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GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate G s heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling.

A faster cruising speed increases drag and thereby the thrust ( T ) needed to fly, while weight and lift ( L ) requirement remains constant. Birds can adjust their wingbeat in multiple ways to accommodate this change in aerodynamic force, but the relative costs of different strategies remain largely unknown. To evaluate the efficiency of several kinematic strategies, I used a robotic wing [E. Ajanic, A. Paolini, C. Coster, D. Floreano, C. Johansson, Adv. Intell. Syst. 5 , 2200148 (2023)] and quantitative flow measurements. I found that, among the tested strategies, changing the mean wingbeat elevation provides the most efficient solution to changing thrust-to-lift ratio ( T / L) , offering insight into why birds tend to beat their wings with a greater ventral than dorsal excursion. I also found that although propulsive efficiency (η p ) may peak at a Strouhal number ( St , measure of relative flapping speed) near 0.3, the overall efficiency of generating force decreases with St . This challenges the expectance of a specific optimal St for flapping flight and instead suggest the chosen St depends on T / L . This may explain variation in preferred St among birds and why bats prefer flying at higher St , 707-711 (2003)], since their body shape imposes relatively higher thrust requirements [F. T. Muijres, L. C. Johansson, M. S. Bowlin, Y. Winter, A. Hedenström, Nature 425 , 707-711 (2003)], since their body shape imposes relatively higher thrust requirements [F. T. Muijres, L. C. Johansson, M. S. Bowlin, Y. Winter, A. Hedenström, PLoS One 7 , e37335 (2012)]. In addition to explaining flapping strategies used by birds, my results suggest alternative, efficient, flapping motions for drones to explore aiming to extend their flight range., Competing Interests: Competing interests statement:The author declares no competing interest.

Background: Complications of orthognathic surgery are quite rare, but they cause suffering in affected individuals. The range of complications is broad and includes both hard and soft tissue., Case Presentation: We here present a case of a fully healthy woman without signs of impaired healing capacity. The patient underwent bimaxillary orthognathic surgery and experienced multiple complications both peri- and post-operatively. During the post operative period, the patient also suffered from soft tissue complications after an orthopaedic injury. Therefore, we referred the patient to her general practitioner for further medical investigation. We also present the result after restorative surgery and endodontic and prosthodontic treatment resulting in a successful rehabilitation., Conclusion: This case report clearly shows the need for a good collaboration between different odontological and medical fields to achieve a good and predictable result. In situations where normal healing processes do not occur, in-depth analysis must be carried out., Highlights: Orthognathic surgery affects soft and hard tissue which can result in adverse healing and complications. It is of great importance to follow up performed surgery to see late complications. Be restrictive with early re-operations when there are signs of necrosis. Always use a multidisciplinary approach when handling complications after surgery., (© 2023. The Author(s).)

The efficiency with which flying animals convert metabolic power to mechanical power dictates an individual's flight behaviour and energy requirements. Despite the significance of this parameter, we lack empirical data on conversion efficiency for most species as in vivo measurements are notoriously difficult to obtain. Furthermore, conversion efficiency is often assumed to be constant across flight speeds, even though the components driving flight power are speed-dependent. We show, through direct measurements of metabolic and aerodynamic power, that conversion efficiency in the migratory bat ( Pipistrellus nathusii ) increases from 7.0 to 10.4% with flight speed. Our findings suggest that peak conversion efficiency in this species occurs near maximum range speed, where the cost of transport is minimized. A meta-analysis of 16 bird and 8 bat species revealed a positive scaling relationship between estimated conversion efficiency and body mass, with no discernible differences between bats and birds. This has profound consequences for modelling flight behaviour as estimates assuming 23% efficiency underestimate metabolic costs for P. nathusii by almost 50% on average (36-62%). Our findings suggest that conversion efficiency may vary around an ecologically relevant optimum speed and provide a crucial baseline for investigating whether this drives variation in conversion efficiency between species.

For all flyers, aeroplanes or animals, making banked turns involve a rolling motion which, due to higher induced drag on the outer than the inner wing, results in a yawing torque opposite to the turn. This adverse yaw torque can be counteracted using a tail, but how animals that lack tail, e.g. all insects, handle this problem is not fully understood. Here, we quantify the performance of turning take-off flights in butterflies and find that they use force vectoring during banked turns without fully compensating for adverse yaw. This lowers their turning performance, increasing turn radius, since thrust becomes misaligned with the flight path. The separation of function between downstroke (lift production) and upstroke (thrust production) in our butterflies, in combination with a more pronounced adverse yaw during the upstroke increases the misalignment of the thrust. This may be a cost the butterflies pay for the efficient thrust-generating upstroke clap, but also other insects fail to rectify adverse yaw during escape manoeuvres, suggesting a general feature in functionally two-winged insect flight. When lacking tail and left with costly approaches to counteract adverse yaw, costs of flying with adverse yaw may be outweighed by the benefits of maintaining thrust and flight speed.

