Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Inner ear sensory system changes as extinct crocodylomorphs transitioned from land to water.

Major evolutionary transitions, in which animals develop new body plans and adapt to dramatically new habitats and lifestyles, have punctuated the history of life. The origin of cetaceans from land-living mammals is among the most famous of these events. Much earlier, during the Mesozoic Era, many reptile groups also moved from land to water, but these transitions are more poorly understood. We use computed tomography to study changes in the inner ear vestibular system, involved in sensing balance and equilibrium, as one of these groups, extinct crocodile relatives called thalattosuchians, transitioned from terrestrial ancestors into pelagic (open ocean) swimmers. We find that the morphology of the vestibular system corresponds to habitat, with pelagic thalattosuchians exhibiting a more compact labyrinth with wider semicircular canal diameters and an enlarged vestibule, reminiscent of modified and miniaturized labyrinths of other marine reptiles and cetaceans. Pelagic thalattosuchians with modified inner ears were the culmination of an evolutionary trend with a long semiaquatic phase, and their pelagic vestibular systems appeared after the first changes to the postcranial skeleton that enhanced their ability to swim. This is strikingly different from cetaceans, which miniaturized their labyrinths soon after entering the water, without a prolonged semiaquatic stage. Thus, thalattosuchians and cetaceans became secondarily aquatic in different ways and at different paces, showing that there are different routes for the same type of transition.

Ontogenetic variation in the crocodylian vestibular system.

Crocodylians today live in tropical to subtropical environments, occupying mostly shallow waters. Their body size changes drastically during ontogeny, as do their skull dimensions and bite forces, which are associated with changes in prey preferences. Endocranial neurosensory structures have also shown to change ontogenetically, but less is known about the vestibular system of the inner ear. Here we use 30 high-resolution computed tomography (CT) scans and three-dimensional geometric morphometrics to investigate the size and shape changes of crocodylian endosseous labyrinths throughout ontogeny, across four stages (hatchling, juvenile, subadult and adult). We find two major patterns of ontogenetic change. First, the labyrinth increases in size during ontogeny, with negative allometry in relation to skull size. Second, labyrinth shape changes significantly, with hatchlings having shorter semicircular canal radii, with thicker diameters and an overall dorsoventrally shorter labyrinth than those of more mature individuals. We argue that the modification of the labyrinth during crocodylian ontogeny is related to constraints imposed by skull growth, due to fundamental changes in the crocodylian braincase during ontogeny (e.g. verticalisation of the basicranium), rather than changes in locomotion, diet, or other biological functions or behaviours.

Identification of Small Molecular Chaperones Binding P23H Mutant Opsin through an In Silico Structure-Based Approach.

N-terminal P23H opsin mutation accounts for most of retinitis pigmentosa (RP) cases. P23H functions and folding can be rescued by small chaperone ligands, which contributes to validate mutant opsin as a suitable target for pharmacological treatment of RP. However, the lack of structural details on P23H mutant opsin strongly impairs drug design, and new chemotypes of effective chaperones of P23H opsin are in high demand. Here, a computational-boosted workflow combining homology modeling with molecular dynamics (MD) simulations and virtual screening was used to select putative P23H opsin chaperones among different libraries through a structure-based approach. In vitro studies corroborated the reliability of the structural model generated in this work and identified a number of novel chemotypes of safe and effective chaperones able to promote P23H opsin trafficking to the outer cell membrane.

Agonist binding evokes extensive conformational changes in the extracellular domain of the ATP-gated human P2X1 receptor ion channel.

P2X receptors for ATP have a wide range of physiological roles and comprise a structurally distinct family of ligand-gated trimeric ion channels. The crystal structure of a P2X4 receptor, in combination with mutagenesis studies, has provided a model of the intersubunit ATP-binding sites and identified an extracellular lateral portal, adjacent to the membrane, that funnels ions to the channel pore. However, little is known about the extent of ATP-induced conformational changes in the extracellular domain of the receptor. To address this issue, we have used MTSEA-biotinylation (N-Biotinoylaminoethyl methanethiosulfonate) to show ATP-sensitive accessibility of cysteine mutants at the human P2X1 receptor. Mapping these data to a P2X1 receptor homology model identifies significant conformational rearrangement. Electron microscopy of purified P2X1 receptors showed marked changes in structure on ATP binding, and introducing disulphide bonds between adjacent subunits to restrict intersubunit movements inhibited channel function. These results are consistent with agonist-induced rotation of the propeller-head domain of the receptor, sliding of adjacent subunits leading to restricted access to the upper vestibule, movement in the ion conducting lateral portals, and gating of the channel pore.

