Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

Hemoglobin and cerebral hypoxic vasodilation in humans:Evidence for nitric oxide-dependent and S-nitrosothiol mediated signal transduction

Cerebral hypoxic vasodilation is poorly understood in humans, which undermines the development of therapeutics to optimize cerebral oxygen delivery. Across four investigations (total n = 195) we investigated the role of nitric oxide (NO) and hemoglobin-based S-nitrosothiol (RSNO) and nitrite ((Formula presented.)) signaling in the regulation of cerebral hypoxic vasodilation. We conducted hemodilution (n = 10) and NO synthase inhibition experiments (n = 11) as well as hemoglobin oxygen desaturation protocols, wherein we measured cerebral blood flow (CBF), intra-arterial blood pressure, and in subsets of participants trans-cerebral release/uptake of RSNO and (Formula presented.). Higher CBF during hypoxia was associated with greater trans-cerebral RSNO release but not (Formula presented.), while NO synthase inhibition reduced cerebral hypoxic vasodilation. Hemodilution increased the magnitude of cerebral hypoxic vasodilation following acute hemodilution, while in 134 participants tested under normal conditions, hypoxic cerebral vasodilation was inversely correlated to arterial hemoglobin concentration. These studies were replicated in a sample of polycythemic high-altitude native Andeans suffering from excessive erythrocytosis (n = 40), where cerebral hypoxic vasodilation was inversely correlated to hemoglobin concentration, and improved with hemodilution (n = 6). Collectively, our data indicate that cerebral hypoxic vasodilation is partially NO-dependent, associated with trans-cerebral RSNO release, and place hemoglobin-based NO signaling as a central mechanism of cerebral hypoxic vasodilation in humans.</p

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Balance recovery following pelvis perturbations during very slow walking

BACKGROUND AND AIM: Healthy humans have the ability to handle balance perturbations during walking very well. The ankle moment, as well as the foot placement location and timing are altered to counteract the perturbations and maintain balance.[1] Previously, healthy subjects have shown a strong linear relation between the body´s centre of mass (COM) velocity at heel contact (HC), and both the foot placement location and centre of pressure (COP) at subsequent toe off (TO) during laterally perturbed walking.[2] The walking speeds were 0.63 and 1.25 m/s.[2] In this study, it is questioned whether this relation also exist during very slow walking, because there will be more time during the double support phase to alter the balance recovery strategy. Therefore, we investigated the relation between the body´s COM velocity, and both the foot placement location and COP during a very slow walking speed. METHODS: Mediolateral (ML) pelvis perturbations were applied to 10 healthy subjects, during very slow (0.36 m/s) and normal (1.25 m/s) treadmill walking at TO of the right foot. An active optical motion capture system was used to record the body kinematics. Ground reaction forces were measured with the built-in force plates in the treadmill. The data was analysed to obtain COM velocities at HC right, foot placement location at HC right, COP locations at TO left and phase durations. RESULTS: Figure 1 presents the durations of the double and single support phases for the different perturbation magnitudes. The ML perturbations significantly affected the double and single support durations during very slow walking, while these durations were not affected during normal walking. Additionally, the COM velocity at HC right showed to have a high predictive value for the foot placement of the leading foot during the normal walking speed, whereas this was considerable lower during the very slow walking speed. The predictive value of the COM velocity was present for the COP location at the subsequent TO for both the normal and very slow walking speed. CONCLUSIONS: The results showed altered recovery strategies in the frontal plane during very slow walking compared to the normal walking speed. These differences were potentially caused by the longer double support phase duration, in which subjects used other strategies to control the distance between the COM and COP. REFERENCES:[1] A. L. Hof, R. M. van Bockel, T. Schoppen, and K. Postema, "Control of lateral balance in walking. Experimental findings in normal subjects and above-knee amputees," Gait Posture, vol. 25, no. 2, pp. 250-258, 2007. [2] M. Vlutters, E. H. F. van Asseldonk, and H. van der Kooij, "Center of mass velocity-based predictions in balance recovery following pelvis perturbations during human walking," J. Exp. Biol., vol. 219, no. 10, pp. 1514-1523,2016