Hovering insects are divided into two categories: 'normal' hoverers that move the wing symmetrically in a horizontal stroke plane, and those with an inclined stroke plane. Normal hoverers have been suggested to support their weight during both downstroke and upstroke, shedding vortex rings each half-stroke. Insects with an inclined stroke plane should, according to theory, produce flight forces only during downstroke, and only generate one set of vortices. The type of hovering is thus linked to the power required to hover. Previous efforts to characterize the wake of hovering insects have used low-resolution experimental techniques or simulated the flow using computational fluid dynamics, and so it remains to be determined whether insect wakes can be represented by any of the suggested models. Here, we used tomographic particle image velocimetry, with a horizontal measurement volume placed below the animals, to show that the wake shed by hovering hawkmoths is best described as a series of bilateral, stacked vortex 'rings'. While the upstroke is aerodynamically active, despite an inclined stroke plane, it produces weaker vortices than the downstroke. In addition, compared with the near wake, the far wake lacks structure and is less concentrated. Both near and far wakes are clearly affected by vortex interactions, suggesting caution is required when interpreting wake topologies. We also estimated induced power (Pind) from downwash velocities in the wake. Standard models predicted a Pind more than double that from our wake measurements. Our results thus question some model assumptions and we propose a reevaluation of the model parameters., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Butterflies look like no other flying animal, with unusually short, broad and large wings relative to their body size. Previous studies have suggested butterflies use several unsteady aerodynamic mechanisms to boost force production with upstroke wing clap being a prominent feature. When the wings clap together at the end of upstroke the air between the wings is pressed out, creating a jet, pushing the animal in the opposite direction. Although viewed, for the last 50 years, as a crucial mechanism in insect flight, quantitative aerodynamic measurements of the clap in freely flying animals are lacking. Using quantitative flow measurements behind freely flying butterflies during take-off and a mechanical clapper, we provide aerodynamic performance estimates for the wing clap. We show that flexible butterfly wings, forming a cupped shape during the upstroke and clap, thrust the butterfly forwards, while the downstroke is used for weight support. We further show that flexible wings dramatically increase the useful impulse (+22%) and efficiency (+28%) of the clap compared to rigid wings. Combined, our results suggest butterflies evolved a highly effective clap, which provides a mechanistic hypothesis for their unique wing morphology. Furthermore, our findings could aid the design of man-made flapping drones, boosting propulsive performance.

Cost of flight at various speeds is a crucial determinant of flight behaviour in birds. Aerodynamic models, predicting that mechanical power ( P mech ) varies with flight speed in a U-shaped manner, have been used together with an energy conversion factor (efficiency) to estimate metabolic power ( P met ) . Despite few empirical studies, efficiency has been assumed constant across flight speeds at 23%. Ideally, efficiency should be estimated from measurements of both P mech and P met in un-instrumented flight . Until recently, progress has been hampered by methodological constraints. The main aim of this study was to evaluate recently developed techniques and estimate flight efficiency across flight speeds. We used the 13 C-labelled sodium bicarbonate method (NaBi) and particle image velocimetry (PIV) to measure P met and P mech in blackcaps flying in a wind tunnel. We also cross-validated measurements made by NaBi with quantitative magnetic resonance (QMR) body composition analysis in yellow-rumped warblers. We found that P met estimated by NaBi was ∼12% lower than corresponding values estimated by QMR. P met varied in a U-shaped manner across flight speeds in blackcaps, but the pattern was not statistically significant. P mech could only be reliably measured for two intermediate speeds and estimated efficiency ranged between 14% and 22% (combining the two speeds for raw and weight/lift-specific power, with and without correction for the ∼12% difference between NaBi and QMR), which were close to the currently used default value. We conclude that NaBi and PIV are viable techniques, allowing researchers to address some of the outstanding questions regarding bird flight energetics., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The long-anticipated high-resolution structures of the human melatonin G protein-coupled receptors MT 1 and MT 2 , involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti-insomnia and antidepression drugs using X-ray free-electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane-buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT 2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT 2 structure allowed accurate mapping of type 2 diabetes-related single-nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein-membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT 1 and MT 2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next-generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects., (© 2019 Federation of European Biochemical Societies.)