P2X receptors: dawn of the post-structure era.

P2X receptors are non-selective cation channels gated by extracellular ATP. They play key roles in various physiological processes such as nerve transmission, pain sensation and the response to inflammation, making them attractive drug targets for the treatment of inflammatory pain. The recent report of the three-dimensional (3D) crystal structure of zebrafish P2X4.1 represents a step change in our understanding of these membrane ion channels, where previously only low-resolution structural data and inferences from indirect structure-function studies were available. The purpose of this review is to place previous work within the context of the new 3D structure, and to summarize the key questions and challenges which await P2X researchers as we move into the post-structure era.

The first occurrence of machimosaurid crocodylomorphs from the Oxfordian of south-central Poland provides new insights into the distribution of macrophagous teleosauroids.

Teleosauroid thalattosuchians were a clade of semi-aquatic crocodylomorphs that achieved a broad geographic distribution during the Mesozoic. While their fossils are well documented in Western European strata, our understanding of teleosauroids (and thalattosuchians in general) is notably poorer in Central-Eastern Europe, and from Poland in particular. Herein, we redescribe a teleosauroid rostrum (MZ VIII Vr-72) from middle Oxfordian strata of Zalecze Wielkie, in south-central Poland. Until now, the specimen has been largely encased in a block of limestone. After preparation, its rostral and dental morphology could be evaluated, showing the specimen to be a non-machimosaurin machimosaurid, similar in morphology to taxa Neosteneosaurus edwardsi and Proexochokefalos heberti. The well-preserved teeth enable us to study the specimen feeding ecology through the means of comparing its teeth to other teleosauroids through PCoA analysis. Comparisons with inferred closely related taxa suggest that the referred specimen was a macrophagous generalist. Notably, MZ VIII Vr-72 displays a prominent pathological distortion of the anterior rostrum, in the form of lateral bending. The pathology affects the nasal passage and tooth size and position, and is fully healed, indicating that, despite its macrophagous diet, it did not prevent the individual from food acquisition.

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum.

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.

Identification of the molecular determinants of antagonist potency in the allosteric binding pocket of human P2X4.

P2X receptors are a family of ATP-gated cation channels comprising seven subtypes in mammals, which play key roles in nerve transmission, pain sensation and inflammation. The P2X4 receptor in particular has attracted significant interest from pharmaceutical companies due to its physiological roles in neuropathic pain and modulation of vascular tone. A number of potent small-molecule P2X4 receptor antagonists have been developed, including the allosteric P2X4 receptor antagonist BX430, which is approximately 30-fold more potent at human P2X4 compared with the rat isoform. A single amino-acid difference between human and rat P2X4 (I312T), located in an allosteric pocket, has previously been identified as critical for BX430 sensitivity, implying that BX430 binds in this pocket. Using a combination of mutagenesis, functional assay in mammalian cells and in silico docking we confirmed these findings. Induced-fit docking, permitting the sidechains of the amino-acids of P2X4 to move, showed that BX430 could access a deeper portion of the allosteric pocket, and that the sidechain of Lys-298 was important for shaping the cavity. We then performed blind docking of 12 additional P2X4 antagonists into the receptor extracellular domain, finding that many of these compounds favored the same pocket as BX430 from their calculated binding energies. Induced-fit docking of these compounds in the allosteric pocket enabled us to show that antagonists with high potency (IC50 ≤ 100 nM) bind deep in the allosteric pocket, disrupting a network of interacting amino acids including Asp-85, Ala-87, Asp-88, and Ala-297, which are vital for transmitting the conformational change following ATP binding to channel gating. Our work confirms the importance of Ile-312 for BX430 sensitivity, demonstrates that the allosteric pocket where BX430 binds is a plausible binding pocket for a series of P2X4 antagonists, and suggests a mode of action for these allosteric antagonists involving disruption of a key structural motif required for the conformational change induced in P2X4 when ATP binds.

