Gait Abnormalities in Parkinson's Disease Are Associated with Extracellular Free-Water Characteristics in the Substantia Nigra.

BACKGROUND: Gait impairments are common in Parkinson's disease (PD). The pathological mechanisms are complex and not thoroughly elucidated, thus quantitative and objective parameters that closely relate to gait characteristics are critically needed to improve the diagnostic assessments and monitor disease progression. The substantia nigra is a relay structure within basal ganglia brainstem loops that is centrally involved in gait modulation. OBJECTIVE: We tested the hypothesis that quantitative gait biomechanics are related to the microstructural integrity of the substantia nigra and PD-relevant gait abnormalities are independent from bradykinesia-linked speed reductions. METHODS: Thirty-eight PD patients and 33 age-matched control participants walked on a treadmill at fixed speeds. Gait parameters were fed into a principal component analysis to delineate relevant features. We applied the neurite orientation dispersion and density imaging (NODDI) model on diffusion-weighted MR-images to calculate the free-water content as an advanced marker of microstructural integrity of the substantia nigra and tested its associations with gait parameters. RESULTS: Patients showed increased duration of stance phase, load response, pre-swing, and double support time, as well as reduced duration of single support and swing time. Gait rhythmic alterations associated positively with the free-water content in the right substantia nigra in PD, indicating that patients with more severe neurodegeneration extend the duration of stance phase, load response, and pre-swing. CONCLUSION: The results provide evidence that gait alterations are not merely a byproduct of bradykinesia-related reduced walking speed. The data-supported association between free-water and the rhythmic component highlights the potential of substantia nigra microstructure imaging as a measure of gait-dysfunction and disease-progression.

ADABase: A Multimodal Dataset for Cognitive Load Estimation.

Driver monitoring systems play an important role in lower to mid-level autonomous vehicles. Our work focuses on the detection of cognitive load as a component of driver-state estimation to improve traffic safety. By inducing single and dual-task workloads of increasing intensity on 51 subjects, while continuously measuring signals from multiple modalities, based on physiological measurements such as ECG, EDA, EMG, PPG, respiration rate, skin temperature and eye tracker data, as well as behavioral measurements such as action units extracted from facial videos, performance metrics like reaction time and subjective feedback using questionnaires, we create ADABase (Autonomous Driving Cognitive Load Assessment Database) As a reference method to induce cognitive load onto subjects, we use the well-established n-back test, in addition to our novel simulator-based k-drive test, motivated by real-world semi-autonomously vehicles. We extract expert features of all measurements and find significant changes in multiple modalities. Ultimately we train and evaluate machine learning algorithms using single and multimodal inputs to distinguish cognitive load levels. We carefully evaluate model behavior and study feature importance. In summary, we introduce a novel cognitive load test, create a cognitive load database, validate changes using statistical tests, introduce novel classification and regression tasks for machine learning and train and evaluate machine learning models.

Validating sweat ammonia as physiological parameter for wearable devices in sports science.

The measurement of physiological parameters in sweat has long been assumed to offer a non-invasive alternative to conventional blood testing. Recently, advances in sensor technology enable the production of printed sweat sensors applicable for the use in wearable devices. However, the remaining challenge is the determination of the physiological correlation between blood and sweat components. In this study, we conducted ammonia measurements in blood and sweat during a stepwise incremental cycle ergometer test in 40 subjects under completely controlled conditions in a clinical environment to determine the correlation between the ammonium concentrations in blood and sweat. Samples were taken for each workload step separately. Sweat was sampled directly from the upper body, blood was taken from an indwelling cannula at the end of each workload step, respectively. For meaningful classification of the measured quantities, blood lactate and heart rate were monitored additionally. The results for blood ammonium concentration show increasing behavior in good accordance with the established indicators for physical exhaustion, whereas sweat ammonium concentration seems to decrease with workload. This is found to be due to dilution, as sweat rate increases. The presented results provide insight in the correlation between blood and sweat parameters and therefore are of high importance for further development of wearable devices.Clinical Relevance-Sweat sensing opens up new possibilities for non-invasive, continuous in-situ monitoring of physiological parameters for healthcare and sports science applications.

Three-dimensional force microscopy of cells in biopolymer networks.

We describe a technique for the quantitative measurement of cell-generated forces in highly nonlinear three-dimensional biopolymer networks that mimic the physiological situation of living cells. We computed forces of MDA-MB-231 breast carcinoma cells from the measured network deformations around the cells using a finite-element approach based on a constitutive equation that captures the complex mechanical properties of diverse biopolymers such as collagen gels, fibrin gels and Matrigel. Our measurements show that breast carcinoma cells cultured in collagen gels generated nearly constant forces regardless of the collagen concentration and matrix stiffness. Furthermore, time-lapse force measurements showed that these cells migrated in a gliding motion with alternating phases of high and low contractility, elongation, migratory speed and persistence.

Alginate-based hydrogels with improved adhesive properties for cell encapsulation.

