Mechanism and spectrum of inhibition of a 4´-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate (TP) metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Auditory sensory range of male mosquitoes for the detection of female flight sound.

Male mosquitoes detect and localize conspecific females by their flight-tones using the Johnston's organ (JO), which detects antennal deflections under the influence of local particle motion. Acoustic behaviours of mosquitoes and their JO physiology have been investigated extensively within the frequency domain, yet the auditory sensory range and the behaviour of males at the initiation of phonotactic flights are not well known. In this study, we predict a maximum spatial sensory envelope for flying Culex quinquefasciatus by integrating the physiological tuning response of the male JO with female aeroacoustic signatures derived from numerical simulations. Our sensory envelope predictions were tested with a behavioural assay of free-flying males responding to a female-like artificial pure tone. The minimum detectable particle velocity observed during flight tests was in good agreement with our theoretical prediction formed by the peak JO sensitivity measured in previous studies. The iso-surface describing the minimal detectable particle velocity represents the quantitative auditory sensory range of males and is directional with respect to the female body orientation. Our results illuminate the intricacy of the mating behaviour and point to the importance of observing the body orientation of flying mosquitoes to understand fully the sensory ecology of conspecific communication.

A semi-empirical model of the aerodynamics of manoeuvring insect flight.

Blade element modelling provides a quick analytical method for estimating the aerodynamic forces produced during insect flight, but such models have yet to be tested rigorously using kinematic data recorded from free-flying insects. This is largely because of the paucity of detailed free-flight kinematic data, but also because analytical limitations in existing blade element models mean that they cannot incorporate the complex three-dimensional movements of the wings and body that occur during insect flight. Here, we present a blade element model with empirically fitted aerodynamic force coefficients that incorporates the full three-dimensional wing kinematics of manoeuvring Eristalis hoverflies, including torsional deformation of their wings. The two free parameters were fitted to a large free-flight dataset comprising N = 26 541 wingbeats, and the fitted model captured approximately 80% of the variation in the stroke-averaged forces in the sagittal plane. We tested the robustness of the model by subsampling the data, and found little variation in the parameter estimates across subsamples comprising 10% of the flight sequences. The simplicity and generality of the model that we present is such that it can be readily applied to kinematic datasets from other insects, and also used for the study of insect flight dynamics.

Aerodynamic imaging by mosquitoes inspires a surface detector for autonomous flying vehicles.

Some flying animals use active sensing to perceive and avoid obstacles. Nocturnal mosquitoes exhibit a behavioral response to divert away from surfaces when vision is unavailable, indicating a short-range, mechanosensory collision-avoidance mechanism. We suggest that this behavior is mediated by perceiving modulations of their self-induced airflow patterns as they enter a ground or wall effect. We used computational fluid dynamics simulations of low-altitude and near-wall flights based on in vivo high-speed kinematic measurements to quantify changes in the self-generated pressure and velocity cues at the sensitive mechanosensory antennae. We validated the principle that encoding aerodynamic information can enable collision avoidance by developing a quadcopter with a sensory system inspired by the mosquito. Such low-power sensing systems have major potential for future use in safer rotorcraft control systems.

The cost-effectiveness of prophylaxis strategies for individuals with advanced HIV starting treatment in Africa.