Reappraisal of the thalattosuchian crocodylomorph record from the Middle-Upper Jurassic Rosso Ammonitico Veronese of northeastern Italy: Age calibration, new specimens and taphonomic biases.

Despite their extremely rare and fragmentary record, aquatic crocodylomorphs from the Middle to Upper Jurassic (Bajocian-Tithonian) Rosso Ammonitico Veronese (RAV) of northeastern Italy have sparked interest since the late 18th century. Among marine reptiles, Thalattosuchia is by far one of the best represented groups from the RAV units, especially in the Middle Jurassic. Although some specimens have been the subject of multiple studies in recent times, most of them still lack precise stratigraphic assignment and taphonomic assessment, while others remain undescribed. Here we provide a comprehensive revision of the thalattosuchian record from the RAV, alongside the most up-to-date age determination, by means of calcareous nannofossils, when available. Three new metriorhynchoid specimens are described for the first time from the Middle Jurassic of Asiago Plateau (Vicenza province). While the taphonomy of the newly described specimens hampers any taxonomic attribution below superfamily/family level, all three were confidently assigned to a precise interval between the upper Bajocian and the upper Bathonian. This revised record has major paleobiogeographical implications: the new specimens confirm an early origin and distribution of Metriorhynchoidea in the Tethys area and suggest a fast colonization of the open-ocean environment since the upper Bajocian.

Thalattosuchian crocodylomorphs from European Russia, and new insights into metriorhynchid tooth serration evolution and their palaeolatitudinal distribution.

From the Middle Jurassic to the Early Cretaceous, metriorhynchid crocodylomorphs inhabited marine ecosystems across the European archipelago. Unfortunately, European metriorhynchids are only well known from Germany, France, and the UK, with the Eastern European fossil record being especially poor. This hinders our understanding of metriorhynchid biodiversity across these continuous seaways, and our ability to investigate provincialism. Here we describe eleven isolated tooth crowns and six vertebrae referable to Metriorhynchidae from the Callovian, Oxfordian, Volgian (Tithonian), and Ryazanian (Berriasian) or Valanginian of European Russia. We also describe an indeterminate thalattosuchian tooth from the lower Bajocian of the Volgograd Oblast, the first discovery of a marine reptile from the Bajocian strata of European Russia. These rare fossils, along with previous reports of Russian thalattosuchians, indicate that thalattosuchians have been common in the Middle Russian Sea since it was formed. Palaeolatitude calculations for worldwide metriorhynchid-bearing localities demonstrate that the occurrences in European Russia are the most northern, located mainly between 44-50 degrees north. However, metriorhynchids appear to be rare at these palaeolatitudes, and are absent from palaeolatitudes higher than 50°. These observations support the hypothesis that metriorhynchids evolved an elevated metabolism but were not endo-homeothermic, especially as endo-homeothermic marine reptiles (ichthyosaurs and plesiosaurs) remained abundant at much higher palaeolatitudes.

Evidence for a novel cranial thermoregulatory pathway in thalattosuchian crocodylomorphs.

Thalattosuchian crocodylomorphs were a diverse clade that lived from the Early Jurassic to the Early Cretaceous. The subclade Metriorhynchoidea underwent a remarkable transition, evolving from semi-aquatic ambush predators into fully aquatic forms living in the open oceans. Thalattosuchians share a peculiar palatal morphology with semi-aquatic and aquatic fossil cetaceans: paired anteroposteriorly aligned grooves along the palatal surface of the bony secondary palate. In extant cetaceans, these grooves are continuous with the greater palatine artery foramina, arteries that supply their oral thermoregulatory structures. Herein, we investigate the origins of thalattosuchian palatal grooves by examining CT scans of six thalattosuchian species (one teleosauroid, two early-diverging metriorhynchoids and three metriorhynchids), and CT scans of eleven extant crocodylian species. All thalattosuchians had paired osseous canals, enclosed by the palatines, that connect the nasal cavity to the oral cavity. These osseous canals open into the oral cavity via foramina at the posterior terminus of the palatal grooves. Extant crocodylians lack both the external grooves and the internal canals. We posit that in thalattosuchians these novel palatal canals transmitted hypertrophied medial nasal vessels (artery and vein), creating a novel heat exchange pathway connecting the palatal vascular plexus to the endocranial region. Given the general hypertrophy of thalattosuchian cephalic vasculature, and their increased blood flow and volume, thalattosuchians would have required a more extensive suite of thermoregulatory pathways to maintain stable temperatures for their neurosensory tissues.

CryoEM structure and Alphafold molecular modelling of a novel molluscan hemocyanin.

Hemocyanins are multimeric oxygen transport proteins present in the blood of arthropods and molluscs, containing up to 8 oxygen-binding functional units per monomer. In molluscs, hemocyanins are assembled in decamer 'building blocks' formed of 5 dimer 'plates', routinely forming didecamer or higher-order assemblies with d5 or c5 symmetry. Here we describe the cryoEM structures of the didecamer (20-mer) and tridecamer (30-mer) forms of a novel hemocyanin from the slipper limpet Crepidula fornicata (SLH) at 7.0 and 4.7 Å resolution respectively. We show that two decamers assemble in a 'tail-tail' configuration, forming a partially capped cylinder, with an additional decamer adding on in 'head-tail' configuration to make the tridecamer. Analysis of SLH samples shows substantial heterogeneity, suggesting the presence of many higher-order multimers including tetra- and pentadecamers, formed by successive addition of decamers in head-tail configuration. Retrieval of sequence data for a full-length isoform of SLH enabled the use of Alphafold to produce a molecular model of SLH, which indicated the formation of dimer slabs with high similarity to those found in keyhole limpet hemocyanin. The fit of the molecular model to the cryoEM density was excellent, showing an overall structure where the final two functional units of the subunit (FU-g and FU-h) form the partial cap at one end of the decamer, and permitting analysis of the subunit interfaces governing the assembly of tail-tail and head-tail decamer interactions as well as potential sites for N-glycosylation. Our work contributes to the understanding of higher-order oligomer formation in molluscan hemocyanins and demonstrates the utility of Alphafold for building accurate structural models of large oligomeric proteins.

Expression, purification, electron microscopy, N-glycosylation mutagenesis and molecular modeling of human P2X4 and Dictyostelium discoideum P2XA.

The recent publication of the apo-, closed-state 3D crystal structure of zebrafish (zf) P2X4.1 has not only revolutionized the P2X research field, but also highlighted the need for further crystal structures, of receptors in different activation states, so that we can gain a complete molecular understanding of ion channel function. zfP2X4.1 was selected as a 3D-crystallization candidate because of its ability to form stable trimers in detergent solution, and purified from over-expression in baculovirus-infected Spodoptera frugiperda (Sf9) insect cells. In this work, we have used a similar approach to express both human P2X4 (hP2X4) and Dictyostelium discoideum P2XA (DdP2XA) in Sf9 cells. Although hP2X4 did not form stable trimers in detergent solution, both receptors bound to ATP-coupled resins, indicating that their extracellular domains were folded correctly. DdP2XA formed strong trimers in detergent solution, and we were able to selectively purify trimers using preparative electrophoresis, and build a 21Å-resolution 3D structure using transmission electron microscopy and single particle analysis. Although the structure of DdP2XA possessed similar dimensions to those of the previously determined low-resolution hP2X4 structure and the zfP2X4.1 crystal structure, N-glycosylation mutagenesis and molecular modeling indicated differences between N-glycan usage and predicted accessibility in models of DdP2XA based on the zfP2X4.1 crystal structure. Our data demonstrate that DdP2XA expressed in insect cells retains ATP-binding capacity after detergent solubilization, is an ideal candidate for structural study, and possesses a significantly different 3D structure to that of both hP2X4 and zfP2X4.1.

Cranial biomechanics of Diplodocus (Dinosauria, Sauropoda): testing hypotheses of feeding behaviour in an extinct megaherbivore.