Melatonin receptors MT 1 and MT 2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC 50 of 0.36 nM. Six of these molecules displayed selectivity for MT 2 over MT 1 . Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28 , had dramatically reduced arrestin recruitment at MT 2 , while compound 37 was devoid of G i signaling at MT 1 , implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists., Competing Interests: NP, XH, JG, LJ, BS, JM, YL, BR, VK No competing interests declared, (© 2020, Patel et al.)

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT 1 and MT 2 . Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle 1-4 . The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep 5,6 and depression 1-4,7-9 . Despite their importance, few in vivo active MT 1 -selective ligands have been reported 2,8,10-12 , hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT 1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT 1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT 1 -selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT 1 - but not in MT 2 -knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT 1 -selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.

The human MT 1 and MT 2 melatonin receptors 1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns 3 . Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer 3 , and MT 2 has also been implicated in type 2 diabetes 4,5 . Here we report X-ray free electron laser (XFEL) structures of the human MT 2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon 6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H208 5.46 A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature 7 ) and N86 2.50 D, obtained in complex with 2-PMT. Comparison of the structures of MT 2 with a published structure 8 of MT 1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [ 3 H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT 1 and MT 2 , but in addition the MT 2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT 2 . Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms 1 by synchronization to environmental cues and is involved in diverse physiological processes 2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function 3 . Melatonin is formed in the pineal gland in a light-regulated manner 4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness 5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT 1 ) and type 1B (MT 2 ) 3,6 . Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden 7 . Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids 8,9 , and is one of the most popular supplements in the United States 10 . Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT 1 in complex with four agonists: the insomnia drug ramelteon 11 , two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine 12,13 . The structure of MT 2 is described in an accompanying paper 14 . Although the MT 1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT 1 , access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT 1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Most flying animals, from insects to seabirds [1], perform flights close to ground or water when taking off or landing [2], drinking, and feeding [3-5] or when traveling near water surfaces [1, 6, 7]. When flying close to a surface within approximately one wingspan, the surface acts as an aerodynamic mirror, interrupting the downwash [8, 9], resulting in increased pressure underneath the wing and suppression of wingtip vortex development [10]. This aerodynamic interaction lowers the energy added to the air by the animal, reducing the cost of flying. Modeling suggests that flapping wings in ground effect can affect the expected power savings compared to gliding flight, either positively or negatively, depending on the wing motion [11-13]. Although aerodynamic theory predicts substantial power reductions when animals fly in ground effect [4-6, 9, 11, 12], quantitative measurements of savings are lacking. Here, we show, through wake-based power measurements, that Daubenton's bats utilize 29% less aerodynamic power when flying in compared to out of ground effect, which is twice the predicted savings. Contrary to theoretical predictions [4-6, 9, 11, 12] we find no variation in savings with distance above ground when in ground effect. Given alterations in kinematics with ground proximity, we hypothesize that modulation of wing kinematics raises the achievable benefit from ground effect relative to current model predictions. The savings from ground effect are comparable to formation flight [14, 15] but are not limited to large bird species. Instead, ground effect is experienced by most flying animals and may have facilitated the evolution of powered animal flight., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

How aerodynamic power required for animal flight varies with flight speed determines optimal speeds during foraging and migratory flight. Despite its relevance, aerodynamic power provides an elusive quantity to measure directly in animal flight. Here, we determine the aerodynamic power from wake velocity fields, measured using tomographical particle image velocimetry, of pied flycatchers flying freely in a wind tunnel. We find a shallow U-shaped power curve, which is flatter than expected by theory. Based on how the birds vary body angle with speed, we speculate that the shallow curve results from increased body drag coefficient and body frontal area at lower flight speeds. Including modulation of body drag in the model results in a more reasonable fit with data than the traditional model. From the wake structure, we also find a single starting vortex generated from the two wings during the downstroke across flight speeds (1-9 m s -1 ). This is accomplished by the arm wings interacting at the beginning of the downstroke, generating a unified starting vortex above the body of the bird. We interpret this as a mechanism resulting in a rather uniform downwash and low induced power, which can help explain the higher aerodynamic performance in birds compared with bats., (© 2018 The Author(s).)

Bats navigate the dark using echolocation. Echolocation is enhanced by external ears, but external ears increase the projected frontal area and reduce the streamlining of the animal. External ears are thus expected to compromise flight efficiency, but research suggests that very large ears may mitigate the cost by producing aerodynamic lift. Here we compare quantitative aerodynamic measures of flight efficiency of two bat species, one large-eared ( Plecotus auritus ) and one small-eared ( Glossophaga soricina ), flying freely in a wind tunnel. We find that the body drag of both species is higher than previously assumed and that the large-eared species has a higher body drag coefficient, but also produces relatively more ear/body lift than the small-eared species, in line with prior studies on model bats. The measured aerodynamic power of P. auritus was higher than predicted from the aerodynamic model, while the small-eared species aligned with predictions. The relatively higher power of the large-eared species results in lower optimal flight speeds and our findings support the notion of a trade-off between the acoustic benefits of large external ears and aerodynamic performance. The result of this trade-off would be the eco-morphological correlation in bat flight, with large-eared bats generally adopting slow-flight feeding strategies., (© 2017 The Author(s).)

