Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

Evaluating a physiologically based pharmacokinetic model for prediction of omeprazole clearance and assessing ethnic sensitivity in CYP2C19 metabolic pathway.

PURPOSE: The purpose of this study is to evaluate the ethnicity-specific population models in the SimCYP Simulator® for prediction of omeprazole clearance with attention to differences in the CYP2C19 metabolic pathway. METHODS: The SimCYP® models incorporating Caucasian, Chinese, and Japanese population-specific demographic, physiological, and enzyme data were applied to simulate omeprazole pharmacokinetics. Published pharmacokinetic data of omeprazole after intravenous or oral administration in Caucasian, Chinese, and Japanese were used for the evaluation. RESULTS: Following oral administration, the ratio of the predicted to observed geometric mean of omeprazole clearance in Caucasian extensive metabolizers (EMs) was 0.88. The ratios in Chinese EMs were 1.16 and 0.99 after intravenous and oral administration, respectively. The ratios in Japanese EMs were 0.88 and 0.71 after intravenous and oral administration, respectively. Significant differences (2-fold) in the observed oral clearance of omeprazole were identified between Caucasian and Asian (Chinese and Japanese) EMs while the observed oral and intravenous clearances of omeprazole were similar between Chinese and Japanese EMs. Physiologically based pharmacokinetics (PBPK) models within SimCYP accurately predicted the difference in the observed oral clearance between Caucasian and Chinese EMs but overpredicted the difference between Caucasians and Japanese EMs due to under-prediction of oral clearance in Japanese EMs. CONCLUSIONS: The PBPK model within SimCYP adequately predicted omeprazole clearance in Caucasian, Chinese, and Japanese EMs and the 2-fold differences in clearance of omeprazole between Caucasian and Asian EMs. This may lead to early identification of ethnic sensitivity in clearance and the need for different dosing regimens in a specific ethnic group for substrates of CYP2C19 which can support the rational design of bridging clinical trials.

Multi-chromatic control of mammalian gene expression and signaling.

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

IL-1α reversibly inhibits skeletal muscle ryanodine receptor. a novel mechanism for critical illness myopathy?

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in "skinned" muscle fibers using force transducers and confocal Ca(2+) fluorescence microscopy for force/Ca(2+) transients and Ca(2+) sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg(2+) inhibition of Ca(2+) release. Low Mg(2+)-induced force and Ca(2+) transients were reversibly abolished by IL-1α. At normal Mg(2+), IL-1α reversibly increased caffeine-induced force and Ca(2+) transients. IL-1α reduced SR Ca(2+) leak via RyR1, as judged by (1) increased SR Ca(2+) retention, (2) increased IL-1α force transients being reproduced by 25 µM tetracaine, and (3) reduced Ca(2+) spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

Motor protein function in skeletal abdominal muscle of cachectic cancer patients.

Cachexia presents with ongoing muscle wasting, altering quality of life in cancer patients. Cachexia is a limiting prognostic factor for patient survival and health care costs. Although animal models and human trials have shown mechanisms of motorprotein proteolysis, not much is known about intrinsic changes of muscle functionality in cancer patients suffering from muscle cachexia, and deeper insights into cachexia pathology in humans are needed. To address this question, rectus abdominis muscle samples were collected from several surgical control, non-cachectic and cachectic cancer patients and processed for skinned fibre biomechanics, molecular in vitro motility assays, myosin isoform protein compositions and quantitative ubiquitin polymer protein analysis. In pre-cachectic and cachectic cancer patient samples, maximum force was significantly compromised compared with controls, but showed an unexpected increase in myofibrillar Ca(2+) sensitivity consistent with a shift from slow to fast myosin isoform expression seen in SDS-PAGE analysis and in vitro motility assays. Force deficit was specific for 'cancer', but not linked to presence of cachexia. Interestingly, quantitative ubiquitin immunoassays revealed no major changes in static ubiquitin polymer protein profiles, whether cachexia was present or not and were shown to mirror profiles in control patients. Our study on muscle function in cachectic patients shows that abdominal wall skeletal muscle in cancer cachexia shows signs of weakness that can be partially attributed to intrinsic changes to contractile motorprotein function. On protein levels, static ubiquitin polymeric distributions were unaltered, pointing towards evenly up-regulated ubiquitin protein turnover with respect to ubiquitin conjugation, proteasome degradation and de-ubiquitination.