INTRODUCTION: Many HIV-positive individuals in Africa have advanced disease when initiating antiretroviral therapy (ART) so have high risks of opportunistic infections and death. The REALITY trial found that an enhanced-prophylaxis package including fluconazole reduced mortality by 27% in individuals starting ART with CD4 <100 cells/mm3 . We investigated the cost-effectiveness of this enhanced-prophylaxis package versus other strategies, including using cryptococcal antigen (CrAg) testing, in individuals with CD4 <200 cells/mm3 or <100 cells/mm3 at ART initiation and all individuals regardless of CD4 count. METHODS: The REALITY trial enrolled from June 2013 to April 2015. A decision-analytic model was developed to estimate the cost-effectiveness of six management strategies in individuals initiating ART in the REALITY trial countries. Strategies included standard-prophylaxis, enhanced-prophylaxis, standard-prophylaxis with fluconazole; and three CrAg testing strategies, the first stratifying individuals to enhanced-prophylaxis (CrAg-positive) or standard-prophylaxis (CrAg-negative), the second to enhanced-prophylaxis (CrAg-positive) or enhanced-prophylaxis without fluconazole (CrAg-negative) and the third to standard-prophylaxis with fluconazole (CrAg-positive) or without fluconazole (CrAg-negative). The model estimated costs, life-years and quality-adjusted life-years (QALY) over 48 weeks using three competing mortality risks: cryptococcal meningitis; tuberculosis, serious bacterial infection or other known cause; and unknown cause. RESULTS: Enhanced-prophylaxis was cost-effective at cost-effectiveness thresholds of US$300 and US$500 per QALY with an incremental cost-effectiveness ratio (ICER) of US$157 per QALY in the CD4 <200 cells/mm3 population providing enhanced-prophylaxis components are sourced at lowest available prices. The ICER reduced in more severely immunosuppressed individuals (US$113 per QALY in the CD4 <100 cells/mm3 population) and increased in all individuals regardless of CD4 count (US$722 per QALY). Results were sensitive to prices of the enhanced-prophylaxis components. Enhanced-prophylaxis was more effective and less costly than all CrAg testing strategies as enhanced-prophylaxis still conveyed health gains in CrAg-negative patients and savings from targeting prophylaxis based on CrAg status did not compensate for costs of CrAg testing. CrAg testing strategies did not become cost-effective unless the price of CrAg testing fell below US$2.30. CONCLUSIONS: The REALITY enhanced-prophylaxis package in individuals with advanced HIV starting ART reduces morbidity and mortality, is practical to administer and is cost-effective. Efforts should continue to ensure that components are accessed at lowest available prices.

Motor output and control input in flapping flight: a compact model of the deforming wing kinematics of manoeuvring hoverflies.

Insects are conventionally modelled as controlling flight by varying a few summary kinematic parameters that are defined on a per-wingbeat basis, such as the stroke amplitude, mean stroke angle and mean wing pitch angle. Nevertheless, as insects have tens of flight muscles and vary their kinematics continuously, the true dimension of their control input space is likely to be much higher. Here, we present a compact description of the deforming wing kinematics of 36 manoeuvring Eristalis hoverflies, applying functional principal components analysis to Fourier series fits of the wingtip position and wing twist measured over 26 541 wingbeats. This analysis offers a high degree of data reduction, in addition to insight into the natural kinematic couplings. We used statistical resampling techniques to verify that the principal components (PCs) were repeatable features of the data, and analysed their coefficient vectors to provide insight into the form of these natural couplings. Conceptually, the dominant PCs provide a natural set of control input variables that span the control input subspace utilized by this species, but they can also be thought of as output states of the flight motor. This functional description of the wing kinematics is appropriate to modelling insect flight as a form of limit cycle control.

Smart wing rotation and trailing-edge vortices enable high frequency mosquito flight.

Mosquitoes exhibit unusual wing kinematics; their long, slender wings flap at remarkably high frequencies for their size (>800 Hz)and with lower stroke amplitudes than any other insect group. This shifts weight support away from the translation-dominated, aerodynamic mechanisms used by most insects, as well as by helicopters and aeroplanes, towards poorly understood rotational mechanisms that occur when pitching at the end of each half-stroke. Here we report free-flight mosquito wing kinematics, solve the full Navier-Stokes equations using computational fluid dynamics with overset grids, and validate our results with in vivo flow measurements. We show that, although mosquitoes use familiar separated flow patterns, much of the aerodynamic force that supports their weight is generated in a manner unlike any previously described for a flying animal. There are three key features: leading-edge vortices (a well-known mechanism that appears to be almost ubiquitous in insect flight), trailing-edge vortices caused by a form of wake capture at stroke reversal, and rotational drag. The two new elements are largely independent of the wing velocity, instead relying on rapid changes in the pitch angle (wing rotation) at the end of each half-stroke, and they are therefore relatively immune to the shallow flapping amplitude. Moreover, these mechanisms are particularly well suited to high aspect ratio mosquito wings.