X-ray free electron lasers (XFELs) have the potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance, and short duration of X-ray pulses. A recently emerged serial femtosecond (fs) crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense fs XFEL pulses enable high-resolution room-temperature structure determination of difficult-to-crystallize biological macromolecules, while simultaneously opening a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods, and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Slotted wing tips of birds are commonly considered an adaptation to improve soaring performance, despite their presence in species that neither soar nor glide. We used particle image velocimetry to measure the airflow around the slotted wing tip of a jackdaw ( Corvus monedula ) as well as in its wake during unrestrained flight in a wind tunnel. The separated primary feathers produce individual wakes, confirming a multi-slotted function, in both gliding and flapping flight. The resulting multi-cored wingtip vortex represents a spreading of vorticity, which has previously been suggested as indicative of increased aerodynamic efficiency. Considering benefits of the slotted wing tips that are specific to flapping flight combined with the wide phylogenetic occurrence of this configuration, we propose the hypothesis that slotted wings evolved initially to improve performance in powered flight., (© 2017 The Author(s).)

Large ears enhance perception of echolocation and prey generated sounds in bats. However, external ears likely impair aerodynamic performance of bats compared to birds. But large ears may generate lift on their own, mitigating the negative effects. We studied flying brown long-eared bats, using high resolution, time resolved particle image velocimetry, to determine the aerodynamics of flying with large ears. We show that the ears and body generate lift at medium to cruising speeds (3-5 m/s), but at the cost of an interaction with the wing root vortices, likely reducing inner wing performance. We also propose that the bats use a novel wing pitch mechanism at the end of the upstroke generating thrust at low speeds, which should provide effective pitch and yaw control. In addition, the wing tip vortices show a distinct spiraling pattern. The tip vortex of the previous wingbeat remains into the next wingbeat and rotates together with a newly formed tip vortex. Several smaller vortices, related to changes in circulation around the wing also spiral the tip vortex. Our results thus show a new level of complexity in bat wakes and suggest large eared bats are less aerodynamically limited than previous wake studies have suggested.

The use of (90)Y resin microspheres (SIR-Spheres® microspheres) in Nuclear Medicine has dramatically increased in recent years due to its favourable outcome in the treatment of liver cancer and liver metastases (Rajekar et al., 2011). The measurement of administered activity before and residual activity after treatment in radionuclide calibrators is required to determine total activity delivered to the patient. In comparison with External Beam Radiotherapy (EBRT) where administered doses are often know to within ±5%, the actual administered activity in nuclear medicine procedures may only be known to within ±20% and subsequent dose calculations can result in even larger uncertainties (Fenwick et al., 2009). It is a well-recognised issue that ion chambers are instruments that are sensitive to the measurement geometry and matrix of a source, in particular for pure beta or low energy (<100keV) x-ray emitters (Gadd et al., 2006). This paper presents new calibration factors for NPL secondary standard ionisation chamber system (Vinten 671) and a Capintec CRC-25R radionuclide calibrator along with a discussion of the measurement problems associated with this radionuclide and matrix. Calibration of the NPL secondary standard system for this measurement matrix will enable NPL to provide standards for the Nuclear Medicine community and consequently increase the measurement capability., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hovering means stationary flight at zero net forward speed, which can be achieved by animals through muscle powered flapping flight. Small bats capable of hovering typically do so with a downstroke in an inclined stroke plane, and with an aerodynamically active outer wing during the upstroke. The magnitude and time history of aerodynamic forces should be reflected by vorticity shed into the wake. We thus expect hovering bats to generate a characteristic wake, but this has until now never been studied. Here we trained nectar-feeding bats, Leptonycteris yerbabuenae, to hover at a feeder and using time-resolved stereoscopic particle image velocimetry in conjunction with high-speed kinematic analysis we show that hovering nectar-feeding bats produce a series of bilateral stacked vortex loops. Vortex visualizations suggest that the downstroke produces the majority of the weight support, but that the upstroke contributes positively to the lift production. However, the relative contributions from downstroke and upstroke could not be determined on the basis of the wake, because wake elements from down- and upstroke mix and interact. We also use a modified actuator disc model to estimate lift force, power and flap efficiency. Based on our quantitative wake-induced velocities, the model accounts for weight support well (108%). Estimates of aerodynamic efficiency suggest hovering flight is less efficient than forward flapping flight, while the overall energy conversion efficiency (mechanical power output/metabolic power) was estimated at 13%., (© 2015 The Author(s).)

Animal flight performance has been studied using models developed for man-made aircraft. For an aeroplane with fixed wings, the energetic cost as a function of flight speed can be expressed in terms of weight, wing span, wing area and body area, where more details are included in proportionality coefficients. Flying animals flap their wings to produce thrust. Adopting the fixed wing flight model implicitly incorporates the effects of wing flapping in the coefficients. However, in practice, these effects have been ignored. In this paper, the effects of reciprocating wing motion on the coefficients of the fixed wing aerodynamic power model for forward flight are explicitly formulated in terms of thrust requirement, wingbeat frequency and stroke-plane angle, for optimized wingbeat amplitudes. The expressions are obtained by simulating flights over a large parameter range using an optimal vortex wake method combined with a low-level blade element method. The results imply that previously assumed acceptable values for the induced power factor might be strongly underestimated. The results also show the dependence of profile power on wing kinematics. The expressions introduced in this paper can be used to significantly improve animal flight models.

Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested., (Copyright © 2015, American Association for the Advancement of Science.)

Bats evolved the ability of powered flight more than 50 million years ago. The modern bat is an efficient flyer and recent research on bat flight has revealed many intriguing facts. By using particle image velocimetry to visualize wake vortices, both the magnitude and time-history of aerodynamic forces can be estimated. At most speeds the downstroke generates both lift and thrust, whereas the function of the upstroke changes with forward flight speed. At hovering and slow speed bats use a leading edge vortex to enhance the lift beyond that allowed by steady aerodynamics and an inverted wing during the upstroke to further aid weight support. The bat wing and its skeleton exhibit many features and control mechanisms that are presumed to improve flight performance. Whereas bats appear aerodynamically less efficient than birds when it comes to cruising flight, they have the edge over birds when it comes to manoeuvring. There is a direct relationship between kinematics and the aerodynamic performance, but there is still a lack of knowledge about how (and if) the bat controls the movements and shape (planform and camber) of the wing. Considering the relatively few bat species whose aerodynamic tracks have been characterized, there is scope for new discoveries and a need to study species representing more extreme positions in the bat morphospace., (© 2015. Published by The Company of Biologists Ltd.)

We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

The Leading Edge Vortex (LEV) is a universal mechanism enhancing lift in flying organisms. LEVs, generally illustrated as a single vortex attached to the wing throughout the downstroke, have not been studied quantitatively in freely flying insects. Previous findings are either qualitative or from flappers and tethered insects. We measure the flow above the wing of freely flying hawkmoths and find multiple simultaneous LEVs of varying strength and structure along the wingspan. At the inner wing there is a single, attached LEV, while at mid wing there are multiple LEVs, and towards the wingtip flow separates. At mid wing the LEV circulation is ~40% higher than in the wake, implying that the circulation unrelated to the LEV may reduce lift. The strong and complex LEV suggests relatively high flight power in hawmoths. The variable LEV structure may result in variable force production, influencing flight control in the animals.

The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.

Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.

The morphology and kinematics of a flying animal determines the resulting aerodynamic lift through the regulation of the speed of the air moving across the wing, the wing area and the lift coefficient. We studied the detailed three-dimensional wingbeat kinematics of the bat, Leptonycteris yerbabuenae, flying in a wind tunnel over a range of flight speeds (0-7 m/s), to determine how factors affecting the lift production vary across flight speed and within wingbeats. We found that the wing area, the angle of attack and the camber, which are determinants of the lift production, decreased with increasing speed. The camber is controlled by multiple mechanisms along the span, including the deflection of the leg relative to the body, the bending of the fifth digit, the deflection of the leading edge flap and the upward bending of the wing tip. All these measures vary throughout the wing beat suggesting active or aeroelastic control. The downstroke Strouhal number, St(d), is kept relatively constant, suggesting that favorable flow characteristics are maintained during the downstroke, across the range of speeds studied. The St(d) is kept constant through changes in the stroke plane, from a strongly inclined stroke plane at low speeds to a more vertical stroke plane at high speeds. The mean angular velocity of the wing correlates with the aerodynamic performance and shows a minimum at the speed of maximum lift to drag ratio, suggesting a simple way to determine the optimal speed from kinematics alone. Taken together our results show the high degree of adjustments that the bats employ to fine tune the aerodynamics of the wings and the correlation between kinematics and aerodynamic performance.

