investigated the influence of different amounts of body weight support on gait in healthy individuals. METHODS: Kinematic and electromyographic data of 19 healthy individuals were recorded during overground walking at different levels of body weight support (0, 10, 20, 30, 40, and 50%). Upper body inclination, lower body joint angles and multi-joint coordination as well as time-distance parameters were calculated. Continuous data were analyzed with regard to distinct changes within a gait cycle across all unloading conditions. RESULTS: Temporal gait parameters were most sensitive to changes in body unloading while spatial variables (step length, joint angles) showed modest responses when unloaded by as much as 50% body weight. The activation of the gastrocnemius muscle showed a gradual decrease with increasing unloading while the biceps femoris muscle showed increased activity levels at 50% unloading. These changes occurred during stance phase while swing phase activity remained unaltered. CONCLUSIONS: Healthy individuals were able to keep their walking kinematics strikingly constant even when unloaded by half of their body weight, suggesting that the weight support system permits a physiological gait pattern. However, maintaining a given walking speed using close-to-normal kinematics while being unloaded was achieved by adapting muscle activity patterns. Interestingly, the required propulsion to maintain speed was not achieved by means of increased gastrocnemius activity at push-off, but rather through elevated biceps femoris activity while retracting the leg during stance phase. It remains to be investigated to what extent neurological patients with gait disorders are able to adapt their gait pattern in response to body unloading.

Multidirectional transparent support for overground gait training.

Gait and balance training is an essential ingredient for locomotor rehabilitation of patients with neurological impairments. Robotic overhead support systems may help these patients train, for example by relieving them of part of their body weight. However, there are only very few systems that provide support during overground gait, and these suffer from limited degrees of freedom and/or undesired interaction forces due to uncompensated robot dynamics, namely inertia. Here, we suggest a novel mechanical concept that is based on cable robot technology and that allows three-dimensional gait training while reducing apparent robot dynamics to a minimum. The solution does not suffer from the conventional drawback of cable robots, which is a limited workspace. Instead, displaceable deflection units follow the human subject above a large walking area. These deflection units are not actuated, instead they are implicitly displaced by means of the forces in the cables they deflect. This leads to an underactuated design, because the deflection units cannot be moved arbitrarily. However, the design still allows accurate control of a three-dimensional force vector acting on a human subject during gait. We describe the mechanical concept, the control concept, and we show first experimental results obtained with the device, including the force control performance during robot-supported overground gait of five human subjects without motor impairments.

Modulation of spinal neuronal excitability by spinal direct currents and locomotion after spinal cord injury.

OBJECTIVE: Spinal neuronal function is impaired after a severe spinal cord injury (SCI) and can be assessed by the analysis of spinal reflex (SR) behavior. We applied transcutaneous spinal direct current stimulation (tsDCS) and locomotor activity, to determine whether the excitability of spinal neuronal circuitries underlying locomotion can be modulated after motor complete SCI. METHOD: SRs were evoked by non-noxious electrical stimulation of the tibial nerve. SR behavior was assessed before, immediately after, and 20 min after four different interventions (anodal, cathodal, sham tsDCS, or locomotion) in subjects with motor complete SCI and healthy subjects. RESULTS: SR amplitudes in SCI subjects were increased after anodal tsDCS by 84% (p < 0.05). Cathodal, sham tsDCS and locomotion had no influence on SR amplitudes. In addition, reflex threshold was lower after anodal tsDCS and locomotion in SCI subjects (p < 0.05). CONCLUSION: Anodal tsDCS is able to modulate spinal neuronal circuitries after SCI. SIGNIFICANCE: This novel, noninvasive approach might be used as a tool to excite spinal neuronal circuitries. If applied repetitively within a training approach, anodal tsDCS might prevent adverse alterations in spinal reflex function in severely affected SCI subjects, i.e., a manifestation of a spinal neuronal dysfunction taking part below the level of a spinal lesion.

Influence of spinal reflexes on the locomotor pattern after spinal cord injury.

