Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Phase-separating pyrenoid proteins form complexes in the dilute phase.

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates.

Joint Detection of Change Points in Multichannel Single-Molecule Measurements.

Recent developments in single-molecule measurement technology have expanded the capability to measure multiple parameters. These emergent modalities provide more holistic observations of complex biomolecular processes and call for new analysis methods to detect state changes in multichannel data. Here we develop an algorithm called MULLR (MUlti-channel Log-Likelihood Ratio test) to jointly identify change points in multichannel single-molecule measurements. MULLR is an extension of the popular single-channel implementation for change point detection based on a binary segmentation and log-likelihood ratio test framework. We validate the algorithm on simulated data and characterize the power of detection and false positive rate. We show that MULLR can identify change points in experimental multichannel data and naturally works with different noise statistics and time resolutions across channels. Further, we quantify the benefit of MULLR compared to single-channel analysis. We envision that the MULLR algorithm will be useful to a range of multiparameter single-molecule measurements.

Evolution of the Wilson-Cowan equations.

The Wilson-Cowan equations were developed to provide a simplified yet powerful description of neural network dynamics. As such, they embraced nonlinear dynamics, but in an interpretable form. Most importantly, it was the first mathematical formulation to emphasize the significance of interactions between excitatory and inhibitory neural populations, thereby incorporating both cooperation and competition. Subsequent research by many has documented the Wilson-Cowan significance in such diverse fields as visual hallucinations, memory, binocular rivalry, and epilepsy. The fact that these equations are still being used to elucidate a wide range of phenomena attests to their validity as a dynamical approximation to more detailed descriptions of complex neural computations.

ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling.

Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes.

Probing DNA-protein interactions using single-molecule diffusivity contrast.

Single-molecule fluorescence investigations of protein-nucleic acid interactions require robust means to identify the binding state of individual substrate molecules in real time. Here, we show that diffusivity contrast, widely used in fluorescence correlation spectroscopy at the ensemble level and in single-particle tracking on individual (but slowly diffusing) species, can be used as a general readout to determine the binding state of single DNA molecules with unlabeled proteins in solution. We first describe the technical basis of drift-free single-molecule diffusivity measurements in an anti-Brownian electrokinetic trap. We then cross-validate our method with protein-induced fluorescence enhancement, a popular technique to detect protein binding on nucleic acid substrates with single-molecule sensitivity. We extend an existing hydrodynamic modeling framework to link measured diffusivity to particular DNA-protein structures and obtain good agreement between the measured and predicted diffusivity values. Finally, we show that combining diffusivity contrast with protein-induced fluorescence enhancement allows simultaneous mapping of binding stoichiometry and location on individual DNA-protein complexes, potentially enhancing single-molecule views of relevant biophysical processes.

Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments.

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments using p-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish that pNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models--including those of our own--in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Faecal immunochemical test to triage patients with abdominal symptoms for suspected colorectal cancer in primary care: review of international use and guidelines.

BACKGROUND: Recently, faecal immunochemical tests (FITs) have been introduced for investigation of primary care patients with low-risk symptoms of colorectal cancer (CRC), but recommendations vary across the world. This systematic review of clinical practice guidelines aimed to determine how FITs are used in symptomatic primary care patients and the underpinning evidence for these guidelines. METHODS: MEDLINE, Embase and TRIP databases were systematically searched, from 1 November 2008 to 1 November 2018 for guidelines on the assessment of patients with symptoms suggestive of CRC. Known guideline databases, websites and references of related literature were searched. The following questions were addressed: (i) which countries use FIT for symptomatic primary care patients; (ii) in which populations is FIT used; (iii) what is the cut-off level used for haemoglobin in the faeces (FIT) and (iv) on what evidence are FIT recommendations based. RESULTS: The search yielded 2433 publications; 25 covered initial diagnostic assessment of patients with symptoms of CRC in 15 countries (Asia, n = 1; Europe, n = 13; Oceania, n = 4; North America, n = 5; and South America, n = 2). In three countries (Australia, Spain and the UK), FIT was recommended for patients with abdominal symptoms, unexplained weight loss, change in bowel habit or anaemia despite a low level of evidence in the symptomatic primary care patient population. CONCLUSIONS: Few countries recommend FITs in symptomatic patients in primary care either because of limited evidence or because symptomatic patients are directly referred to secondary care without triage. These results demonstrate a clear need for research on FIT in the symptomatic primary care population.

