Molecular architecture of potassium chloride co-transporter KCC2

KCC2 is a neuron specific K+-Cl− co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into “head” and “core” domains connected by a flexible “linker”. Dimers are asymmetrical and display a bent “S-shape” architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2

Comparison of embedded and added motor imagery training in patients after stroke: Study protocol of a randomised controlled pilot trial using a mixed methods approach

Copyright @ 2009 Schuster et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Two different approaches have been adopted when applying motor imagery (MI) to stroke patients. MI can be conducted either added to conventional physiotherapy or integrated within therapy sessions. The proposed study aims to compare the efficacy of embedded MI to an added MI intervention. Evidence from pilot studies reported in the literature suggests that both approaches can improve performance of a complex motor skill involving whole body movements, however, it remains to be demonstrated, which is the more effective one.Methods/Design: A single blinded, randomised controlled trial (RCT) with a pre-post intervention design will be carried out. The study design includes two experimental groups and a control group (CG). Both experimental groups (EG1, EG2) will receive physical practice of a clinical relevant motor task ('Going down, laying on the floor, and getting up again') over a two week intervention period: EG1 with embedded MI training, EG2 with MI training added after physiotherapy. The CG will receive standard physiotherapy intervention and an additional control intervention not related to MI.The primary study outcome is the time difference to perform the task from pre to post-intervention. Secondary outcomes include level of help needed, stages of motor task completion, degree of motor impairment, balance ability, fear of falling measure, motivation score, and motor imagery ability score. Four data collection points are proposed: twice during baseline phase, once following the intervention period, and once after a two week follow up. A nested qualitative part should add an important insight into patients' experience and attitudes towards MI. Semi-structured interviews of six to ten patients, who participate in the RCT, will be conducted to investigate patients' previous experience with MI and their expectations towards the MI intervention in the study. Patients will be interviewed prior and after the intervention period.Discussion: Results will determine whether embedded MI is superior to added MI. Findings of the semi-structured interviews will help to integrate patient's expectations of MI interventions in the design of research studies to improve practical applicability using MI as an adjunct therapy technique

How to improve walking, balance and social participation following stroke: a comparison of the long term effects of two walking aids--canes and an orthosis TheraTogs--on the recovery of gait following acute stroke. A study protocol for a multi-centre, single blind, randomised control trial

<p>Abstract</p> <p>Background</p> <p>Annually, some 9000 people in Switzerland suffer a first time stroke. Of these 60% are left with moderate to severe walking disability. Evidence shows that rehabilitation techniques which emphasise activity of the hemiplegic side increase ipsilesional cortical plasticity and improve functional outcomes. Canes are commonly used in gait rehabilitation although they significantly reduce hemiplegic muscle activity. We have shown that an orthosis "TheraTogs" (a corset with elasticated strapping) significantly increases hemiplegic muscle activity during gait. The aim of the present study is to investigate the long term effects on the recovery of gait, balance and social participation of gait rehabilitation with TheraTogs compared to gait rehabilitation with a cane following first time acute stroke.</p> <p>Methods/Design</p> <p>Multi-centre, single blind, randomised trial with 120 patients after first stroke. When subjects have reached Functional Ambulation Category 3 they will be randomly allocated into TheraTogs or cane group. TheraTogs will be applied to support hip extensor and abductor musculature according to a standardised procedure. Cane walking held at the level of the radial styloid of the sound wrist. Subjects will walk throughout the day with only the assigned walking aid. Standard therapy treatments and usual care will remain unchanged and documented. The intervention will continue for five weeks or until patients have reached Functional Ambulation category 5. Outcome measures will be assessed the day before begin of intervention, the day after completion, 3 months, 6 months and 2 years. Primary outcome: Timed "up and go" test, secondary outcomes: peak surface EMG of gluteus maximus and gluteus medius, activation patterns of hemiplegic leg musculature, temporo-spatial gait parameters, hemiplegic hip kinematics in the frontal and sagittal planes, dynamic balance, daily activity measured by accelerometry, Stroke Impact Scale. Significance levels will be 5% with 95% CI's. IntentionToTreat analyses will be performed. Descriptive statistics will be presented.</p> <p>Discussion</p> <p>This study could have significant implications for the clinical practice of gait rehabilitation after stroke, particularly the effect and appropriate use of walking aids.</p> <p>The results could be important for the development of clinical guidelines and for the socio-economic costs of post-stroke care</p> <p>Trial registration number</p> <p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01366729">NCT01366729</a>.</p