In complete spinal cord injured (cSCI) subjects a shift from dominant early (60-120ms latency) to dominant late (120-450ms latency) spinal reflex (SR) components occurs over time after injury. This shift is assumed to reflect a spinal neuronal dysfunction below the level of a spinal lesion. The neuronal pathways of SR are suggested to be closely connected with spinal locomotor circuits. The aim of this study was to explore the influence of the two SR components on the electromyographic (EMG) pattern induced by assisted locomotion in cSCI subjects. Leg muscle EMG activity was analysed during assisted locomotion in both healthy and motor cSCI subjects. SR were evoked by non-noxious tibial nerve stimulation during mid-stance phase of the gait cycle. Early and late SR components had a differential influence on the locomotor pattern. In healthy and cSCI subjects with a dominant early SR component the locomotor EMG pattern was modulated in the form of a short increase in leg flexors activity in the stance phase (tibialis anterior, biceps femoris). In contrast, in chronic cSCI subjects with a dominant late SR component no activation in biceps femoris but a long-lasting activation of tibialis anterior and rectus femoris muscles during the stance phase was evoked. It is concluded that the same tibial nerve stimuli activated two different neuronal pathways, resulting in divergent interactions with spinal locomotor circuitries. It is proposed that the two SR components have different physiological roles during locomotion.

Characterization of neurological recovery following traumatic sensorimotor complete thoracic spinal cord injury.

STUDY DESIGN: Retrospective, longitudinal analysis of sensory, motor and functional outcomes from individuals with thoracic (T2-T12) sensorimotor complete spinal cord injury (SCI). OBJECTIVES: To characterize neurological changes over the first year after traumatic thoracic sensorimotor complete SCI. METHODS: A dataset of 399 thoracic complete SCI subjects from the European Multi-center study about SCI (EMSCI) was examined for neurological level, sensory levels and sensory scores (pin-prick and light touch), lower extremity motor score (LEMS), ASIA Impairment Scale (AIS) grade, and Spinal Cord Independence Measure (SCIM) over the first year after SCI. RESULTS: AIS grade conversions were limited. Sensory scores exhibited minimal mean change, but high variability in both rostral and caudal directions. Pin-prick and light touch sensory levels, as well as neurological level, exhibited minor changes (improvement or deterioration), but most subjects remained within one segment of their initial injury level after 1 year. Recovery of LEMS occurred predominantly in subjects with low thoracic SCI. The sensory zone of partial preservation (ZPP) had no prognostic value for subsequent recovery of sensory levels or LEMS. However, after mid or low thoracic SCI, ≥3 segments of sensory ZPP correlated with an increased likelihood for AIS grade conversion. CONCLUSION: The data suggest that a sustained deterioration of three or more thoracic sensory levels or loss of upper extremity motor function are rare events and may be useful for tracking the safety of a therapeutic intervention in early phase acute SCI clinical trials, if a significant proportion of study subjects exhibit such an ascent.

Neuronal dysfunction in chronic spinal cord injury.

This review describes the changes of spinal neuronal function that occur after a motor complete spinal cord injury (cSCI) in humans. In healthy subjects, polysynaptic spinal reflex (SR) evoked by non-noxious tibial nerve stimulation consists of an early SR component and rarely a late SR component. Soon after a cSCI, SR and locomotor activity are absent. After spinal shock; however, an early SR component re-appears associated with the recovery of locomotor activity in response to appropriate peripheral afferent input. Clinical signs of spasticity take place in the following months, largely as a result of non-neuronal changes. After around 1 year, the locomotor and SR activity undergo fundamental changes, that is, the electromyographic amplitude in the leg muscles during assisted locomotion exhaust rapidly, accompanied by a shift from early to dominant late SR components. The exhaustion of locomotor activity is also observed in non-ambulatory patients with an incomplete spinal cord injury (SCI). At about 1 year after injury, in most cSCI subjects the neuronal dysfunction is fully established and remains more or less stable in the following years. It is assumed that in chronic SCI, the patient's immobility resulting in a reduced input from supraspinal and peripheral sources leads to a predominance of inhibitory drive within spinal neuronal circuitries underlying locomotor pattern and SR generation. Training of spinal interneuronal circuits including the enhancement of an appropriate afferent input might serve as an intervention to prevent neuronal dysfunction after an SCI.

A hybrid tool for reaching and grasping rehabilitation: the ArmeoFES.

Many research groups are currently working with robotic devices for hand grasp rehabilitation and restoration. A common problem in this area is the fact that existing and commercially available robotic exoskeletons are able to provide gravity compensation of the shoulder and elbow but do not provide any support for the grasping and releasing movements of the hand. The lack of a flexible support technology for the hand reduces the possible ways in which clinicians can deal with the issue of a personalized, effective rehabilitation. This paper presents new software that allows FES assisted grasping to integrate with the ArmeoSpring (Hocoma AG). The system uses a Man-In-The-Loop control approach, whereby surface EMG signals from proximal muscles are used to trigger and modulate multichannel FES applied to distal muscles, thus allowing patient induced and strength adapted movement control of the hand. Combining volitionally controlled FES with arm-weight-compensation allows early adoption of FES assisted therapy for patients, augmenting their functionalities and extending training capabilities with the ArmeoSpring.