Hydrogel-based biomaterials are ideal scaffolding matrices for microencapsulation, but they need to be modified to resemble the mechanical, structural and chemical properties of the native extracellular matrix. Here, we compare the mechanical properties and the degradation behavior of unmodified and modified alginate hydrogels in which cell adhesive functionality is conferred either by blending or covalently cross-linking with gelatin. Furthermore, we measure the spreading and proliferation of encapsulated osteoblast-like MG-63 cells. Alginate hydrogels covalently crosslinked with gelatin show the highest degree of cell adhesion, spreading, migration, and proliferation, as well as a faster degradation rate, and are therefore a particularly suitable material for microencapsulation.

Biphasic response of cell invasion to matrix stiffness in three-dimensional biopolymer networks.

When cells come in contact with an adhesive matrix, they begin to spread and migrate with a speed that depends on the stiffness of the extracellular matrix. On a flat surface, migration speed decreases with matrix stiffness mainly due to an increased stability of focal adhesions. In a three-dimensional (3-D) environment, cell migration is thought to be additionally impaired by the steric hindrance imposed by the surrounding matrix. For porous 3-D biopolymer networks such as collagen gels, however, the effect of matrix stiffness on cell migration is difficult to separate from effects of matrix pore size and adhesive ligand density, and is therefore unknown. Here we used glutaraldehyde as a crosslinker to increase the stiffness of self-assembled collagen biopolymer networks independently of collagen concentration or pore size. Breast carcinoma cells were seeded onto the surface of 3-D collagen gels, and the invasion depth was measured after 3 days of culture. Cell invasion in gels with pore sizes >5 µm increased with higher gel stiffness, whereas invasion in gels with smaller pores decreased with higher gel stiffness. These data show that 3-D cell invasion is enhanced by higher matrix stiffness, opposite to cell behavior in two dimensions, as long as the pore size does not fall below a critical value where it causes excessive steric hindrance. These findings may be important for optimizing the recellularization of soft tissue implants or for the design of 3-D invasion models in cancer research.

Temporal correction of detected R-peaks in ECG signals: A crucial step to improve QRS detection algorithms.

In the last decade the interest for heart rate variability analysis has increased tremendously. Related algorithms depend on accurate temporal localization of the heartbeat, e.g. the R-peak in electrocardiogram signals, especially in the presence of arrhythmia. This localization can be delivered by numerous solutions found in the literature which all lack an exact specification of their temporal precision. We implemented three different state-of-the-art algorithms and evaluated the precision of their R-peak localization. We suggest a method to estimate the overall R-peak temporal inaccuracy-dubbed beat slackness-of QRS detectors with respect to normal and abnormal beats. We also propose a simple algorithm that can complement existing detectors to reduce this slackness. Furthermore we define improvements to one of the three detectors allowing it to be used in real-time on mobile devices or embedded hardware. Across the entire MIT-BIH Arrhythmia Database, the average slackness of all the tested algorithms was 9ms for normal beats and 13ms for abnormal beats. Using our complementing algorithm this could be reduced to 4ms for normal beats and to 7ms for abnormal beats. The presented methods can be used to significantly improve the precision of R-peak detection and provide an additional measurement for QRS detector performance.

Activation and regulation of endogenous retroviral genes in the human pituitary gland and related endocrine tumours.

AIMS: Adenohypophysis (AH) hormone-producing cells represent the origin of diverse groups of pituitary adenomas (PA). Deregulation of hypothalamic hormone receptors, growth factors and cAMP signalling have been implicated in the aetiology of PA. Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections and represent more than 8% of the human genome. Some ERV genes encode open reading frames and produce functional proteins, for example, the ERVW-1 envelope gene Syncytin-1, essential for placentogenesis, but also deregulated in human tumours. Data concerning ERV expression in the AH and related endocrine tumours are missing. METHODS: Syncytin-1 protein was analysed in normal AH (n = 15) and compared with five PA subtypes (n = 117) by immunohistochemistry. Absolute gene expression of 20 ERV functional envelope genes and ERVW-5 gag was measured. PA tissues were examined for Syncytin-1 and the cAMP signalling marker phospho-CREB-Ser133 using immunohistochemistry. Isolated primary human PA cells were treated with different hormones. Murine embryonic and adult pituitary gland ERV expressions were compared with human AH. RESULTS: Syncytin-1 protein colocalized with corticotropic cells of AH. In contrast, all PA demonstrated significant Syncytin-1 protein overexpression, supporting deregulation. All other ERV genes showed significant up-regulations in different PA subtypes. Phospho-CREB-Ser133 and Syncytin-1 colocalized in PA cells. Cultivated primary PA cells with ACTH or CRH induced their respective receptors and ERV genes. Syncytin-A/-B, murine orthologues to human Syncytin-1/-2, localized to embryonic and adult pituitary glands demonstrating functional mammalian conservation. CONCLUSIONS: Deregulated ERV genes may contribute to PA development via cAMP signalling.