Sauropod dinosaurs were the largest terrestrial herbivores and pushed at the limits of vertebrate biomechanics and physiology. Sauropods exhibit high craniodental diversity in ecosystems where numerous species co-existed, leading to the hypothesis that this biodiversity is linked to niche subdivision driven by ecological specialisation. Here, we quantitatively investigate feeding behaviour hypotheses for the iconic sauropod Diplodocus. Biomechanical modelling, using finite element analysis, was used to examine the performance of the Diplodocus skull. Three feeding behaviours were modelled: muscle-driven static biting, branch stripping and bark stripping. The skull was found to be 'over engineered' for static biting, overall experiencing low stress with only the dentition enduring high stress. When branch stripping, the skull, similarly, is under low stress, with little appreciable difference between those models. When simulated for bark stripping, the skull experiences far greater stresses, especially in the teeth and at the jaw joint. Therefore, we refute the bark-stripping hypothesis, while the hypotheses of branch stripping and/or precision biting are both consistent with our findings, showing that branch stripping is a biomechanically plausible feeding behaviour for diplodocids. Interestingly, in all simulations, peak stress is observed in the premaxillary-maxillary 'lateral plates', supporting the hypothesis that these structures evolved to dissipate stress induced while feeding. These results lead us to conclude that the aberrant craniodental form of Diplodocus was adapted for food procurement rather than resisting high bite forces.

Identification of a novel P2X7 antagonist using structure-based virtual screening.

P2X4 and P2X7 receptors are ATP-gated ion channels, which play important roles in neuropathic and inflammatory pain, and as such they are important drug targets in diseases of inflammatory origin. While several compounds targeting P2X4 and P2X7 receptors have been developed using traditional high-throughput screening approaches, relatively few compounds have been developed using structure-based design. We initially set out to develop compounds targeting human P2X4, by performing virtual screening on the orthosteric (ATP-binding) pocket of a molecular model of human P2X4 based on the crystal structure of the Danio rerio receptor. The screening of a library of approximately 300,000 commercially available drug-like compounds led to the initial selection of 17 compounds; however, none of these compounds displayed a significant antagonist effect at P2X4 in a Fluo-4 ATP-induced calcium influx assay. When the same set of compounds was tested against human P2X7 in an ATP-stimulated Yo-Pro1 dye uptake assay, one compound (an indeno(1,2-b)pyridine derivative; GP-25) reduced the response by greater than 50% when applied at a concentration of 30 µM. GP-25 displayed an IC50 value of 8.7 µM at human P2X7 and 24.4 µM at rat P2X7, and was confirmed to be active using whole-cell patch clamp electrophysiology and not cytotoxic. Schild analysis suggested that mode of action of GP-25 was orthosteric. Screening of a further 16 commercially available analogues of GP-25 led to the discovery of five additional compounds with antagonist activity at human P2X7, enabling us to investigate the structure-activity relationship. Finally, docking of the R- and S-enantiomers of GP-25 into the orthosteric pocket of molecular models of human P2X4 and human P2X7 revealed that, while both enantiomers were able to make multiple interactions between their carboxyl moieties and conserved positively charged amino-acids in human P2X7, only the S-enantiomer of GP-25 was able to do this in human P2X4, potentially explaining the lack of activity of GP-25 at this receptor.

Optimising expression and extraction of recombinant proteins in plants.

Recombinant proteins are of paramount importance for research, industrial and medical use. Numerous expression chassis are available for recombinant protein production, and while bacterial and mammalian cell cultures are the most widely used, recent developments have positioned transgenic plant chassis as viable and often preferential options. Plant chassis are easily maintained at low cost, are hugely scalable, and capable of producing large quantities of protein bearing complex post-translational modification. Several protein targets, including antibodies and vaccines against human disease, have been successfully produced in plants, highlighting the significant potential of plant chassis. The aim of this review is to act as a guide to producing recombinant protein in plants, discussing recent progress in the field and summarising the factors that must be considered when utilising plants as recombinant protein expression systems, with a focus on optimising recombinant protein expression at the genetic level, and the subsequent extraction and purification of target proteins, which can lead to substantial improvements in protein stability, yield and purity.

The ecological diversification and evolution of Teleosauroidea (Crocodylomorpha, Thalattosuchia), with insights into their mandibular biomechanics.