Hyperchaos in Wilson-Cowan oscillator circuits.

The Wilson-Cowan equations were originally shown to produce limit cycle oscillations for a range of parameters. Others subsequently showed that two coupled Wilson-Cowan oscillators could produce chaos, especially if the oscillator coupling was from inhibitory interneurons of one oscillator to excitatory neurons of the other. Here this is extended to show that chains, grids, and sparse networks of Wilson-Cowan oscillators generate hyperchaos with linearly increasing complexity as the number of oscillators increases. As there is now evidence that humans can voluntarily generate hyperchaotic visuomotor sequences, these results are particularly relevant to the unpredictability of a range of human behaviors. These also include incipient senescence in aging, effects of concussive brain injuries, autism, and perhaps also intelligence and creativity.NEW & NOTEWORTHY This paper represents an exploration of hyperchaos in coupled Wilson-Cowan equations. Results show that hyperchaos (number of positive Lyapunov exponents) grows linearly with the number of oscillators in the array and leads to high levels of unpredictability in the neural response.

Voluntary Generation of Hyperchaotic Visuo-Motor Patterns.

Unpredictable escape behaviour is an integral part of the repertoire of many prey species. Many social species, including humans, also employ unpredictability to enhance their chances of success in social interactions with conspecifics. However, it is unclear what mechanisms provide the behavioural and neural bases of this unpredictability, which might result either from randomness or from chaotic dynamics. A novel paradigm described here demonstrates that unpredictable behaviour generated voluntarily in a simple visuomotor task represents high dimensional chaos, or hyperchaos, but not randomness. The exponential decay of predictability was also shown to be longer in older adults. As chaos has been observed among cortical neurons, this provides a plausible neural basis for hyperchaos. These results thus provide evidence that voluntary unpredictable behaviour can result from neural hyperchaos. This work has implications for the study of autism, aftereffects of concussions, early dementia, and the concept of free will.

Binocular contrast, stereopsis, and rivalry: Toward a dynamical synthesis.

It is well known that small orientation differences between two monocular gratings fuse to generate a stereoscopic perception of tilt, while large differences trigger binocular rivalry. In addition, unequal monocular contrasts combine nonlinearly to generate binocular contrast. A nonlinear neural model is developed here to account for binocular contrast, fusion at small orientation differences, and rivalry at large differences. The model also accounts for hysteresis in the transition between fusion and rivalry. Finally, the model predicts that interocular contrast differences between fusible gratings will produce a reduced tilt percept, and experiments reported here support this. Key to the model is the presence of two classes of inhibitory interneurons: one operating on similar orientations to normalize interocular contrast (IN), and one operating across large orientation differences to generate rivalry (IR). Critically, the IN neurons switch off the IR neurons driven by the other eye, thus permitting fusion of binocular plaids.

Face perception develops similarly across viewpoint in children and adolescents with and without autism spectrum disorder.

Atypical face perception has been associated with the socio-communicative difficulties that characterize autism spectrum disorder (ASD). Growing evidence, however, suggests that a widespread impairment in face perception is not as common as once thought. One important issue arising with the interpretation of this literature is the relationship between face processing and a more general perceptual tendency to focus on local rather than global information. Previous work has demonstrated that when discriminating faces presented from the same view, older adolescents and adults with ASD perform similarly to typically developing individuals. When faces are presented from different views, however, they perform more poorly-specifically, when access to local cues is minimized. In this study, we assessed the cross-sectional development of face identity discrimination across viewpoint using same- and different-view conditions in children and adolescents with and without ASD. Contrary to the findings in adults, our results revealed that all participants experienced greater difficulty identifying faces from different views than from same views, and demonstrated similar age-expected improvements in performance across tasks. These results suggest that differences in face discrimination across views may only emerge beyond the age of 15 years in ASD.

