Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.This work was supported by a Sir Henry Dale Fellowship (098406), jointly funded by the Wellcome Trust and the Royal Society (to SCG). BGB is a Wellcome Trust PhD student. Remote synchrotron access was supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (Contract No. 283570

A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella

CymA (tetrahaem cytochrome c) is a member of the NapC/NirT family of quinol dehydrogenases. Essential for the anaerobic respiratory flexibility of shewanellae, CymA transfers electrons from menaquinol to various dedicated systems for the reduction of terminal electron acceptors including fumarate and insoluble minerals of Fe(III). Spectroscopic characterization of CymA from Shewanella oneidensis strain MR-1 identifies three low-spin His/His co-ordinated c-haems and a single high-spin c-haem with His/H2O co-ordination lying adjacent to the quinol-binding site. At pH 7, binding of the menaquinol analogue, 2-heptyl-4-hydroxyquinoline-N-oxide, does not alter the mid-point potentials of the high-spin (approximately −240 mV) and low-spin (approximately −110, −190 and −265 mV) haems that appear biased to transfer electrons from the high- to low-spin centres following quinol oxidation. CymA is reduced with menadiol (Em=−80 mV) in the presence of NADH (Em=−320 mV) and an NADH–menadione (2-methyl-1,4-naphthoquinone) oxidoreductase, but not by menadiol alone. In cytoplasmic membranes reduction of CymA may then require the thermodynamic driving force from NADH, formate or H2 oxidation as the redox poise of the menaquinol pool in isolation is insufficient. Spectroscopic studies suggest that CymA requires a non-haem co-factor for quinol oxidation and that the reduced enzyme forms a 1:1 complex with its redox partner Fcc3 (flavocytochrome c3 fumarate reductase). The implications for CymA supporting the respiratory flexibility of shewanellae are discussed.</jats:p

Motor kinematic differences in children with autism sepectrum disorder : ecological gameplay with a sensorised toy

Background Evidence suggests gross motor differences are present in children with Autism Spectrum Disorder (ASD) from birth. Trevarthen and Delafield-Butt (2013) proposed that one of the early markers of ASD are abnormalities in the development of intentional movements, which are present before the manifestations of symptoms typically associated with autism, like deficiencies in social interaction and communication. A growing body of literature demonstrates kinematic and action patterns differences in children and adults with ASD. However, these experiments typically require expensive laboratory-based optical motion tracking systems. Here, we developed bespoke, sensorised wooden cubes for motor assessment of children’s play and report on the kinematic and action pattern differences of the children with autism compared to children developing typically. Objectives A description of ASD-specific action patterns and kinematics using sensorised toys. Methods Participants. Children 3 to 5 years diagnosed with ASD (n = 15) recruited from the Scottish Centre for Autism, Glasgow, UK. Children 3 to 5 years old developing typically recruited from nurseries in Glasgow, UK. Adults 20 to 25 years old without ASD recruited from Glasgow, UK. The study was approved by the University of Strathclyde Ethics Committee and consent obtained from the parents of children or the adults. In the case of the children with ASD, pre-screening with Vineland-II, AQ-Child and Leiter-R Brief IQ was performed. Procedure. The children were seated at a table and instructed to play two simple games that involved moving the cube from one position to another: a Serially Organized Action (SOA) game and a Single Repetitive Action (SRA) game. The first required complex motor sequencing and engagement with the experimenter, while the second consisted of a simple repetitive movement. Each game produced a single measured movement to a goal with 25 iterations or repetitions to yield 50 movements in total. An electronic board inside the cubes was equipped with tri-axial magnetometer, gyroscope and accelerometer wirelessly transferred the cube’s motion data to a laptop. The signal (raw motion data) was extracted through a Matlab-based platform and analysed. Data Analysis. Kinematic features of movement duration; maximum value of acceleration, velocity, and jerk during each movement; time to maximum value; % duration to maximum value; and the acceleration, velocity, and jerk action patterns profiles were calculated. Results The jerk profile of children with ASD was significantly different, showing increased maximum jerk, reduced time to maximum value and & duration to maximum value, and lower variability than typically developing children. Further, movement duration was shorter compared to age-matched typically developing children, and maximum velocity was significantly higher in children with ASD compared to children developing typically. Conclusion The increased jerk values and onset times in the ASD group are a particularly interesting finding that support new data appearing by other groups. It appeared, especially in the SRA game, that when moving the cube from one position to the next, the children with autism impacted on the surface of the table with greater velocity and typically included the resulting force immediately into to the next movement, giving it a greater jerk value in a shorter span of time that typically children. Typically developing children, on the other hand, paused for a moment (>100 ms) before commencing the next movement. Further, children with autism did not enjoy the SRA game, but they did enjoy the simpler, more repetitive SOA one. The repetitive simplicity of the SOA game and its resulting jerk profile appears to report on a particular behavioural motor feature distinct to ASD, namely stopping an action and starting a new one, while also describing an underlying motor difference that may contribute to it

Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a “nested trap” and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins

Rabbit and Human Non-Keratinising Stratified Squamous Oesophageal Epithelium Displays Similar Microridge Structure by Scanning Electron Microscopy

Since the oesophageal epithelium of common laboratory animals, rats and mice, is keratinized it is unsuitable for comparison with typical non-keratinized stratified squamous human epithelium. It is thus important to find a suitable animal model for the study of human oesophageal tissue changes. This study investigated the microridge structure of immature and adult rabbit specimens, and adult human biopsies by scanning electron microscopy and morphometry. The investigation revealed a similarity between typical squamous human and adult rabbit oesophageal mucosal epithelium. While human epithelium specimens subdivided into two other groups (non-typical squamous and non-squamous); all typical squamous human biopsies were from patients who had normal endoscopy reports and no reflux symptoms. The surface cells of typical squamous human epithelium displayed complex microridge patterns (64% of cell surface) but patterns in non-typical squamous specimens were more variable (38%) (P \u3c 0.001) and cell boundaries less obvious. Rabbit squames displayed clear microridge patterns with an elevation in the percentage of cell surface covered by microridges, with increasing age, from immature to adult specimens (P \u3c 0.001). There was no statistically significant differences between adult rabbit, and \u27typical squamous\u27 human biopsies (range 51-65%), results which suggest potential use of a rabbit model to study changes in human oesophageal tissue

Objective and automatic classification of Parkinson disease with Leap Motion controller

Background: The main objective of this paper is to develop and test the ability of the Leap Motion controller (LMC) to assess the motor dysfunction in patients with Parkinson disease (PwPD) based on the MDS-UPDRSIII exercises. Four exercises (thumb forefinger tapping, hand opening/closing, pronation/supination, postural tremor) were used to evaluate the characteristics described in MDS-UPDRSIII. Clinical ratings according to the MDS/UPDRS-section III items were used as target. For that purpose, 16 participants with PD and 12 healthy people were recruited in Ospedale Cisanello, Pisa, Italy. The participants performed standardized hand movements with camera-based marker. Time and frequency domain features related to velocity, angle, amplitude, and frequency were derived from the LMC data. Results: Different machine learning techniques were used to classify the PD and healthy subjects by comparing the subjective scale given by neurologists against the predicted diagnosis from the machine learning classifiers. Feature selection methods were used to choose the most significant features. Logistic regression (LR), naive Bayes (NB), and support vector machine (SVM) were trained with tenfold cross validation with selected features. The maximum obtained classification accuracy with LR was 70.37%; the average area under the ROC curve (AUC) was 0.831. The obtained classification accuracy with NB was 81.4%, with AUC of 0.811. The obtained classification accuracy with SVM was 74.07%, with AUC of 0.675. Conclusions: Results revealed that the system did not return clinically meaningful data for measuring postural tremor in PwPD. In addition, it showed limited potential to measure the forearm pronation/supination. In contrast, for finger tapping and hand opening/closing, the derived parameters showed statistical and clinical significance. Future studies should continue to validate the LMC as updated versions of the software are developed. The obtained results support the fact that most of the set of selected features contributed significantly to classify the PwPD and healthy subjects

Discrete breathers in a two-dimensional hexagonal Fermi-Pasta-Ulam lattice

We consider a two-dimensional Fermi-Pasta-Ulam (FPU) lattice with hexagonal symmetry. Using asymptotic methods based on small amplitude ansatz, at third order we obtain a reduction to a cubic nonlinear Schrodinger equation (NLS) for the breather envelope. However, this does not support stable soliton solutions, so we pursue a higher-order analysis yielding a generalised NLS, which includes known stabilising terms. We present numerical results which suggest that long-lived stationary and moving breathers are supported by the lattice. We find breather solutions which move in an arbitrary direction, an ellipticity criterion for the wavenumbers of the carrier wave, asymptotic estimates for the breather energy, and a minimum threshold energy below which breathers cannot be found. This energy threshold is maximised for stationary breathers, and becomes vanishingly small near the boundary of the elliptic domain where breathers attain a maximum speed. Several of the results obtained are similar to those obtained for the square FPU lattice (Butt & Wattis, J Phys A, 39, 4955, (2006)), though we find that the square and hexagonal lattices exhibit different properties in regard to the generation of harmonics, and the isotropy of the generalised NLS equation.Comment: 29 pages, 14 Figure

An auxin-inducible, GAL4-compatible, gene expression system for Drosophila

The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila