Modifiable Clinical Dental Impression Methods to Obtain Whole-Mouth and Detailed Dental Traits From Vertebrates.

Dental impressions, developed for accurate capture of oral characteristics in human clinical settings, are seldom used in research on nonlivestock, nonprimate, and especially nonmammalian vertebrates due to a lack of appropriate tools. Studies of dentitions in most vertebrate species usually require euthanasia and specimen dissection, microCT and other scans with size and resolution tradeoffs, and/or ad-hoc individual impressions or removal of single teeth. These approaches prevent in-vivo studies that factor in growth and other chronological changes and separate teeth from the context of the whole mouth.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response.

Living-Cell Diffracted X-ray Tracking Analysis Confirmed Internal Salt Bridge Is Critical for Ligand-Induced Twisting Motion of Serotonin Receptors.

Serotonin receptors play important roles in neuronal excitation, emotion, platelet aggregation, and vasoconstriction. The serotonin receptor subtype 2A (5-HTR) is a Gq-coupled GPCR, which activate phospholipase C. Although the structures and functions of 5-HTRs have been well studied, little has been known about their real-time dynamics.

Vertebrae-Based Body Length Estimation in Crocodylians and Its Implication for Sexual Maturity and the Maximum Sizes.

Body size is fundamental to the physiology and ecology of organisms. Crocodyliforms are no exception, and several methods have been developed to estimate their absolute body sizes from bone measurements. However, species-specific sizes, such as sexually mature sizes and the maximum sizes were not taken into account due to the challenging maturity assessment of osteological specimens.

Dental microwear of a basal ankylosaurine dinosaur, Jinyunpelta and its implication on evolution of chewing mechanism in ankylosaurs.

Jinyunpelta sinensis is a basal ankylosaurine dinosaur excavated from the mid Cretaceous Liangtoutang Formation of Jinyun County, Zhejiang Province, China. In the present study, its dental microwear was observed using a confocal laser microscope. Jinyunpelta had steep wear facets that covered most of buccal surfaces of posterior dentary teeth.

Agonist and Antagonist-Diverted Twisting Motions of a Single TRPV1 Channel.

Transient receptor potential vanilloid type 1 (TRPV1) channels are activated by heat, vanilloids, and extracellular protons. Cryo-EM has revealed various conformations of TRPV1, and these structures suggest an intramolecular twisting motion in response to ligand binding. However, limited experimental data support this observation.

X-ray-based living-cell motion analysis of individual serotonin receptors.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins, which transmit extracellular signals inside cells via activating G proteins. GPCRs are involved in a wide variety of physiological functions, such as signal sensing, immune system processes, and neurotransmission. Although the structures and functions of GPCRs have been well studied, little has been known about their real-time dynamics on live cells.

cDNA Display of Disulfide-Containing Peptide Library and In Vitro Evolution.

Directed in vitro evolution (IVE) is now a widely applied technology to obtain molecules that have designed new features of one's demands. We describe here experimental procedures for a cDNA display IVE to select peptide aptamers from a scaffold-based random peptide library. A three-finger (3-F) peptide library is exemplified, which has been shown its pluripotency to various target molecules.

Biogeographical Network Analysis of Cretaceous Terrestrial Tetrapods: A Phylogeny-Based Approach.

Network methods are widely used to represent and analyze biogeography. It is difficult, however, to convert occurrence data of fossil vertebrates to a biogeographical network, as most species were known from a single locality. A new method for creating a biogeographical network that can incorporate phylogenetic information is proposed in this study, which increases the number of edges in the network of fossil vertebrates and enables the application of various network methods.

Transitions between foot postures are associated with elevated rates of body size evolution in mammals.

Terrestrial mammals have evolved various foot postures: flat-footed (plantigrady), tiptoed (digitigrady), and hooved (unguligrady) postures. Although the importance of foot posture on ecology and body size of mammalian species has been widely recognized, its evolutionary trajectory and influence on body size evolution across mammalian phylogeny remain untested. Taking a Bayesian phylogenetic approach combined with a comprehensive dataset of foot postures in 880 extant mammalian species, we investigated the evolutionary history of foot postures and rates of body size evolution, within the same posture and at transitions between postures.

Diffracted X-ray Blinking Tracks Single Protein Motions.

Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities.

Three-dimensional tooth surface texture analysis on stall-fed and wild boars (Sus scrofa).