Modulation of spinal reflex by assisted locomotion in humans with chronic complete spinal cord injury.

OBJECTIVE: In healthy subjects, spinal reflexes (SR) evoked by non-noxious tibial nerve stimulation consist of an early (60-120ms latency) and an occasional late-appearing (120-450ms latency) component in the ipsilateral tibialis anterior. In chronic (>1year) complete spinal cord injured (cSCI) subjects early components are small or lacking while late components are dominant. Here we report on the modulation of SR by assisted locomotion in healthy and chronic motor cSCI subjects. METHODS: SR was evoked by tibial nerve stimulation at the terminal stance phase during assisted locomotion and was compared to SR recorded during upright stance. RESULTS: In chronic cSCI subjects only a late SR component was consistently present during upright stance. However during assisted locomotion, an early SR component appeared, while amplitude of the late SR component became small. In contrast, in healthy subjects the early SR component dominated in all conditions, but a small late component appeared during assisted locomotion. CONCLUSION: A more balanced activity of early and late SR components occurred in both subject groups if an appropriate proprioceptive input was provided. SIGNIFICANCE: Early and late SR components are assumed to reflect the activity of separate neuronal circuits, which are associated with the locomotor circuitry possibly by shaping the pattern.

Changes in spinal reflex and locomotor activity after a complete spinal cord injury: a common mechanism?

Locomotor activity and spinal reflexes (SRs) show common features in different mammals, including humans. Here we report the time-course of the development of locomotor activity and SRs after a complete spinal cord injury in humans. SRs evoked by tibial nerve stimulation were studied, as was the leg muscle electromyography activity evoked by mechanically assisted locomotion (Lokomat) in biceps femoris, rectus femoris, tibialis anterior and gastrocenmius medialis. Around 8 weeks after the injury, an early SR component (latency 60-120 ms) appeared, as in healthy subjects, and a well-organized leg muscle activity was present during assisted locomotion. At around 6 months after injury an additional, late reflex component (latency 120-450 ms) appeared, which remained even 15 years after the spinal cord injury. In contrast, the early component had markedly decreased at 18 months after injury. These changes in SR were associated with a loss of electromyography activity and a successively stronger electromyography exhaustion (i.e. decline of electromyography amplitude), when comparing the level of electromyography activity at 2 and 10 min, respectively, during assisted locomotion. These changes in electromyography activity affected mainly the biceps femoris, gastrocenmius medialis and tibialis anterior but less so the rectus femoris. When the amplitude relationship of the early to late SR component was calculated, there was a temporal relationship between the decrease of the early component and an increase of the late component and the degree of exhaustion of locomotor activity. In chronic, severely affected but sensori-motor incomplete spinal cord injury subjects a late SR component, associated with an electromyography exhaustion, was present in subjects who did not regularly perform stepping movements. Our data are consistent with the proposal of a common mechanism underlying the changes in SR activity and locomotor activity after spinal cord injury. These findings should be taken into consideration in the development of novel rehabilitation schemes and programs to facilitate regeneration-inducing therapies in spinal cord injury subjects.

Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2.

Neuroligins (NL) are postsynaptic cell adhesion molecules that are thought to specify synapse properties. Previous studies showed that mutant mice carrying an autism-associated point mutation in NL3 exhibit social interaction deficits, enhanced inhibitory synaptic function and increased staining of inhibitory synaptic puncta without changes in overall inhibitory synapse numbers. In contrast, mutant mice lacking NL2 displayed decreased inhibitory synaptic function. These studies raised two relevant questions. First, does NL2 deletion impair inhibitory synaptic function by altering the number of inhibitory synapses, or by changing their efficacy? Second, does this effect of NL2 deletion on inhibition produce behavioral changes? We now show that although NL2-deficient mice exhibit an apparent decrease in number of inhibitory synaptic puncta, the number of symmetric synapses as determined by electron microscopy is unaltered, suggesting that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers. This decrease in inhibitory synaptic function in NL2-deficient mice correlates with a discrete behavioral phenotype that includes a marked increase in anxiety-like behavior, a decrease in pain sensitivity and a slight decrease in motor co-ordination. This work confirms that NL2 modulates inhibitory synaptic function and is the first demonstration that global deletion of NL2 can lead to a selective behavioral phenotype.

Modulation of locomotor activity in complete spinal cord injury.