Four-dimensional in vivo X-ray microscopy with projection-guided gating.

Visualizing fast micrometer scale internal movements of small animals is a key challenge for functional anatomy, physiology and biomechanics. We combine phase contrast tomographic microscopy (down to 3.3 µm voxel size) with retrospective, projection-based gating (in the order of hundreds of microseconds) to improve the spatiotemporal resolution by an order of magnitude over previous studies. We demonstrate our method by visualizing 20 three-dimensional snapshots through the 150 Hz oscillations of the blowfly flight motor.

In vivo time-resolved microtomography reveals the mechanics of the blowfly flight motor.

Dipteran flies are amongst the smallest and most agile of flying animals. Their wings are driven indirectly by large power muscles, which cause cyclical deformations of the thorax that are amplified through the intricate wing hinge. Asymmetric flight manoeuvres are controlled by 13 pairs of steering muscles acting directly on the wing articulations. Collectively the steering muscles account for <3% of total flight muscle mass, raising the question of how they can modulate the vastly greater output of the power muscles during manoeuvres. Here we present the results of a synchrotron-based study performing micrometre-resolution, time-resolved microtomography on the 145 Hz wingbeat of blowflies. These data represent the first four-dimensional visualizations of an organism's internal movements on sub-millisecond and micrometre scales. This technique allows us to visualize and measure the three-dimensional movements of five of the largest steering muscles, and to place these in the context of the deforming thoracic mechanism that the muscles actuate. Our visualizations show that the steering muscles operate through a diverse range of nonlinear mechanisms, revealing several unexpected features that could not have been identified using any other technique. The tendons of some steering muscles buckle on every wingbeat to accommodate high amplitude movements of the wing hinge. Other steering muscles absorb kinetic energy from an oscillating control linkage, which rotates at low wingbeat amplitude but translates at high wingbeat amplitude. Kinetic energy is distributed differently in these two modes of oscillation, which may play a role in asymmetric power management during flight control. Structural flexibility is known to be important to the aerodynamic efficiency of insect wings, and to the function of their indirect power muscles. We show that it is integral also to the operation of the steering muscles, and so to the functional flexibility of the insect flight motor.

Operation of the alula as an indicator of gear change in hoverflies.

The alula is a hinged flap found at the base of the wings of most brachyceran Diptera. The alula accounts for up to 10 per cent of the total wing area in hoverflies (Syrphidae), and its hinged arrangement allows the wings to be swept back over the thorax and abdomen at rest. The alula is actuated via the third axillary sclerite, which is a component of the wing hinge that is involved in wing retraction and control. The third axillary sclerite has also been implicated in the gear change mechanism of flies. This mechanism allows rapid switching between different modes of wing kinematics, by imposing or removing contact with a mechanical stop limiting movement of the wing during the lower half of the downstroke. The alula operates in two distinct states during flight-flipped or flat-and we hypothesize that its state indicates switching between different flight modes. We used high-speed digital video of free-flying hoverflies (Eristalis tenax and Eristalis pertinax) to investigate whether flipping of the alula was associated with changes in wing and body kinematics. We found that alula state was associated with different distributions of multiple wing kinematic parameters, including stroke amplitude, stroke deviation angle, downstroke angle of incidence and timing of supination. Changes in all of these parameters have previously been linked to gear change in flies. Symmetric flipping of the alulae was associated with changes in the symmetric linear acceleration of the body, while asymmetric flipping of the alulae was associated with asymmetric angular acceleration of the body. We conclude that the wings produce less aerodynamic force when the alula is flipped, largely as a result of the accompanying changes in wing kinematics. The alula changes state at mid-downstroke, which is the point at which the gear change mechanism is known to come into effect. This transition is accompanied by changes in the other wing kinematic parameters. We therefore find that the state of the alula is linked to the same parameters as are affected by the gear change mechanism. We conclude that the state of the alula does indeed indicate the operation of different flight modes in Eristalis, and infer that a likely mechanism for these changes in flight mode is the gear change mechanism.