Impact of distributions on the archetypes and prototypes in heterogeneous nanoparticle ensembles.

The magnitude and complexity of the structural and functional data available on nanomaterials requires data analytics, statistical analysis and information technology to drive discovery. We demonstrate that multivariate statistical analysis can recognise the sets of truly significant nanostructures and their most relevant properties in heterogeneous ensembles with different probability distributions. The prototypical and archetypal nanostructures of five virtual ensembles of Si quantum dots (SiQDs) with Boltzmann, frequency, normal, Poisson and random distributions are identified using clustering and archetypal analysis, where we find that their diversity is defined by size and shape, regardless of the type of distribution. At the complex hull of the SiQD ensembles, simple configuration archetypes can efficiently describe a large number of SiQDs, whereas more complex shapes are needed to represent the average ordering of the ensembles. This approach provides a route towards the characterisation of computationally intractable virtual nanomaterial spaces, which can convert big data into smart data, and significantly reduce the workload to simulate experimentally relevant virtual samples.

Position selectivity in face-sensitive visual cortex to facial and nonfacial stimuli: an fMRI study.

BACKGROUND: Evidence for position sensitivity in object-selective visual areas has been building. On one hand, most of the relevant studies have utilized stimuli for which the areas are optimally selective and examine small sections of cortex. On the other hand, visual field maps established with nonspecific stimuli have been found in increasingly large areas of visual cortex, though generally not in areas primarily responsive to faces. METHODS: fMRI was used to study the position sensitivity of the occipital face area (OFA) and the fusiform face area (FFA) to both standard rotating wedge retinotopic mapping stimuli and quadrant presentations of synthetic facial stimuli. Analysis methods utilized were both typical, that is, mean univariate BOLD signals and multivoxel pattern analysis (MVPA), and novel, that is, distribution of voxels to pattern classifiers and use of responses to nonfacial retinotopic mapping stimuli to classify responses to facial stimuli. RESULTS: Polar angle sensitivity was exhibited to standard retinotopic mapping stimuli with a stronger contralateral bias in OFA than in FFA, a stronger bias toward the vertical meridian in FFA than in OFA, and a bias across both areas toward the inferior visual field. Contralateral hemispheric lateralization of both areas was again shown using synthetic face stimuli based on univariate BOLD signals, MVPA, and the biased contribution of voxels toward multivariate classifiers discriminating the contralateral visual field. Classifiers based on polar angle responsivity were used to classify the patterns of activation above chance levels to face stimuli in the OFA but not in the FFA. CONCLUSIONS: Both the OFA and FFA exhibit quadrant sensitivity to face stimuli, though the OFA exhibits greater position responsivity across stimuli than the FFA and includes overlap in the response pattern to the disparate stimulus types. Such biases are consistent with varying position sensitivity along different surfaces of occipito-temporal cortex.

A closer look at four-dot masking of a foveated target.

Four-dot masking with a common onset mask was recently demonstrated in a fully attended and foveated target (Filmer, Mattingley & Dux, 2015). Here, we replicate and extend this finding by directly comparing a four-dot mask with an annulus mask while probing masking as a function of mask duration, and target-mask separation. Our results suggest that while an annulus mask operates via spatially local contour interactions, a four-dot mask operates through spatially global mechanisms. We also measure how the visual system's representation of an oriented bar is impacted by a four-dot mask, and find that masking here does not degrade the precision of perceived targets, but instead appears to be driven exclusively by rendering the target completely invisible.

Detection of periodic motion trajectories: Effects of frequency and radius.

Periodic trajectories are an important component of biological motion. Or, Thabet, Wilkinson, and Wilson (2011) studied radial frequency (RF) motion trajectory detection and concluded that, for RF2-5 trajectories, the threshold function paralleled that of static RF patterns. We have extended Or et al.'s (2011) findings to a broader range of RFs (three to 24 cycles) and across a 4-fold range of radii (1°-4°). We report that (a) thresholds for RF trajectories decrease as a power function of RF for low RF trajectories (three to six cycles) before approaching an asymptote at high RFs (12-24 cycles); (b) detection thresholds for RF trajectories scale proportionally with radius; and (c) there is no lower versus upper field advantage in the parafoveal field for stimuli displaced from fixation on the vertical midline. The results are compared to earlier findings for static RF thresholds, and we argue that our findings support the existence of parallel spatial and temporal processing channels that may contribute to both action perception and production.