Recovering linear and angular momentum during walking

For most individuals maintaining balance during walking goes natural. We are not continuously thinking of how to maintain balance, and still we usually do not fall. However, maintaining balance is not a given for everyone. Both aging and various neuromuscular disorders affect the ability to maintain balance, resulting in an increased incidence of falls and the associated consequences. Also, in the situation of someone walking with an assistive device such as a powered lower-limb exoskeleton, for example due to a spinal cord injury (SCI), maintaining balance may be challenging. To provide better care and training programs and to improve balance support with assistive devices, a better understanding is needed of human balance recovery. Previous research often focused on the recovery of linear perturbations disturbing the body's linear momentum. However, in daily life we also encounter perturbations resulting in a rotational effect, disturbing the body's angular momentum. The aim of this work was to gain insights in the use of human balance strategies to recover whole-body linear and angular momentum. To provoke balance recovery responses we performed experiments in which perturbations were applied during standing and treadmill walking. These studies were performed with healthy participants, since they can serve as a source of inspiration for how balance recovery strategies are being used successfully. Perturbations of the linear and/or angular momentum were induced by the application of forces to the body at shoulder and/or pelvis height, provided by a haptic robot. This allowed for a controlled duration, magnitude and onset of the perturbations. Thereafter we analyzed how modulations of the ground reaction force and its point of application were used in order to maintain balance. We studied this in 1) situations that are relevant for SCI individuals walking with a powered lower limb exoskeleton and 2) situations that have not extensively been studied before, and therefore fill a gap in the current knowledge on human balance recovery. In several chapters we address the topic of balance recovery during very slow walking, since this is a relevant speed for walking with a lower limb exoskeleton. Walking very slowly increases the time spent in the double support phase. Studying the responses to perturbations of the whole-body linear momentum (WBLM) while standing in a static double support phase, also called a staggered stance posture, provided insights in the coupling between the frontal- and sagittal-plane. A large base of support (BoS) enables opportunities for centre of pressure (CoP) modulation. Therefore, the large dimension of the BoS in the anteroposterior direction during staggered stance could also be used in the recovery from perturbations that were perpendicular to this direction. Focusing on the double support phase, with simulations based on a simple linear inverted pendulum model, we showed the effects of modulations of the CoP trajectory on the control of the centre of mass position and velocity. Comparing the simulated opportunities with the strategies that healthy individuals used, it turned out that we do not fully exploit the available options for a quick balance recovery. A specific type of perturbation that we used for several studies is a perturbation of the whole-body angular momentum (WBAM). This was obtained by applying two perturbations at the same time in opposite direction on the pelvis and upper body respectively. The responses to these perturbations revealed a high priority in recovery of the WBAM. This was done even at the expense of the WBLM. The WBAM recovery comprised a modulation of the horizontal ground reaction force, affecting the WBLM while this was not perturbed initially. This effect was independent of the instant of the gait cycle at which the perturbation was given and holds for very slow and normal walking speeds. The results emphasize the importance and prioritization of WBAM regulation in balance recovery. To conclude, the studies presented in this thesis provide insights into the human balance strategies used to recover from perturbations of the WBLM and WBAM during walking at very low and normal speeds. These insights can be considered in the development of controllers to assist balance or to improve balance training for those experiencing difficulties with balance control. Finally, the recorded data itself is valuable for validating whether proposed recovery strategies are human-like

Balance recovery in the double support during perturbed walking

I. INTRODUCTION Exoskeleton walking increases the relative time spent in the double support phase (DSP). It is therefore crucial to control balance when both feet are on the ground. Healthy humans have excellent balance capabilities to avoid falling. The centre of pressure (CoP) describes the control of the centre of mass (CoM) movement [1]. The range of possible CoP locations in the DSP is determined by the foot placement at the end of the preceding single support phase. This study focuses on the CoP modulation during the DSP in the control of the CoM state. II. METHODS CoP trajectories in response to pelvis perturbations were extracted from an existing data set by Vlutters et al [2]. Anteroposterior and mediolateral perturbations with magnitudes up to 16% of the body weight were given at the moment of toe off. Parameterized CoP trajectories were generated with a spline function based on the experimental CoP trajectories, examples are shown in figure 1. Parameterization was done as a function of 1) the duration of the DSP, 2) the amplitude of the CoP, and 3) percentage of the amplitude reached halfway the DSP (= midpoint). The parameters were varied within a range equal to the standard deviation around the mean value obtained from the experimental data. The generated trajectories were used in model simulations of the CoM during the first DSP following the perturbation. A simple inverted pendulum model, relating the horizontal distance between the CoP and CoM to CoM acceleration, was used to assess the effectiveness of the CoP modulation in counteracting perturbation induced CoM velocity changes [3]. III. RESULTS The model outcome corresponds with the experimental data, figure 1. All the three CoP parameters are linearly related to the change in CoM velocity over the DSP, in both the experimental and modelled data. Changes of the midpoint resulted in larger variations in the modelled Δ CoM velocity, compared to those resulting from changes in the duration or amplitude, see figure 2. IV. DISCUSSION A simple inverted pendulum model was able to model representative CoM trajectories from the generated CoP trajectories as input. To control the CoM velocity after a perturbation, subjects used all CoP parameters. However, in the experimental data these parameters were also related with each other. When uncoupling the effect of these parameters in the model, the shape of the CoP trajectory, represented by the CoP shift that is reached halfway the DSP, had the largest influence on the changes of the CoM velocity during the DSP. Shifting the load earlier or later to the leading leg helps in increasing or decreasing the CoM velocity. This will help in counteracting the effect of the perturbation and returning to the baseline CoM velocity. REFERENCES [1] H. Reimann, T. D. Fettrow, E. D. Thompson, P. Agada, B. J. McFadyen, and J. J. Jeka, “Complementary mechanisms for upright balance during walking,” PLoS One, vol. 12, no. 2, pp. 1–16, 2017. [2] M. Vlutters, E. H. F. van Asseldonk, and H. van der Kooij, “Center of mass velocity-based predictions in balance recovery following pelvis perturbations during human walking,” J. Exp. Biol., vol. 219, no. 10, pp. 1514–1523, 2016. [3] Y. Jian, D. Winter, M. Ishac, and L. Gilchrist, “Trajectory of the body COG and COP during initiation and termination of gait,” Gait Posture, vol. 1, no. 1, pp. 9–2