Flying insects typically possess two pairs of wings. In beetles, the front pair has evolved into short, hardened structures, the elytra, which protect the second pair of wings and the abdomen. This allows beetles to exploit habitats that would otherwise cause damage to the wings and body. Many beetles fly with the elytra extended, suggesting that they influence aerodynamic performance, but little is known about their role in flight. Using quantitative measurements of the beetle's wake, we show that the presence of the elytra increases vertical force production by approximately 40 per cent, indicating that they contribute to weight support. The wing-elytra combination creates a complex wake compared with previously studied animal wakes. At mid-downstroke, multiple vortices are visible behind each wing. These include a wingtip and an elytron vortex with the same sense of rotation, a body vortex and an additional vortex of the opposite sense of rotation. This latter vortex reflects a negative interaction between the wing and the elytron, resulting in a single wing span efficiency of approximately 0.77 at mid downstroke. This is lower than that found in birds and bats, suggesting that the extra weight support of the elytra comes at the price of reduced efficiency.

Pulses from detectors used for radioactivity measurement can vary in size by several orders of magnitude. Large pulses will lead to saturation at the preamplifier output and extension of the pulse length. As a consequence, the dead time of the system increases and pulses may be lost. Electronic design techniques employed to protect against overloading tend to increase the amplifier noise level. However, an optoelectronic method of overload protection has been devised which has only a negligible effect on noise. An infrared light emitting diode interfaced to the output of the preamplifier is linked by fibre optic cable to an ultra-low leakage photodiode at the input. The conduction of the photodiode increases with the amplitude of the preamplifier output signal. Excess current is thereby prevented from entering the preamplifier and causing saturation. The preamplifier has been tested on 4π beta-gamma and gas counting systems and found to give good protection against overloading., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

The activity concentration of gaseous beta-emitting radionuclides such as (3)H, (85)Kr and, more recently, (11)C, is measured at NPL using a set of length-compensated proportional counters. The active gas is mixed with argon-methane (P-10) and passed to the counters. Adding gases to P-10 changes the mean ionisation energy, W, of the gas mixture. Estimation of the counting losses using the Monte Carlo model requires a knowledge of W. Unfortunately, only a limited amount of published data is available. This paper describes the initial experimental studies performed to enable the extension of the MC model based loss correction method to gases other than carbon dioxide in P-10. Preliminary measurements have been made to determine the W value for a gas mixture containing (85)Kr in nitrogen and P-10. The DC current through the counters is measured; the counters are also operated in the normal way with pulse amplifiers, discriminators and scalers. The value of W is derived from a knowledge of activity, counter current and mean beta energy., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Most hovering animals, such as insects and hummingbirds, enhance lift by producing leading edge vortices (LEVs) and by using both the downstroke and upstroke for lift production. By contrast, most hovering passerine birds primarily use the downstroke to generate lift. To compensate for the nearly inactive upstroke, weight support during the downstroke needs to be relatively higher in passerines when compared with, e.g. hummingbirds. Here we show, by capturing the airflow around the wing of a freely flying pied flycatcher, that passerines may use LEVs during the downstroke to increase lift. The LEV contributes up to 49 per cent to weight support, which is three times higher than in hummingbirds, suggesting that avian hoverers compensate for the nearly inactive upstroke by generating stronger LEVs. Contrary to other animals, the LEV strength in the flycatcher is lowest near the wing tip, instead of highest. This is correlated with a spanwise reduction of the wing's angle-of-attack, partly owing to upward bending of primary feathers. We suggest that this helps to delay bursting and shedding of the particularly strong LEV in passerines.

Many small passerines regularly fly slowly when catching prey, flying in cluttered environments or landing on a perch or nest. While flying slowly, passerines generate most of the flight forces during the downstroke, and have a 'feathered upstroke' during which they make their wing inactive by retracting it close to the body and by spreading the primary wing feathers. How this flight mode relates aerodynamically to the cruising flight and so-called 'normal hovering' as used in hummingbirds is not yet known. Here, we present time-resolved fluid dynamics data in combination with wingbeat kinematics data for three pied flycatchers flying across a range of speeds from near hovering to their calculated minimum power speed. Flycatchers are adapted to low speed flight, which they habitually use when catching insects on the wing. From the wake dynamics data, we constructed average wingbeat wakes and determined the time-resolved flight forces, the time-resolved downwash distributions and the resulting lift-to-drag ratios, span efficiencies and flap efficiencies. During the downstroke, slow-flying flycatchers generate a single-vortex loop wake, which is much more similar to that generated by birds at cruising flight speeds than it is to the double loop vortex wake in hovering hummingbirds. This wake structure results in a relatively high downwash behind the body, which can be explained by the relatively active tail in flycatchers. As a result of this, slow-flying flycatchers have a span efficiency which is similar to that of the birds in cruising flight and which can be assumed to be higher than in hovering hummingbirds. During the upstroke, the wings of slowly flying flycatchers generated no significant forces, but the body-tail configuration added 23 per cent to weight support. This is strikingly similar to the 25 per cent weight support generated by the wing upstroke in hovering hummingbirds. Thus, for slow-flying passerines, the upstroke cannot be regarded as inactive, and the tail may be of importance for flight efficiency and possibly manoeuvrability.