Estimating the 3D pore size distribution of biopolymer networks from directionally biased data.

The pore size of biopolymer networks governs their mechanical properties and strongly impacts the behavior of embedded cells. Confocal reflection microscopy and second harmonic generation microscopy are widely used to image biopolymer networks; however, both techniques fail to resolve vertically oriented fibers. Here, we describe how such directionally biased data can be used to estimate the network pore size. We first determine the distribution of distances from random points in the fluid phase to the nearest fiber. This distribution follows a Rayleigh distribution, regardless of isotropy and data bias, and is fully described by a single parameter--the characteristic pore size of the network. The bias of the pore size estimate due to the missing fibers can be corrected by multiplication with the square root of the visible network fraction. We experimentally verify the validity of this approach by comparing our estimates with data obtained using confocal fluorescence microscopy, which represents the full structure of the network. As an important application, we investigate the pore size dependence of collagen and fibrin networks on protein concentration. We find that the pore size decreases with the square root of the concentration, consistent with a total fiber length that scales linearly with concentration.

Regulation of murine placentogenesis by the retroviral genes Syncytin-A, Syncytin-B and Peg10.

The murine placenta has a trichorial structure with two multinucleated syncytiotrophoblast (SCT) layers representing a barrier between the maternal and fetal blood system. Genes of endogenous retroviruses and retrotransposon-derived paternally expressed genes (Peg), remnants of past infections and integrations in the genome, have essential functions in placentogenesis. Previous studies showed that the envelope genes Syncytin-A and Syncytin-B were essential for cell-cell fusion of the SCT. The goal of this study was to analyze the temporal localization and expression of nine genes throughout placental development from embryonic day (E)8.5 to E18.5 using in situ-hybridization and absolute RNA-quantification. These included a comparison of previously characterized genes from the labyrinth Syncytin-A, Syncytin-B, Gcm1, the junctional zone PL-1, PL-2, Plf, Tpbpa with two further characterized genes Peg10 and Tpbpb. Syncytin-A and Syncytin-B RNA localized to SCT-I and SCT-II, respectively. Peg10 RNA localized to all extraembryonic tissues, specifically to the parietal and sinusoidal TGC of the labyrinth layer, which is in contact with SCT-I and the maternal blood. All three retroviral/retrotransposon-derived genes showed the highest expression at E16.5, but Peg10 with 188,917.1 molecules/ng cDNA was 208-fold and 106.8-fold higher expressed than Syncytin-A and Syncytin-B, respectively. Tpbpb localized to the junctional zone and showed the highest expression at E16.5 along with PL-2, Plf, Tpbpa, but not PL-1, which decreased in expression at E10.5. To investigate a role of Syncytin-A, Syncytin-B and Peg10 in cell-cell fusion, we established a cell culture system with fractionated primary trophoblasts from murine placentae. Culturing trophoblasts for up to 72h partly resembled trophoblast development in vivo according to the nine marker genes. Knockdown of Syncytin-A demonstrated a functional regulation of cell-cell fusion, where knockdown of Peg10 showed no involvement in cell fusion. Due to the expression of Peg10 in TGCs, we propose an essential functional role in the fetal-maternal blood system.

Parameter-free binarization and skeletonization of fiber networks from confocal image stacks.

We present a method to reconstruct a disordered network of thin biopolymers, such as collagen gels, from three-dimensional (3D) image stacks recorded with a confocal microscope. The method is based on a template matching algorithm that simultaneously performs a binarization and skeletonization of the network. The size and intensity pattern of the template is automatically adapted to the input data so that the method is scale invariant and generic. Furthermore, the template matching threshold is iteratively optimized to ensure that the final skeletonized network obeys a universal property of voxelized random line networks, namely, solid-phase voxels have most likely three solid-phase neighbors in a 3 x 3 x 3 neighborhood. This optimization criterion makes our method free of user-defined parameters and the output exceptionally robust against imaging noise.

Tyrosine phosphorylation of vinculin at position 1065 modifies focal adhesion dynamics and cell tractions.

Focal adhesions (FAs) connect the cellular actin cytoskeleton via integrin with the extracellular matrix. They comprise of many structural and signaling proteins which are highly dynamic, well regulated, and responsible for the sensing of physical properties from the environment. Vinculin is a protein that incorporates all these functions. Here, we investigated the phosphorylation of Y1065 in the activation/regulation of vinculin. We used different vinculin mutants mimicking either a permanently activated or inhibited phosphorylation site at position 1065. Using these mutants, we determined their influence on the exchange dynamics and cell forces using fluorescence recovery after photobleaching and traction microscopy. The results indicate that phosphorylation at Y1065 significantly increases the amount of freely exchanging vinculin within FAs whereas inhibition of this phosphorylation site leads to an uncontrolled exchange of vinculin and reduced adhesive cell forces. In conclusion, we show that phosphorylation on position Y1065 is essential for accurate incorporation of vinculin into FAs and mechanical behavior of cells.