Screening TLR4 Binding Peptide from Naja atra Venom Glands Based on Phage Display.

Toll-like receptor 4 (TLR4) is a crucial inflammatory signaling pathway that can serve as a potential treatment target for various disorders. A number of inhibitors have been developed for the TLR4 pathway, and although no inhibitors have been approved for clinical use, most have been screened against the TLR4-MD2 conformation. The venom gland is the organ of venomous snakes that secretes substances that are toxic to other animals. The level of gene transcription in venom glands is different from that in other tissues, includes a large number of biologically active ingredients, and is an important natural resource for the development of new drugs. We constructed a T7 phage display library using the cobra (Naja atra) venom gland from the Guangdong Snake Breeding Plant and performed three rounds of screening with TLR4 as the target, randomly selecting monoclonal phage spots for PCR followed by Sanger sequencing. The obtained sequences were subjected to length analysis, molecular docking, solubility prediction, and stability prediction, and a peptide containing 39 amino acids (NA39) was finally screened out. The BLAST results indicated that NA39 was a sequence in RPL19 (Ribosomal Protein L19). After peptide synthesis, the binding ability of NA39 to TLR4 was verified by the surface plasmon resonance (SPR) technique. In this study, a new peptide that can specifically bind TLR4 was successfully screened from the cobra venom gland cDNA library, further demonstrating the effectiveness of phage display technology in the field of drug discovery.

High-Frequency Local Field Potential Oscillations for Pigeons in Effective Turning.

Flexible turning behavior endows Homing Pigeons (Columba livia domestica) with high adaptability and intelligence in long-distance flight, foraging, hazard avoidance, and social interactions. The present study recorded the activity pattern of their local field potential (LFP) oscillations and explored the relationship between different bands of oscillations and turning behaviors in the formatio reticularis medialis mesencephali (FRM). The results showed that the C (13-60 Hz) and D (61-130 Hz) bands derived from FRM nuclei oscillated significantly in active turning, while the D and E (131-200 Hz) bands oscillated significantly in passive turning. Additionally, compared with lower-frequency stimulation (40 Hz and 60 Hz), 80 Hz stimulation can effectively activate the turning function of FRM nuclei. Electrical stimulation elicited stronger oscillations of neural activity, which strengthened the pigeons' turning locomotion willingness, showing an enhanced neural activation effect. These findings suggest that different band oscillations play different roles in the turning behavior; in particular, higher-frequency oscillations (D and E bands) enhance the turning behavior. These findings will help us decode the complex relationship between bird brains and behaviors and are expected to facilitate the development of neuromodulation techniques for animal robotics.

Image-free recognition of moderate ROP from mild with machine learning algorithm on plasma Raman spectrum.

The retinopathy of prematurity (ROP) can cause serious clinical consequences and, fortunately, it is remediable while the time window for treatment is relatively narrow. Therefore, it is urgent to screen all premature infants and diagnose ROP degree timely, which has become a large workload for pediatric ophthalmologists. We developed a retinal image-free procedure using small amount of blood samples based on the plasma Raman spectrum with the machine learning model to automatically classify ROP cases before medical intervention was performed. Statistical differences in infrared Raman spectra of plasma samples were found among the control, mild (ZIIIS1), moderate (ZIIIS2 & ZIIS1), and advanced (ZIIS2) ROP groups. With the different wave points of Raman spectra as the inputs, the outputs of our support vector machine showed that the area under the curves in the receiver operating characteristic (AUC) were 0.763 for the pair comparisons of the control with the mild groups, 0.821 between moderate and advanced groups (ZIIS2), while more than 90% in comparisons of the other four pairs: control vs. moderate (0.981), control vs. advanced (0.963), mild vs. moderate (0.936), and mild vs. advanced (0.953), respectively. Our study could advance principally the ROP diagnosis in two dimensions: the moderate ROPs have been classified remarkably from the mild ones, which leaves more time for the medical treatments, and the procedure of Raman spectrum with a machine learning model based on blood samples can be conveniently promoted to those hospitals lacking of the pediatric ophthalmologists with experience in reading retinal images.

Grade-control outdoor turning flight of robo-pigeon with quantitative stimulus parameters.

Introduction: The robo-pigeon using homing pigeons as a motion carrier has great potential in search and rescue operations due to its superior weight-bearing capacity and sustained flight capabilities. However, before deploying such robo-pigeons, it is necessary to establish a safe, stable, and long-term effective neuro-electrical stimulation interface and quantify the motion responses to various stimuli. Methods: In this study, we investigated the effects of stimulation variables such as stimulation frequency (SF), stimulation duration (SD), and inter-stimulus interval (ISI) on the turning flight control of robo-pigeons outdoors, and evaluated the efficiency and accuracy of turning flight behavior accordingly. Results: The results showed that the turning angle can be significantly controlled by appropriately increasing SF and SD. Increasing ISI can significantly control the turning radius of robotic pigeons. The success rate of turning flight control decreases significantly when the stimulation parameters exceed SF > 100 Hz or SD > 5 s. Thus, the robo-pigeon's turning angle from 15 to 55° and turning radius from 25 to 135 m could be controlled in a graded manner by selecting varying stimulus variables. Discussion: These findings can be used to optimize the stimulation strategy of robo-pigeons to achieve precise control of their turning flight behavior outdoors. The results also suggest that robo-pigeons have potential for use in search and rescue operations where precise control of flight behavior is required.