The aim of this study was to evaluate the modulation of muscle activity during locomotor-like movements by different walking speeds in subjects with a motor complete spinal cord injury (SCI) compared to actively--and passively-walking control subjects without neurological deficit. Stepping movements on a treadmill were induced and assisted by a driven gait orthosis. Electromyographic (EMG) muscle activity of one leg (rectus and biceps femoris, tibialis anterior and gastrocnemius) was recorded and analyzed at three stepping velocities with similar body weight support in both subject groups. In SCI subjects, the EMG amplitude of biceps femoris, tibialis anterior and gastrocnemius was in general similar or weaker than in passively- and actively-stepping control subjects, but that of rectus femoris was larger. The degree of co-activation between tibialis anterior and gastrocnemius was higher in SCI than in control subjects. A significant velocity-dependent EMG modulation was present in all four-leg muscles in both subject groups. In SCI subjects, this EMG modulation was similar to that in actively stepping control subjects. It is concluded that in complete spastic SCI subjects, spinal neuronal circuits underlying locomotion can to a large extent adequately respond to a change in external drive to adapt the neuronal pattern to a new locomotion speed. The application of various speeds might enhance the effect of locomotor training in incomplete SCI subjects.

Molecular and cellular permeability control at the blood-brain barrier.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells. These cells have at least three properties which distinguish them from their peripheral counterparts: (1) tight junctions (TJs) of extremely low permeability; (2) low rates of fluid-phase endocytosis; (3) specific transport and carrier molecules. In combination, these features restrict the nonspecific flux of ions, proteins, and other substances into the central nervous system (CNS) environment. The restriction protects neurons from harmful compositional fluctuations occurring in the blood and allows uptake of essential molecules. Breakdown of the BBB is associated with a variety of CNS disorders and results in aggravation of the condition. Restoration of the BBB is thus one strategy during therapy of CNS diseases. Its success depends on a precise knowledge of the structural and functional principles underlying BBB functionality. In this review we have tried to summarise the current knowledge of TJs, including information gained from non-neuronal systems, and describe selected mechanisms involved in permeability regulation.

Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-kappaB activation.

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.

Identification of a novel neuroligin in humans which binds to PSD-95 and has a widespread expression.

Neuroligins, first discovered in rat brain, form a family of three synaptically enriched membrane proteins. Using reverse transcription-PCR of human brain polyadenylated RNA and extensive database searches, we identified the human homologues of the three rat neuroligins and a cDNA encoding a fourth member, which we named neuroligin 4. Neuroligin 4 has 63-73% amino acid identity with the other members of the human neuroligin family, and the same predicted domain structure. DNA database analyses, furthermore, indicated that a possible fifth neuroligin gene may be present in the human genome. Northern-blot analysis revealed expression of neuroligin 4 in heart, liver, skeletal muscle and pancreas, but barely at all in brain. Overexpression of neuroligin 4 cDNA in COS-7 cells led to the production of a 110 kDa protein. Immunofluorescence analysis demonstrated that the protein was integrated into the plasma membrane. Overexpression of cDNAs encoding neuroligin 4 and the PDZ-domain protein, PSD-95, in COS-7 cells resulted in the formation of detergent-resistant complexes. Neuroligin 4 did not bind to ZO-1, another PDZ-domain protein. Together, our data show that the human neuroligin family is composed of at least one additional member, and suggest that neuroligin 4 may also be produced outside the central nervous system.

Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain.

Further genetic evidence is provided here that Bradyrhizobium japonicum possesses a mitochondria-like electron-transport pathway: 2[H]----UQ----bc1----c----aa3----O2. Two Tn5-induced mutants, COX122 and COX132, having cytochrome c oxidase-negative phenotypes, were obtained and characterized. Mutant COX122 was defective in a novel gene, named cycM, which was responsible for the synthesis of a c-type cytochrome with an Mr of 20,000 (20K). This 20K cytochrome c appeared to catalyse electron transport from the cytochrome bc1 complex to the aa3-type terminal oxidase and, unlike mitochondrial cytochrome c, was membrane-bound in B. japonicum. The Tn5 insertion of mutant COX132 was localized in coxA, the structural gene for subunit I of cytochrome aa3. This finding also led to the cloning and sequencing of the corresponding wild-type coxA gene that encoded a 541-amino-acid protein with a predicted Mr of 59,247. The CoxA protein shared about 60% sequence identity with the cytochrome aa3 subunit I of mitochondria. The B. japonicum cycM and coxA mutants were able to fix nitrogen in symbiosis with soybean (Fix+). In contrast, mutants described previously which lacked the bc1 complex did not develop into endosymbiotic bacteroids and were thus Fix-. The data suggest that a symbiosis-specific respiratory chain exists in B. japonicum in which the electrons branch off at the bc1 complex.