Flight is one of the energetically most costly activities in the animal kingdom, suggesting that natural selection should work to optimize flight performance. The similar size and flight speed of birds and bats may therefore suggest convergent aerodynamic performance; alternatively, flight performance could be restricted by phylogenetic constraints. We test which of these scenarios fit to two measures of aerodynamic flight efficiency in two passerine bird species and two New World leaf-nosed bat species. Using time-resolved particle image velocimetry measurements of the wake of the animals flying in a wind tunnel, we derived the span efficiency, a metric for the efficiency of generating lift, and the lift-to-drag ratio, a metric for mechanical energetic flight efficiency. We show that the birds significantly outperform the bats in both metrics, which we ascribe to variation in aerodynamic function of body and wing upstroke: Bird bodies generated relatively more lift than bat bodies, resulting in a more uniform spanwise lift distribution and higher span efficiency. A likely explanation would be that the bat ears and nose leaf, associated with echolocation, disturb the flow over the body. During the upstroke, the birds retract their wings to make them aerodynamically inactive, while the membranous bat wings generate thrust and negative lift. Despite the differences in performance, the wake morphology of both birds and bats resemble the optimal wake for their respective lift-to-drag ratio regimes. This suggests that evolution has optimized performance relative to the respective conditions of birds and bats, but that maximum performance is possibly limited by phylogenetic constraints. Although ecological differences between birds and bats are subjected to many conspiring variables, the different aerodynamic flight efficiency for the bird and bat species studied here may help explain why birds typically fly faster, migrate more frequently and migrate longer distances than bats.

Bats are unique among extant actively flying animals in having very flexible wings, controlled by multi-jointed fingers. This gives the potential for fine-tuned active control to optimize aerodynamic performance throughout the wingbeat and thus a more efficient flight. But how bat wing performance scales with size, morphology and ecology is not yet known. Here, we present time-resolved fluid wake data of two species of bats flying freely across a range of flight speeds using stereoscopic digital particle image velocimetry in a wind tunnel. From these data, we construct an average wake for each bat species and speed combination, which is used to estimate the flight forces throughout the wingbeat and resulting flight performance properties such as lift-to-drag ratio (L/D). The results show that the wake dynamics and flight performance of both bat species are similar, as was expected since both species operate at similar Reynolds numbers (Re) and Strouhal numbers (St). However, maximum L/D is achieved at a significant higher flight speed for the larger, highly mobile and migratory bat species than for the smaller non-migratory species. Although the flight performance of these bats may depend on a range of morphological and ecological factors, the differences in optimal flight speeds between the species could at least partly be explained by differences in their movement ecology.

Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A standard of (210)Pb in solution was produced at the National Physical Laboratory by a novel technique combining Čerenkov counting with the established liquid scintillation efficiency tracing technique known as the CIEMAT/NIST method. Coincidence counting was applied in order to validate the measurements and the activity concentrations of the solution determined with each technique are shown to be in agreement. Radiochemical separation of the (210)Pb from its daughters was also necessary and the scheme for the separation is described. After performing this two-stage standardisation, the uncertainty was successfully lowered to 0.66% (k=1). This uncertainty is approximately a factor of four lower than previously achieved at NPL by the classical method of standardisation of radionuclides, i.e. coincidence counting., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

A new triple to double coincidence ratio (TDCR) system has been established at NPL. The system incorporates a spherical optical chamber, a manual sample changing facility and an integral light-tight housing. A 6 in diameter NaI(Tl) detector has been incorporated to allow 4pibeta-gamma coincidence measurements to be performed in parallel. Details of the detectors, the electronics and the overall TDCR system are given. Validation measurements using suitable low-energy beta and electron capture nuclides, i.e. (3)H and (55)Fe have been performed. The highest efficiency achieved with a (3)H solution in Ultima Gold AB in a glass vial was 53% and in a high-efficiency LS cocktail 65%. This indicates that the optical chamber is performing well. After varying the efficiency by de-focusing the PM tubes, the activity of the sources was determined. The activity concentration determined with TDCR agreed with certified values within the range of uncertainties. Further results from validation measurements and the corresponding uncertainty budgets are presented., (Crown Copyright 2009. Published by Elsevier Ltd. All rights reserved.)