From chaos to split-ups--SHG microscopy reveals a specific remodelling mechanism in ageing dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a common inherited muscle disease showing chronic inflammation and progressive muscle weakness. Absent dystrophin renders sarcolemma more Ca(2+) -permeable, disturbs signalling and triggers inflammation. Sustained degeneration/regeneration cycles render muscle cytoarchitecture susceptible to remodelling. Quantitative morphometry was introduced in living cells using second-harmonic generation (SHG) microscopy of myosin. As the time course of cellular remodelling is not known, we used SHG microscopy in mdx muscle fibres over a wide age range for three-dimensional (3D) rendering and detection of verniers and cosine angle sums (CASs). Wild-type (wt) and transgenic mini-dystrophin mice (MinD) were also studied. Vernier densities (VDs) declined in wt and MinD fibres until adulthood, while in mdx fibres, VDs remained significantly elevated during the life span. CAS values were close to unity in adult wt and MinD fibres, in agreement with tight regular myofibril orientation, while always smaller in mdx fibres. Using SHG 3D morphometry, we identified two types of altered ultrastructure: branched fibres and a novel, previously undetected 'chaotic' fibre type, both of which can be classified by distinct CAS and VD combinations. We present a novel model of tissue remodelling in dystrophic progression with age that involves the transition from normal to chaotic to branched fibres. Our model predicts a ~50% contribution of altered cytoarchitecture to progressive force loss with age. We also provide an improved automated image algorithm that is suitable for future ageing studies in human myopathies.

Parietofrontal integrity determines neural modulation associated with grasping imagery after stroke.

Chronic stroke patients with heterogeneous lesions, but no direct damage to the primary sensorimotor cortex, are capable of longitudinally acquiring the ability to modulate sensorimotor rhythms using grasping imagery of the affected hand. Volitional modulation of neural activity can be used to drive grasping functions of the paralyzed hand through a brain-computer interface. The neural substrates underlying this skill are not known. Here, we investigated the impact of individual patient's lesion pathology on functional and structural network integrity related to this volitional skill. Magnetoencephalography data acquired throughout training was used to derive functional networks. Structural network models and local estimates of extralesional white matter microstructure were constructed using T(1)-weighted and diffusion-weighted magnetic resonance imaging data. We employed a graph theoretical approach to characterize emergent properties of distributed interactions between nodal brain regions of these networks. We report that interindividual variability in patients' lesions led to differential impairment of functional and structural network characteristics related to successful post-training sensorimotor rhythm modulation skill. Patients displaying greater magnetoencephalography global cost-efficiency, a measure of information integration within the distributed functional network, achieved greater levels of skill. Analysis of lesion damage to structural network connectivity revealed that the impact on nodal betweenness centrality of the ipsilesional primary motor cortex, a measure that characterizes the importance of a brain region for integrating visuomotor information between frontal and parietal cortical regions and related thalamic nuclei, correlated with skill. Edge betweenness centrality, an analogous measure, which assesses the role of specific white matter fibre pathways in network integration, showed a similar relationship between skill and a portion of the ipsilesional superior longitudinal fascicle connecting premotor and posterior parietal visuomotor regions known to be crucially involved in normal grasping behaviour. Finally, estimated white matter microstructure integrity in regions of the contralesional superior longitudinal fascicle adjacent to primary sensorimotor and posterior parietal cortex, as well as grey matter volume co-localized to these specific regions, positively correlated with sensorimotor rhythm modulation leading to successful brain-computer interface control. Thus, volitional modulation of ipsilesional neural activity leading to control of paralyzed hand grasping function through a brain-computer interface after longitudinal training relies on structural and functional connectivity in both ipsilesional and contralesional parietofrontal pathways involved in visuomotor information processing. Extant integrity of this structural network may serve as a future predictor of response to longitudinal therapeutic interventions geared towards training sensorimotor rhythms in the lesioned brain, secondarily improving grasping function through brain-computer interface applications.

Artificial Cytoskeleton Assembly for Synthetic Cell Motility.

Imitation of cellular processes in cell-like compartments is a current research focus in synthetic biology. Here, a method is introduced for assembling an artificial cytoskeleton in a synthetic cell model system based on a poly(N-isopropyl acrylamide) (PNIPAM) composite material. Toward this end, a PNIPAM-based composite material inside water-in-oil droplets that are stabilized with PNIPAM-functionalized and commercial fluorosurfactants is introduced. The temperature-mediated contraction/release behavior of the PNIPAM-based cytoskeleton is investigated. The reversibility of the PNIPAM transition is further examined in bulk and in droplets and it could be shown that hydrogel induced deformation could be used to controllably manipulate droplet-based synthetic cell motility upon temperature changes. It is envisioned that a combination of the presented artificial cytoskeleton with naturally occurring components might expand the bandwidth of the bottom-up synthetic biology.