Single-molecule biosensors: Recent advances and applications

Single-molecule biosensors serve the unmet need for real time detection of individual biological molecules in the molecular crowd with high specificity and accuracy, uncovering unique properties of individual molecules which are hidden when measured using ensemble averaging methods. Measuring a signal generated by an individual molecule or its interaction with biological partners is not only crucial for early diagnosis of various diseases such as cancer and to follow medical treatments but also offers a great potential for future point-of-care devices and personalized medicine. This review summarizes and discusses recent advances in nanosensors for both in vitro and in vivo detection of biological molecules offering single-molecule sensitivity. In the first part, we focus on label-free platforms, including electrochemical, plasmonic, SERS-based and spectroelectrochemical biosensors. We review fluorescent single-molecule biosensors in the second part, highlighting nanoparticle-amplified assays, digital platforms and the utilization of CRISPR technology. We finally discuss recent advances in the emerging nanosensor technology of important biological species as well as future perspectives of these sensors

The influence of in-cylinder flows and bulk gas density on early Spray G injection in an optical research engine

A well-characterized multi-hole gasoline injector, the Engine Combustion Network’s (ECN) Spray G injector, was investigated in an optically accessible research engine under four motored operating conditions with early injection. The experiments were conducted at intake pressures of 0.4 bar and 0.95 bar, nearly matching the ECN’s standard early injection operating conditions, Spray G2 (flash boiling) and Spray G3 (early injection), respectively. This was combined with two engine speeds at 800 rpm and 1500 rpm. Using particle image velocimetry and volumetric Mie scatter imaging, the in-cylinder flows were evaluated and the effects on the spray morphology were characterized. The in-cylinder flow was evaluated to understand the spray-flow interaction, including the turbulent kinetic energy. Little effect on turbulent energy was observed in the region examined near the exit of the fuel injector nozzle shortly after injection. Mie scatter imaging was used to characterize the spray morphology and wall wetting was clearly visible on the spark plug. Cyclic variability of the sprays was found to be insignificant; and major differences in spray morphology are attributed to the in-cylinder velocity and intake pressure at the time of injection. Decreasing the bulk gas density by decreasing the intake pressure had a number of effects on the evolution of the spray including faster evaporation, increased axial liquid penetration, and decreased spray angle. Increasing the in-cylinder flow magnitudes by increasing the engine speed had a similar effect on spray morphology by also increasing the evaporation rate, increasing the axial penetration, and decreasing the spray opening angle. Comparison of the motored spray cases with a no-flow case (when the fuel is sprayed into the engine without the piston present) further illustrated the extent to which the intake flow influenced the spray shape

The influence of in-cylinder flows and bulk gas density on early Spray G injection in an optical research engine

A well-characterized multi-hole gasoline injector, the Engine Combustion Network's (ECN) Spray G injector, was investigated in an optically accessible research engine under four motored operating conditions with early injection. The experiments were conducted at intake pressures of 0.4bar and 0.95bar, nearly matching the ECN's standard early injection operating conditions, Spray G2 (flash boiling) and Spray G3 (early injection), respectively. This was combined with two engine speeds at 800rpm and 1500rpm. Using particle image velocimetry and volumetric Mie scatter imaging, the in-cylinder flows were evaluated and the effects on the spray morphology were characterized. The in-cylinder flow was evaluated to understand the spray-flow interaction, including the turbulent kinetic energy. Little effect on turbulent energy was observed in the region examined near the exit of the fuel injector nozzle shortly after injection. Mie scatter imaging was used to characterize the spray morphology and wall wetting was clearly visible on the spark plug. Cyclic variability of the sprays was found to be insignificant; and major differences in spray morphology are attributed to the in-cylinder velocity and intake pressure at the time of injection. Decreasing the bulk gas density by decreasing the intake pressure had a number of effects on the evolution of the spray including faster evaporation, increased axial liquid penetration, and decreased spray angle. Increasing the in-cylinder flow magnitudes by increasing the engine speed had a similar effect on spray morphology by also increasing the evaporation rate, increasing the axial penetration, and decreasing the spray opening angle. Comparison of the motored spray cases with a no-flow case (when the fuel is sprayed into the engine without the piston present) further illustrated the extent to which the intake flow influenced the spray shape

Drug Discovery at the Single Molecule Level: Inhibition-in-Solution Assay of Membrane-Reconstituted beta-Secretase Using Single-Molecule Imaging

Inhibition-in-solution assays (ISA) employing surface-based biosensors such as surface plasmon resonance (SPR) are an effective screening approach in drug discovery. However, analysis of potent binders remains a significant hurdle due to limited sensitivity and accompanied depletion of the inhibiting compounds due to high protein concentrations needed for detectable binding signals. To overcome this limitation, we explored a microscopy-based single-molecule ISA compatible with liposome-reconstituted membrane proteins. Using a set of validated small molecule inhibitors against beta-secretase 1 (BACE1), the assay was benchmarked with respect to sensitivity and dynamic range against SPR. We demonstrate that the dynamic range of measurable affinities is greatly extended by more than 2 orders of magnitude as compared to SPR, thus facilitating measurements of highly potent (K-