As part of a BIPM key-comparison of (85)Kr, a primary standardisation using internal gas proportional counting was performed. The supplied activity for the comparison was approximately 40 MBq and a two-stage dilution was required to reduce the activity concentration to a level suitable for gas counting. A new gas-handling rig was constructed for performing the dilutions. The dilutions, however, introduced significant uncertainties in the final result, so that additional methods suitable for measurement at higher activity levels were also used. A series of dilution ampoules with activities of 6 and 1 MBq were prepared in the new NPL gas-handling rig using inactive krypton as a diluent. Internal gas proportional counting was performed on each of the 1 MBq dilution ampoules. The proportion of the activity transferred to the counting system was estimated using pressure and volume data and the total ampoule activity calculated. Counting losses below the threshold were assumed to be 2%. The effect of changing the composition of the counting gas by inclusion of krypton was evaluated and found to not significantly change the gas gain, i.e. losses below the noise threshold (approximately 120 eV) remained essentially constant. The proportional counters were assumed to be 100% efficient with an uncertainty of 0.5% (k=1). Both 1 and 6 MBq ampoules were assayed by gamma-spectrometry using HPGe and NaI(Tl) detectors. This method resulted in an activity value with a smaller uncertainty than the primary method. Activity values for the three methods employed were consistent within the uncertainty of measurement., (Crown Copyright 2009. Published by Elsevier Ltd. All rights reserved.)

To obtain a full understanding of the aerodynamics of animal flight, the movement of the wings, the kinematics, needs to be connected to the wake left behind the animal. Here the detailed 3D wingbeat kinematics of bats, Glossophaga soricina, flying in a wind tunnel over a range of flight speeds (1-7 m s(-1)) was determined from high-speed video. The results were compared with the wake geometry and quantitative wake measurements obtained simultaneously to the kinematics. The wingbeat kinematics varied gradually with flight speed and reflected the changes observed in the wake of the bats. In particular, several of the kinematic parameters reflected the differences in the function of the upstroke at low and high flight speeds. At lower flight speeds the bats use a pitch-up rotation to produce a backward flick which creates thrust and some weight support. At higher speeds this mechanism disappears and the upstroke generates weight support but no thrust. This is reflected by the changes in e.g. angle of attack, span ratio, camber and downstroke ratio. We also determined how different parameters vary throughout a wingbeat over the flight speeds studied. Both the camber and the angle of attack varied over the wingbeat differently at different speeds, suggesting active control of these parameters to adjust to the changing aerodynamic conditions. This study of the kinematics strongly indicates that the flight of bats is governed by an unsteady high-lift mechanism at low flight speeds and points to differences between birds and bats.

Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.

Qualitative comparison of bird and bat wakes has demonstrated significant differences in the structure of the far wake. Birds have been found to have a unified vortex wake of the two wings, while bats have a more complex wake with gradients in the circulation along the wingspan, and with each wing generating its own vortex structure. Here, we compare quantitative measures of the circulation in the far wake of three bird and one bat species. We find that bats have a significantly stronger normalized circulation of the start vortex than birds. We also find differences in how the circulation develops during the wingbeat as demonstrated by the ratio of the circulation of the dominant start vortex and the total circulation of the same sense. Birds show a more prominent change with changing flight speed and a relatively weaker start vortex at minimum power speed than bats. We also find that bats have a higher normalized wake loading based on the start vortex, indicating higher relative induced drag and therefore less efficient lift generation than birds. Our results thus indicate fundamental differences in the aerodynamics of bird and bat flight that will further our understanding of the evolution of vertebrate flight.

Membrane proteins are embedded in a lipid bilayer and maintain strong interactions with lipid molecules. Tightly bound lipids are responsible for vertical positioning and integration of proteins in the membrane and for assembly of multisubunit complexes and occasionally act as substrates. In this work we present the lipidic sponge phase crystal structure of the reaction center from Blastochloris viridis to 1.86 A, which reveals lipid molecules interacting with the protein surface. A diacylglycerol molecule is bound, through a thioether bond, to the N-terminus of the tetraheme cytochrome c subunit. From the electron density recovered at the Q(B) site and the observed change in recombination kinetics in lipidic sponge phase-grown crystals, the mobile ubiquinone appears to be displaced by a monoolein molecule. A 36 A long electron density feature is observed at the interface of transmembrane helices belonging to the H- and M-subunits, probably arising from an unidentified lipid.