Sensing the (digital) pulse. Future steps for improving the secondary use of data for research in Switzerland.

Introduction: Ensuring that the health data infrastructure and governance permits an efficient secondary use of data for research is a policy priority for many countries. Switzerland is no exception and many initiatives have been launched to improve its health data landscape. The country now stands at an important crossroad, debating the right way forward. We aimed to explore which specific elements of data governance can facilitate - from ethico-legal and socio-cultural perspectives - the sharing and reuse of data for research purposes in Switzerland. Methods: A modified Delphi methodology was used to collect and structure input from a panel of experts via successive rounds of mediated interaction on the topic of health data governance in Switzerland. Results: First, we suggested techniques to facilitate data sharing practices, especially when data are shared between researchers or from healthcare institutions to researchers. Second, we identified ways to improve the interaction between data protection law and the reuse of data for research, and the ways of implementing informed consent in this context. Third, we put forth ideas on policy changes, such as the steps necessary to improve coordination between different actors of the data landscape and to win the defensive and risk-adverse attitudes widespread when it comes to health data. Conclusions: After having engaged with these topics, we highlighted the importance of focusing on non-technical aspects to improve the data-readiness of a country (e.g., attitudes of stakeholders involved) and of having a pro-active debate between the different institutional actors, ethico-legal experts and society at large.

The heart in Duchenne muscular dystrophy: early detection of contractile performance alteration.

Progressive cardiomyopathy is a major cause of death in Duchenne muscular dystrophy (DMD) patients. Coupling between Ca(2+) handling and contractile properties in dystrophic hearts is poorly understood. It is also not clear whether developing cardiac failure is dominated by alterations in Ca(2+) pathways or more related to the contractile apparatus. We simultaneously recorded force and Ca(2+) transients in field-stimulated papillary muscles from young (10-14 weeks) wild-type (wt) and dystrophic mdx mice. Force amplitudes were fivefold reduced in mdx muscles despite only 30% reduction in fura-2 ratio amplitudes. This indicated mechanisms other than systolic Ca(2+) to additionally account for force decrements in mdx muscles. pCa-force relations revealed decreased mdx myofibrillar Ca(2+) sensitivity. 'In vitro' motility assays, studied in mdx hearts here for the first time, showed significantly slower sliding velocities. mdx MLC/MHC isoforms were not grossly altered. Dystrophic hearts showed echocardiography signs of early ventricular wall hypertrophy with a significantly enlarged end-diastolic diameter 'in vivo'. However, fractional shortening was still comparable to wt mice. Changes in the contractile apparatus satisfactorily explained force drop in mdx hearts. We give first evidence of early hypertrophy in mdx mice and possible mechanisms for already functional impairment of cardiac muscle in DMD.

Disease Modeling and Model-Based Meta-Analyses to Define a New Direction for a Phase III Program of Gantenerumab in Alzheimer's Disease.

Selecting the right dose is a significant challenge in designing clinical development programs, especially for slowly progressing diseases lacking predictive biomarkers of efficacy that may require long-term treatment to assess clinical benefit. Gantenerumab, a fully human monoclonal antibody (mAb) that binds to aggregated amyloid-beta, was tested in two 24-month phase III studies (NCT01224106, NCT02051608) in participants with prodromal and mild Alzheimer's disease (AD), respectively. Dosing in the first phase III study was suspended after a preplanned interim futility analysis in 2014. Subsequently, a dose-response relationship was observed in a subgroup of fast AD progressors that, together with contemporary aducanumab (another anti-amyloid-beta mAb) data, indicated higher doses may be needed for clinical efficacy. The gantenerumab phase III studies were therefore transformed into dose-finding, open-label extension (OLE) trials. Two exposure-response models were developed to support dose selection via simulations for the OLEs: a pharmacokinetics (PK)/PET (positron emission tomography) model describing amyloid removal using PET data from low-dose gantenerumab and high-dose aducanumab, and a PK/ARIA-E (amyloid-related imaging abnormalities-edema) model describing the occurrence of ARIA-E events leveraging an existing bapineuzumab model. Multiple regimens were designed to gradually up-titrate participants to the target dose of 1,200 mg gantenerumab every 4 weeks to mitigate the increased risk of ARIA-E events that may be associated with higher doses of anti-amyloid-beta antibodies. Favorable OLE data that matched well with model predictions supported the decision to continue the gantenerumab clinical development program and further apply model-based analytical techniques to optimize the design of new phase III studies.