Deformable wing kinematics in free-flying hoverflies.

Here, we present a detailed analysis of the deforming wing kinematics of free-flying hoverflies (Eristalis tenax, Linnaeus) during hovering flight. We used four high-speed digital video cameras to reconstruct the motion of approximately 22 points on each wing using photogrammetric techniques. While the root-flapping motion of the wing is similar in both the downstroke and upstroke, and is well modelled as a simple harmonic motion, other wing kinematic parameters show substantial variation between the downstroke and upstroke. Whereas the magnitude of the angle of incidence varies considerably within and between different hoverflies, the twist distribution along the wing is highly stereotyped. The angle of incidence and camber both show a recoil effect as they change abruptly at stroke reversal. Pronation occurs consistently after stroke reversal, which is perhaps surprising, because this has been found to reduce lift production in modelling studies. We find that the alula, a hinged flap near the base of the wing, operates in two discrete states: either in plane with the wing, or flipped approximately normal to it. We hypothesize that the alula may be acting as a flow-control device.

The typical flight performance of blowflies: measuring the normal performance envelope of Calliphora vicina using a novel corner-cube arena.

Despite a wealth of evidence demonstrating extraordinary maximal performance, little is known about the routine flight performance of insects. We present a set of techniques for benchmarking performance characteristics of insects in free flight, demonstrated using a model species, and comment on the significance of the performance observed. Free-flying blowflies (Calliphora vicina) were filmed inside a novel mirrored arena comprising a large (1.6 m1.6 m1.6 m) corner-cube reflector using a single high-speed digital video camera (250 or 500 fps). This arrangement permitted accurate reconstruction of the flies' 3-dimensional trajectories without the need for synchronisation hardware, by virtue of the multiple reflections of a subject within the arena. Image sequences were analysed using custom-written automated tracking software, and processed using a self-calibrating bundle adjustment procedure to determine the subject's instantaneous 3-dimensional position. We illustrate our method by using these trajectory data to benchmark the routine flight performance envelope of our flies. Flight speeds were most commonly observed between 1.2 ms(-1) and 2.3 ms(-1), with a maximum of 2.5 ms(-1). Our flies tended to dive faster than they climbed, with a maximum descent rate (-2.4 ms(-1)) almost double the maximum climb rate (1.2 ms(-1)). Modal turn rate was around 240 degrees s(-1), with maximal rates in excess of 1700 degrees s(-1). We used the maximal flight performance we observed during normal flight to construct notional physical limits on the blowfly flight envelope, and used the distribution of observations within that notional envelope to postulate behavioural preferences or physiological and anatomical constraints. The flight trajectories we recorded were never steady: rather they were constantly accelerating or decelerating, with maximum tangential accelerations and maximum centripetal accelerations on the order of 3 g.

Details of insect wing design and deformation enhance aerodynamic function and flight efficiency.

Insect wings are complex structures that deform dramatically in flight. We analyzed the aerodynamic consequences of wing deformation in locusts using a three-dimensional computational fluid dynamics simulation based on detailed wing kinematics. We validated the simulation against smoke visualizations and digital particle image velocimetry on real locusts. We then used the validated model to explore the effects of wing topography and deformation, first by removing camber while keeping the same time-varying twist distribution, and second by removing camber and spanwise twist. The full-fidelity model achieved greater power economy than the uncambered model, which performed better than the untwisted model, showing that the details of insect wing topography and deformation are important aerodynamically. Such details are likely to be important in engineering applications of flapping flight.

Deformable wing kinematics in the desert locust: how and why do camber, twist and topography vary through the stroke?