Progresses of animal robots: A historical review and perspectiveness.

Animal robots have remarkable advantages over traditional mechatronic ones in terms of energy supply, self-orientation, and natural concealment and can provide remarkable theoretical and practical values for scientific investigation, community service, military detection and other fields. Given these features, animal robots have become high-profile research objects and have recently attracted extensive attention. Herein, we have defined animal robots, reviewed the main types of animal robots, and discussed the potential developing directions. We have also detailed the mechanisms underlying the regulation of animal robots and introduced key methods for manipulating them. We have further proposed several application prospects for different types of animal robots. Finally, we have presented research directions for their further improvement.

Hierarchical auditory perception for species discrimination and individual recognition in the music frog.

The ability to discriminate species and recognize individuals is crucial for reproductive success and/or survival in most animals. However, the temporal order and neural localization of these decision-making processes has remained unclear. In this study, event-related potentials (ERPs) were measured in the telencephalon, diencephalon, and mesencephalon of the music frog Nidirana daunchina. These ERPs were elicited by calls from 1 group of heterospecifics (recorded from a sympatric anuran species) and 2 groups of conspecifics that differed in their fundamental frequencies. In terms of the polarity and position within the ERP waveform, auditory ERPs generally consist of 4 main components that link to selective attention (N1), stimulus evaluation (P2), identification (N2), and classification (P3). These occur around 100, 200, 250, and 300 ms after stimulus onset, respectively. Our results show that the N1 amplitudes differed significantly between the heterospecific and conspecific calls, but not between the 2 groups of conspecific calls that differed in fundamental frequency. On the other hand, the N2 amplitudes were significantly different between the 2 groups of conspecific calls, suggesting that the music frogs discriminated the species first, followed by individual identification, since N1 and N2 relate to selective attention and stimuli identification, respectively. Moreover, the P2 amplitudes evoked in females were significantly greater than those in males, indicating the existence of sexual dimorphism in auditory discrimination. In addition, both the N1 amplitudes in the left diencephalon and the P2 amplitudes in the left telencephalon were greater than in other brain areas, suggesting left hemispheric dominance in auditory perception. Taken together, our results support the hypothesis that species discrimination and identification of individual characteristics are accomplished sequentially, and that auditory perception exhibits differences between sexes and in spatial dominance.

The Screening of Therapeutic Peptides for Anti-Inflammation through Phage Display Technology.

For the treatment of inflammatory illnesses such as rheumatoid arthritis and carditis, as well as cancer, several anti-inflammatory medications have been created over the years to lower the concentrations of inflammatory mediators in the body. Peptides are a class of medication with the advantages of weak immunogenicity and strong activity, and the phage display technique is an effective method for screening various therapeutic peptides, with a high affinity and selectivity, including anti-inflammation peptides. It enables the selection of high-affinity target-binding peptides from a complex pool of billions of peptides displayed on phages in a combinatorial library. In this review, we will discuss the regular process of using phage display technology to screen therapeutic peptides, and the peptides screened for anti-inflammation properties in recent years according to the target. We will describe how these peptides were screened and how they worked in vitro and in vivo. We will also discuss the current challenges and future outlook of using phage display to obtain anti-inflammatory therapeutic peptides.

Neural activities in music frogs reveal call variations and phylogenetic relationships within the genus Nidirana.

The characteristics of acoustic signals co-evolve with preferences of the auditory sensory system. However, how the brain perceives call variations and whether it can reveal phylogenetic relationships among signalers remains poorly understood. Here, we recorded the neural signals from the Emei music frogs (Nidirana daunchina) in response to broadcasted calls of five different species of the same genus. We found that responses in terms of the different amplitudes of various event-related potential (ERP) components were correlated with diversification trends in acoustic signals, as well as phylogenetic relationships between N. daunchina and heterospecific callers. Specifically, P2 decreased gradually along the ordinal decline of similarities in acoustic characteristics of calls compared with those from conspecifics. Moreover, P3a amplitudes showed increasing trends in correspondence with callers' genetic distances from the subject species. These observations collectively support the view that neural activities in music frogs can reflect call variations and phylogenetic relationships within the genus Nidirana.

Parasite defensive limb movements enhance acoustic signal attraction in male little torrent frogs.