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells.

By using electrostatic interactions as driving force to assemble vesicles, the droplet-stabilized method was recently applied to reconstitute and encapsulate proteins, or compartments, inside giant unilamellar vesicles (GUVs) to act as minimal synthetic cells. However, the droplet-stabilized approach exhibits low production efficiency associated with the troublesome release of the GUVs from the stabilized droplets, corresponding to a major hurdle for the droplet-stabilized approach. Herein, we report the use of pH as a potential trigger to self-assemble droplet-stabilized GUVs (dsGUVs) by either bulk or droplet-based microfluidics. Moreover, pH enables the generation of compartmentalized GUVs with flexibility and robustness. By co-encapsulating pH-sensitive small unilamellar vesicles (SUVs), negatively charged SUVs, and/or proteins, we show that acidification of the droplets efficiently produces dsGUVs while sequestrating the co-encapsulated material. Most importantly, the pH-mediated assembly of dsGUVs significantly improves the production efficiency of free-standing GUVs (i.e., released from the stabilizing-droplets) compared to its previous implementation.

Dendritic oligothiophenes terminated with tris(alkyloxy)phenylethynyl tails: synthesis, physical properties, and self-assembly.

Three-dimensional (3D) π-conjugated dendritic oligothiophenes up to a third generation have been functionalized with tris(decyloxy)phenylethynyl tails at the periphery. The first-generation compounds (3 T-p-Ph-C10 and 6 T-p-Ph-C10) were synthesized by palladium-catalyzed Sonogashira coupling reactions, whereas the higher generation products were synthesized by palladium-catalyzed Suzuki coupling reactions in a divergent approach. The optical and electrochemical properties were investigated by UV/Vis absorption, fluorescence spectroscopy, and cyclic voltammetry. The results revealed that the terminal tris(alkyloxy)phenylethynyl groups are conjugated to the branched oligothiophene core, yielding redshifted absorption and fluorescence spectra and reduced optical band gaps relative to the dendritic oligothiophene core. A structural study revealed a close relationship between the type of supramolecular organization and the size of the oligothiophene core. The first-generation compounds 3 T-p-Ph-C10 and 6 T-p-Ph-C10 displayed columnar phases in the bulk state, which was confirmed by two-dimensional wide-angle X-ray scattering (2D WAXS) measurements. The self-assembly into columnar stacks has mainly been attributed to phase separation between the rigid thiophene cores and the flexible side-chains assisted by minor π-stacking interactions between the conjugated dendritic oligothiophene units. The high-generation compounds, however, showed less ordered structures in the solid state.

Influence of CYP2C19 genotype on the pharmacokinetics of R483, a CYP2C19 substrate, in healthy subjects and type 2 diabetes patients.

OBJECTIVES: R483 is a thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist with anti-diabetic properties and also a cytochrome P450 2C19 (CYP2C19) substrate. The aim of the clinical studies reported here was to investigate the influence of the CYP2C19 genotype on the pharmacokinetics (PK) of R483 in healthy subjects and in type 2 diabetes mellitus (T2DM) patients. METHODS: data came from two clinical studies, one including 58 Japanese and Caucasian healthy subjects and another including 93 Asian T2DM patients. All subjects received multiple doses of R483, 20 mg once daily for the healthy subjects and 12 mg once daily for the T2DM patients. Blood samples were taken up to 24 h after the last dose to determine plasma concentrations of R483 and its major metabolites. RESULTS: poor metabolizers (PMs; CYP2C19*2/*2 or *2/*3 or *3/*3) had a higher plasma exposure and a lower clearance of the parent drug than extensive metabolizers (EMs; CYP2C19*1/*1 or *1/*2 or *1/*3). The homozygous PM/EM ratio for the area under the plasma concentration-time curve (AUC(0-24)) [95% confidence interval] was 3.9 [2.7-5.7] for healthy subjects versus 2.0 [1.5-2.6] in T2DM patients. The heterozygous EMs were all T2DM patients, the PM/EM ratio for AUC(0-24) was 1.5 [1.2-1.8]. The dose-normalized exposure to R483 was 1.2- (for PMs) and 2.4-fold (for EMs) higher in T2DM patients than in healthy subjects. R483 was well tolerated in both studies. CONCLUSIONS: the plasma exposure to R483 was significantly higher in PMs than in EMs. R483 exhibits different PK in healthy subjects and T2DM patients, and the difference in exposure between EM and PM was less pronounced in T2DM patients than in healthy subjects. Factors such as diabetes condition and age may influence the metabolism of R483. This knowledge allowed for a realistic dose recommendation for poor metabolizer T2DM patients.