Throughout the Jurassic, a plethora of marine reptiles dominated ocean waters, including ichthyosaurs, plesiosaurs and thalattosuchian crocodylomorphs. These Jurassic ecosystems were characterized by high niche partitioning and spatial variation in dietary ecology. However, while the ecological diversity of many marine reptile lineages is well known, the overall ecological diversification of Teleosauroidea (one of the two major groups within thalattosuchian crocodylomorphs) has never been explored. Teleosauroids were previously deemed to have a morphologically conservative body plan; however, they were in actuality morphofunctionally more diverse than previously thought. Here we investigate the ecology and feeding specializations of teleosauroids, using morphological and functional cranio-dental characteristics. We assembled the most comprehensive dataset to date of teleosauroid taxa (approximately 20 species) and ran a series of principal component analyses (PC) to categorize them into various feeding ecomorphotypes based on 17 dental characteristics (38 specimens) and 16 functionally significant mandibular characters (18 specimens). The results were examined in conjunction with a comprehensive thalattosuchian phylogeny (153 taxa and 502 characters) to evaluate macroevolutionary patterns and significant ecological shifts. Machimosaurids display a well-developed ecological shift from: (1) slender, pointed tooth apices and an elongate gracile mandible; to (2) more robust, pointed teeth with a slightly deeper mandible; and finally, (3) rounded teeth and a deep-set, shortened mandible with enlarged musculature. Overall, there is limited mandibular functional variability in teleosaurids and machimosaurids, despite differing cranial morphologies and habitat preferences in certain taxa. This suggests a narrow feeding ecological divide between teleosaurids and machimosaurids. Resource partitioning was primarily related to snout and skull length as well as habitat; only twice did teleosauroids manage to make a major evolutionary leap to feed distinctly differently, with only the derived machimosaurines successfully radiating into new feeding ecologies.

Solid-State Structural Properties of Alloxazine Determined from Powder XRD Data in Conjunction with DFT-D Calculations and Solid-State NMR Spectroscopy: Unraveling the Tautomeric Identity and Pathways for Tautomeric Interconversion.

We report the solid-state structural properties of alloxazine, a tricyclic ring system found in many biologically important molecules, with structure determination carried out directly from powder X-ray diffraction (XRD) data. As the crystal structures containing the alloxazine and isoalloxazine tautomers both give a high-quality fit to the powder XRD data in Rietveld refinement, other techniques are required to establish the tautomeric form in the solid state. In particular, high-resolution solid-state 15N NMR data support the presence of the alloxazine tautomer, based on comparison between isotropic chemical shifts in the experimental 15N NMR spectrum and the corresponding values calculated for the crystal structures containing the alloxazine and isoalloxazine tautomers. Furthermore, periodic DFT-D calculations at the PBE0-MBD level indicate that the crystal structure containing the alloxazine tautomer has significantly lower energy. We also report computational investigations of the interconversion between the tautomeric forms in the crystal structure via proton transfer along two intermolecular N-H···N hydrogen bonds; DFT-D calculations at the PBE0-MBD level indicate that the tautomeric interconversion is associated with a lower energy transition state for a mechanism involving concerted (rather than sequential) proton transfer along the two hydrogen bonds. However, based on the relative energies of the crystal structures containing the alloxazine and isoalloxazine tautomers, it is estimated that under conditions of thermal equilibrium at ambient temperature, more than 99.9% of the molecules in the crystal structure will exist as the alloxazine tautomer.

'Ear stones' in crocodylians: a cross-species comparative and ontogenetic survey of otolith structures.

The vestibular system of the inner ear is a crucial sensory organ, involved in the sensation of balance and equilibrium. It consists of three semicircular canals that sense angular rotations of the head and the vestibule that detects linear acceleration and gravity. The vestibule often contains structures, known as the otoliths or 'ear stones'. Otoliths are present in many vertebrates and are particularly well known from the fossil record of fish, but surprisingly have not been described in detail in most tetrapods, living or extinct. Here, we present for the first time a survey of the otoliths of a broad sample of extant crocodylian species, based on computed tomography scans. We find that otoliths are present in numerous crocodylian species of different growth stages, and they continue to increase in size during ontogeny, with positive allometry compared to skull length. Our results confirm that otoliths are a common component of the crocodylian vestibular system, and suggest they play an important role in sensory detection. Otoliths are likely common, but overlooked, constituents of the inner ear in tetrapods, and a broader study of their size, shape and distribution promises insight into sensory abilities.