Here, we present a detailed analysis of the wing kinematics and wing deformations of desert locusts (Schistocerca gregaria, Forskål) flying tethered in a wind tunnel. We filmed them using four high-speed digital video cameras, and used photogrammetry to reconstruct the motion of more than 100 identified points. Whereas the hindwing motions were highly stereotyped, the forewing motions showed considerable variation, consistent with a role in flight control. Both wings were positively cambered on the downstroke. The hindwing was cambered through an 'umbrella effect' whereby the trailing edge tension compressed the radial veins during the downstroke. Hindwing camber was reversed on the upstroke as the wing fan corrugated, reducing the projected area by 30 per cent, and releasing the tension in the trailing edge. Both the wings were strongly twisted from the root to the tip. The linear decrease in incidence along the hindwing on the downstroke precisely counteracts the linear increase in the angle of attack that would otherwise occur in root flapping for an untwisted wing. The consequent near-constant angle of attack is reminiscent of the optimum for a propeller of constant aerofoil section, wherein a linear twist distribution allows each section to operate at the unique angle of attack maximizing the lift to drag ratio. This implies tuning of the structural, morphological and kinematic parameters of the hindwing for efficient aerodynamic force production.

Photogrammetric reconstruction of high-resolution surface topographies and deformable wing kinematics of tethered locusts and free-flying hoverflies.

Here, we present a suite of photogrammetric methods for reconstructing insect wing kinematics, to provide instantaneous topographic maps of the wing surface. We filmed tethered locusts (Schistocerca gregaria) and free-flying hoverflies (Eristalis tenax) using four high-speed digital video cameras. We digitized multiple natural features and marked points on the wings using manual and automated tracking. Epipolar geometry was used to identify additional points on the hoverfly wing outline which were anatomically indistinguishable. The cameras were calibrated using a bundle adjustment technique that provides an estimate of the error associated with each individual data point. The mean absolute three-dimensional measurement error was 0.11 mm for the locust and 0.03 mm for the hoverfly. The error in the angle of incidence was at worst 0.51 degrees (s.d.) for the locust and 0.88 degrees (s.d.) for the hoverfly. The results we present are of unprecedented spatio-temporal resolution, and represent the most detailed measurements of insect wing kinematics to date. Variable spanwise twist and camber are prominent in the wingbeats of both the species, and are of such complexity that they would not be adequately captured by lower resolution techniques. The role of spanwise twist and camber in insect flight has yet to be fully understood, and accurate insect wing kinematics such as we present here are required to be sure of making valid predictions about their aerodynamic effects.

New experimental approaches to the biology of flight control systems.

Here we consider how new experimental approaches in biomechanics can be used to attain a systems-level understanding of the dynamics of animal flight control. Our aim in this paper is not to provide detailed results and analysis, but rather to tackle several conceptual and methodological issues that have stood in the way of experimentalists in achieving this goal, and to offer tools for overcoming these. We begin by discussing the interplay between analytical and empirical methods, emphasizing that the structure of the models we use to analyse flight control dictates the empirical measurements we must make in order to parameterize them. We then provide a conceptual overview of tethered-flight paradigms, comparing classical ;open-loop' and ;closed-loop' setups, and describe a flight simulator that we have recently developed for making flight dynamics measurements on tethered insects. Next, we provide a conceptual overview of free-flight paradigms, focusing on the need to use system identification techniques in order to analyse the data they provide, and describe two new techniques that we have developed for making flight dynamics measurements on freely flying birds. First, we describe a technique for obtaining inertial measurements of the orientation, angular velocity and acceleration of a steppe eagle Aquila nipalensis in wide-ranging free flight, together with synchronized measurements of wing and tail kinematics using onboard instrumentation and video cameras. Second, we describe a photogrammetric method to measure the 3D wing kinematics of the eagle during take-off and landing. In each case, we provide demonstration data to illustrate the kinds of information available from each method. We conclude by discussing the prospects for systems-level analyses of flight control using these techniques and others like them.