Engineering Light-Responsive Contractile Actomyosin Networks with DNA Nanotechnology.

External control and precise manipulation is key for the bottom-up engineering of complex synthetic cells. Minimal actomyosin networks have been reconstituted into synthetic cells; however, their light-triggered symmetry breaking contraction has not yet been demonstrated. Here, light-activated directional contractility of a minimal synthetic actomyosin network inside microfluidic cell-sized compartments is engineered. Actin filaments, heavy-meromyosin-coated beads, and caged ATP are co-encapsulated into water-in-oil droplets. ATP is released upon illumination, leading to a myosin-generated force which results in a motion of the beads along the filaments and hence a contraction of the network. Symmetry breaking is achieved using DNA nanotechnology to establish a link between the network and the compartment periphery. It is demonstrated that the DNA-linked actin filaments contract to one side of the compartment forming actin asters and quantify the dynamics of this process. This work exemplifies that an engineering approach to bottom-up synthetic biology, combining biological and artificial elements, can circumvent challenges related to active multi-component systems and thereby greatly enrich the complexity of synthetic cellular systems.

A general strategy for the production of difficult-to-express inducer-dependent bacterial repressor proteins in Escherichia coli.

Inducer-dependent prokaryotic transcriptional repressor proteins that originally evolved to orchestrate the transcriptome with intracellular and extracellular metabolite pools, have become universal tools in synthetic biology, drug discovery, diagnostics and functional genomics. Production of the repressor proteins is often limited due to inhibiting effects on the production host and requires iterative process optimization for each individual repressor. At the example of the Streptomyces pristinaespiralis-derived streptogramin-dependent repressor PIP, the expression of which was shown to inhibit growth of Escherichia coli BL21*, we demonstrate that the addition of the PIP-specific streptogramin antibiotic pristinamycin I neutralizes the growth-inhibiting effect and results in >100-fold increased PIP titers. The yield of PIP was further increased 2.5-fold by the engineering of a new E. coli host suitable for the production of growth-inhibiting proteins encoded by an unfavorable codon usage. PIP produced in the presence of pristinamycin I was purified and was shown to retain the antibiotic-dependent binding to its operator pir as demonstrated by a fluorescence resonance energy transfer (FRET)-based approach. At the example of the macrolide-, tetracycline- and arsenic-dependent repressors MphR(A), TetR and ArsR, we further demonstrate that the production yields can be increased 2- to 3-fold by the addition of the cognate inducer molecules erythromycin, tetracycline and As(3+), respectively. Therefore, the addition of inducer molecules specific to the target repressor protein seems to be a general strategy to increase the yield of this interesting protein class.

Developmental aspects of amperometric ATP biosensors based on entrapped enzymes.

A novel concept for a dual-enzyme-based microbiosensor for the detection of adenosine-5'-triphosphate (ATP) was developed. The employed enzymes pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and hexokinase were entrapped, using pH-shift-induced precipitation of electrodeposition paint (EDP) at platinum microelectrodes (diameter of 25 microm). PQQ-GDH is known showing a superior activity for glucose conversion at the relevant conditions (low oxygen concentration) for ATP detection in targeted biomedical studies. For immobilizing the two enzymes PQQ-GDH and hexokinase, the deposition conditions of EDP Resydrol AY498w/35WA were adapted to ensure high immobilization rates. Prior to ATP sensing, the conversion of glucose, which is the co-substrate for both enzymatic reactions, was optimized. Optimization was targeted towards ATP measurements in biomedical environments by optimizing the PQQ-GDH sensor for glucose. Therefore, different mediators were tested regarding their electron transfer rate and their compatibility with the enzyme: free-diffusing N-methylphenazonium methyl sulfate (PMS) and ferrocenemethanol, and an immobilized chromium hexacyanoferrate layer at platinum electrode. Free-diffusing ferrocenemethanol reveals high sensitivity towards glucose of 1.5 +/- 0.4 nA/mM. In a next step, hexokinase was co-entrapped in the polymer film resulting in a sensitivity of up to 290 pA/microM.