Categorizing the archaeological remains of Sus scrofa as domesticated "pigs" or wild "boars" is often difficult because of their morphological and genetic similarities. For this purpose, we tested whether feeding ecological change of S. scrofa that accompanied their domestication can be detected based on the three-dimensional texture created on the tooth enamel surface by mastication.

Comparative limb proportions reveal differential locomotor morphofunctions of alligatoroids and crocodyloids.

Although two major clades of crocodylians (Alligatoroidea and Crocodyloidea) were split during the Cretaceous period, relatively few morphological and functional differences between them have been known. In addition, interaction of multiple morphofunctional systems that differentiated their ecology has barely been assessed. In this study, we examined the limb proportions of crocodylians to infer the differences of locomotor functions between alligatoroids and crocodyloids, and tested the correlation of locomotor and feeding morphofunctions.

Masticatory jaw movement of Exaeretodon argentinus (Therapsida: Cynodontia) inferred from its dental microwear.

Dental microwear of four postcanine teeth of Exaeretodon argentinus was analyzed using both two dimensional (2D) and three dimensional (3D) methods to infer their masticatory jaw movements. Results of both methods were congruent, showing that linear microwear features (scratches) were well aligned and mostly directed to the antero-posterior direction in all four teeth examined. These findings support the palinal masticatory jaw movement, which was inferred in previous studies based on the observation of gross morphology of wear facets.

A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.

We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers.

Nonplantigrade Foot Posture: A Constraint on Dinosaur Body Size.

Dinosaurs had functionally digitigrade or sub-unguligrade foot postures. With their immediate ancestors, dinosaurs were the only terrestrial nonplantigrades during the Mesozoic. Extant terrestrial mammals have different optimal body sizes according to their foot posture (plantigrade, digitigrade, and unguligrade), yet the relationship of nonplantigrade foot posture with dinosaur body size has never been investigated, even though the body size of dinosaurs has been studied intensively.

Pr-SNTX, a short-chain three-finger toxin from Papuan pigmy mulga snake, is an antagonist of muscle-type nicotinic acetylcholine receptor (α2βδε).

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis.

Real time ligand-induced motion mappings of AChBP and nAChR using X-ray single molecule tracking.

We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes.

Versatile C-terminal specific biotinylation of proteins using both a puromycin-linker and a cell-free translation system for studying high-throughput protein-molecule interactions.

Immobilization of a protein in a functionally active form and correct orientation for high-throughput analysis is crucial for surface-based protein-molecular interaction studies and should aid progress in associated nanotechnologies. Here, we present a general method for controlled and oriented immobilization of proteins by a puromycin-linker for cDNA display technology. The utility and potential of this method was demonstrated by examining the interaction between the B domain of protein A and immunoglobulin G (IgG) by surface plasmon resonance.

Directed evolution of three-finger toxin to produce serine protease inhibitors.

Directed evolution is a very popular strategy for improving biophysical properties and even for generating proteins with novel functions. Recent advances in combinatorial protein engineering mean it is now possible to develop protein scaffolds that could substitute for whole antibody-associated properties as emerging therapeutic proteins. In particular, disulfide-rich proteins are attractive templates for directed evolution in the search for novel molecules because they can regulate the activities of receptors, enzymes, and other molecules.

Molecular Cloning and Sequence Analysis of the cDNAs Encoding Toxin-Like Peptides from the Venom Glands of Tarantula Grammostola rosea.

Tarantula venom glands produce a large variety of bioactive peptides. Here we present the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared from the venom glands of the Chilean common tarantula, Grammostola rosea. The cDNA sequences of about 1500 clones out of 4000 clones were analyzed after selection using several criteria.

Functional characterization of Kunitz-type protease inhibitor Pr-mulgins identified from New Guinean Pseudechis australis.

Kunitz-type protease inhibitors, which consist of around 60 amino acid residues and three distinctive disulfide bridges, exhibit a broad range of physiological functions such as protease inhibitor and ion channel blocker. In this study, we identified cDNAs encoding Kunitz-type protease inhibitors, Pr-mulgins 1, 2 and 3, from the venom gland cDNA library of Papuan pigmy mulga snake (New Guinean Pseudechis australis). The deduced amino acid sequences of the Pr-mulgins are 92.

Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.

We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor.

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea.

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system.

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.

Background: Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F).