Effect of motion discontinuities on discrimination of periodic trajectories.

Many biologically important motions are described by periodic trajectories. Radial frequency (RF) trajectories are one example, in which the motion of a difference of Gaussians (DOG) target moves along a path described by a sinusoidal deviation of the radius from a perfect circle (Or, Thabet, Wilkinson, & Wilson, 2011). Here we explore the hypothesis that visual processing of RF trajectories involves global spatio-temporal processes that are disrupted by motion discontinuity. To test this hypothesis, RF trajectories were used that interspersed smooth, continuous motion with three or four discontinuous jumps to other portions of the trajectory. These jumps were arranged so that the entire trajectory was traversed in the same amount of time as in the continuous motion control condition. The motion discontinuities increased thresholds by a factor of approximately 2.1 relative to continuous motion. This result provides support for global spatio-temporal processing of RF motion trajectories. Comparison with previous results suggests that motion discontinuities erase memory for earlier parts of the trajectory, thereby causing thresholds to be based on only the final segment viewed. Finally, it is shown that RF trajectories obey the 1/3 power law characteristic of biological motion.

The Caledonian face test: A new test of face discrimination.

This study aimed to develop a clinical test of face perception which is applicable to a wide range of patients and can capture normal variability. The Caledonian face test utilises synthetic faces which combine simplicity with sufficient realism to permit individual identification. Face discrimination thresholds (i.e. minimum difference between faces required for accurate discrimination) were determined in an "odd-one-out" task. The difference between faces was controlled by an adaptive QUEST procedure. A broad range of face discrimination sensitivity was determined from a group (N=52) of young adults (mean 5.75%; SD 1.18; range 3.33-8.84%). The test is fast (3-4 min), repeatable (test-re-test r(2)=0.795) and demonstrates a significant inversion effect. The potential to identify impairments of face discrimination was evaluated by testing LM who reported a lifelong difficulty with face perception. While LM's impairment for two established face tests was close to the criterion for significance (Z-scores of -2.20 and -2.27) for the Caledonian face test, her Z-score was -7.26, implying a more than threefold higher sensitivity. The new face test provides a quantifiable and repeatable assessment of face discrimination ability. The enhanced sensitivity suggests that the Caledonian face test may be capable of detecting more subtle impairments of face perception than available tests.

Catalytic potential of highly defective (211) surfaces of zinc blende ZnO.

Zinc blende (ZB) ZnO has gained increasing research interest due to its favorable properties and its stabilization on the nanoscale. While surface properties are important on the nanoscale, the studies on ZB ZnO surface properties are rare. Here we have performed first principles calculations of the energies and structures of ZB and wurtzite (WZ) ZnO surfaces. Our results indicate that, among the four surfaces parallel to the polar axes, such as (101̄0) and (112̄0) of the WZ phase and (110) and (211) of the ZB phase, the polar (211) surface has substantially lower surface vacancy formation energies than the others, which makes ZB ZnO promising for catalytic applications. Our results also imply that the stabilization of ZB ZnO on the nanoscale is due to some mechanisms other than surface energies.

Masking with faces in central visual field under a variety of temporal schedules.

With a few exceptions, previous studies have explored masking using either a backward mask or a common onset trailing mask, but not both. In a series of experiments, we demonstrate the use of faces in central visual field as a viable method to study the relationship between these two types of mask schedule. We tested observers in a two alternative forced choice face identification task, where both target and mask comprised synthetic faces, and show that a simple model can successfully predict masking across a variety of masking schedules ranging from a backward mask to a common onset trailing mask and a number of intermediate variations. Our data are well accounted for by a window of sensitivity to mask interference that is centered at around 100 ms.