Think to move: a neuromagnetic brain-computer interface (BCI) system for chronic stroke.

BACKGROUND AND PURPOSE: Stroke is a leading cause of long-term motor disability among adults. Present rehabilitative interventions are largely unsuccessful in improving the most severe cases of motor impairment, particularly in relation to hand function. Here we tested the hypothesis that patients experiencing hand plegia as a result of a single, unilateral subcortical, cortical or mixed stroke occurring at least 1 year previously, could be trained to operate a mechanical hand orthosis through a brain-computer interface (BCI). METHODS: Eight patients with chronic hand plegia resulting from stroke (residual finger extension function rated on the Medical Research Council scale=0/5) were recruited from the Stroke Neurorehabilitation Clinic, Human Cortical Physiology Section of the National Institute for Neurological Disorders and Stroke (NINDS) (n=5) and the Clinic of Neurology of the University of Tübingen (n=3). Diagnostic MRIs revealed single, unilateral subcortical, cortical or mixed lesions in all patients. A magnetoencephalography-based BCI system was used for this study. Patients participated in between 13 to 22 training sessions geared to volitionally modulate micro rhythm amplitude originating in sensorimotor areas of the cortex, which in turn raised or lowered a screen cursor in the direction of a target displayed on the screen through the BCI interface. Performance feedback was provided visually in real-time. Successful trials (in which the cursor made contact with the target) resulted in opening/closing of an orthosis attached to the paralyzed hand. RESULTS: Training resulted in successful BCI control in 6 of 8 patients. This control was associated with increased range and specificity of mu rhythm modulation as recorded from sensors overlying central ipsilesional (4 patients) or contralesional (2 patients) regions of the array. Clinical scales used to rate hand function showed no significant improvement after training. CONCLUSIONS: These results suggest that volitional control of neuromagnetic activity features recorded over central scalp regions can be achieved with BCI training after stroke, and used to control grasping actions through a mechanical hand orthosis.

Velocity distributions of single F-actin trajectories from a fluorescence image series using trajectory reconstruction and optical flow mapping.

We present an approach for the computation of single-object velocity statistics in a noisy fluorescence image series. The algorithm is applied to molecular imaging data from an in vitro actin-myosin motility assay. We compare the relative efficiency of wavelet and curvelet transform denoising in terms of noise reduction and object restoration. It is shown that while both algorithms reduce background noise efficiently, curvelet denoising restores the curved edges of actin filaments more reliably. Noncrossing spatiotemporal actin trajectories are unambiguously identified using a novel segmentation scheme that locally combines the information of 2-D and 3-D segmentation. Finally, the optical flow vector field for the image sequence is computed via the 3-D structure tensor and mapped to the segmented trajectories. Using single-trajectory statistics, the global velocity distribution extracted from an image sequence is decomposed into the contributions of individual trajectories. The technique is further used to analyze the distribution of the x and y components of the velocity vectors separately, and it is shown that directed actin motion is found in myosin extracts from single skeletal muscle fibers. The presented approach may prove helpful to identify actin filament subpopulations and to analyze actin-myosin interaction kinetics under biochemical regulation.

Gas-inducible transgene expression in mammalian cells and mice.

We describe the design and detailed characterization of a gas-inducible transgene control system functional in different mammalian cells, mice and prototype biopharmaceutical manufacturing. The acetaldehyde-inducible AlcR-P(alcA) transactivator-promoter interaction of the Aspergillus nidulans ethanol-catabolizing regulon was engineered for gas-adjustable transgene expression in mammalian cells. Fungal AlcR retained its transactivation characteristics in a variety of mammalian cell lines and reversibly adjusted transgene transcription from chimeric mammalian promoters (P(AIR)) containing P(alcA)-derived operators in a gaseous acetaldehyde-dependent manner. Mice implanted with microencapsulated cells engineered for acetaldehyde-inducible regulation (AIR) of the human glycoprotein secreted placental alkaline phosphatase showed adjustable serum phosphatase levels after exposure to different gaseous acetaldehyde concentrations. AIR-controlled interferon-beta production in transgenic CHO-K1-derived serum-free suspension cultures could be modulated by fine-tuning inflow and outflow of acetaldehyde-containing gas during standard bioreactor operation. AIR technology could serve as a tool for therapeutic transgene dosing as well as biopharmaceutical manufacturing.