Many animals rely on complex signals that target multiple senses to attract mates and repel rivals. These multimodal displays can however also attract unintended receivers, which can be an important driver of signal complexity. Despite being taxonomically widespread, we often lack insight into how multimodal signals evolve from unimodal signals and in particular what roles unintended eavesdroppers play. Here, we assess whether the physical movements of parasite defense behavior increase the complexity and attractiveness of an acoustic sexual signal in the little torrent frog (Amolops torrentis). Calling males of this species often display limb movements in order to defend against blood-sucking parasites such as frog-biting midges that eavesdrop on their acoustic signal. Through mate choice tests we show that some of these midge-evoked movements influence female preference for acoustic signals. Our data suggest that midge-induced movements may be incorporated into a sexual display, targeting both hearing and vision in the intended receiver. Females may play an important role in incorporating these multiple components because they prefer signals which combine multiple modalities. Our results thus help to understand the relationship between natural and sexual selection pressure operating on signalers and how in turn this may influence multimodal signal evolution.

Screening of TNFR1 Binding Peptides from Deinagkistrodon acutus Venom through Phage Display.

The venomous species Deinagkistrodon acutus has been used as anti-inflammatory medicine in China for a long time. It has been proven to have anti-inflammatory activity, but its specific anti-inflammatory components have not yet been fully elucidated. Tumor necrosis factor receptor-1 (TNFR1), which participates in important intracellular signaling pathways, mediates apoptosis, and functions as a regulator of inflammation, is often used as the target to develop anti-inflammatory drugs. The small peptides of snake venom have the advantages of weak immunogenicity and strong activity. To obtain the specific TNFR1 binding peptides, we constructed a T7 phage library of D. acutus venom glands, and then performed biopanning against TNFR1 on the constructed library. After biopanning three times, several sequences with potential binding capacity were obtained and one 41-amino acid peptide was selected through a series of biological analyses including sequence length, solubility, and simulated affinity, named DAvp-1. After synthesis, the binding capacity of DAvp-1 and TNFR1 was verified using surface plasmon resonance technology (SPR). Conclusively, by applying phage display technology, this work depicts the successful screening of a promising peptide DAvp-1 from D. acutus venom that binds to TNFR1. Additionally, our study emphasizes the usefulness of phage display technology for studies on screening natural product components.

Noise constrains the evolution of call frequency contours in flowing water frogs: a comparative analysis in two clades.

BACKGROUND: The acoustic adaptation hypothesis (AAH) states that signals should evolve towards an optimal transmission of the intended information from senders to intended receivers given the environmental constraints of the medium that they traverse. To date, most AAH studies have focused on the effect of stratified vegetation on signal propagation. These studies, based on the AAH, predict that acoustic signals should experience less attenuation and degradation where habitats are less acoustically complex. Here, we explored this effect by including an environmental noise dimension to test some AAH predictions in two clades of widespread amphibians (Bufonidae and Ranidae) that actively use acoustic signals for communication. By using data from 106 species in these clades, we focused on the characterization of the differences in dominant frequency (DF) and frequency contour (i.e., frequency modulation [FM] and harmonic performances) of mating calls and compared them between species that inhabit flowing-water or still-water environments. RESULTS: After including temperature, body size, habitat type and phylogenetic relationships, we found that DF differences among species were explained mostly by body size and habitat structure. We also showed that species living in lentic habitats tend to have advertisement calls characterized by well-defined FM and harmonics. Likewise, our results suggest that flowing-water habitats can constrain the evolutionary trajectories of the frequency-contour traits of advertisement calls in these anurans. CONCLUSIONS: Our results may support AAH predictions in frogs that vocalize in noisy habitats because flowing-water environments often produce persistent ambient noise. For instance, these anurans tend to generate vocalizations with less well-defined FM and harmonic traits. These findings may help us understand how noise in the environment can influence natural selection as it shapes acoustic signals in affected species.

Do pit vipers assess their venom? Defensive tactics of Deinagkistrodon acutus shift with changed venom reserve.

To maximize survival probability, animals must assess predation risks and adopt flexible defensive strategies based on specific conditions. Pit vipers utilize venom for predation and self-defense, and venom status significantly influences its effectiveness. Thus, pit vipers may evaluate their venom reserve and adopt corresponding defensive tactics. Twenty-three sharp-snouted pit vipers (Deinagkistrodon acutus) were grouped by different venom status and were subjected to eight behavior trials. Subjects' defensive behaviors were recorded and analyzed. Results showed that the normal venom group displayed stable responses across the trials. The low venom group showed fewer strikes and more fleeing behaviors at the end of experiments. After given prolonged intervals for replenishing the venom, significant increases of strike behaviors were observed in the replenishing venom group. These results demonstrated the capability of adopting flexible defensive tactics based on varied venom reserve and provided new evidence for venom-status-recognition.

Laterality in Responses to Acoustic Stimuli in Giant Pandas.

Cerebral lateralization is a common feature present in many vertebrates and is often observed in response to various sensory stimuli. Numerous studies have proposed that some vertebrate species have a right hemisphere or left hemisphere dominance in response to specific types of acoustic stimuli. We investigated lateralization of eight giant pandas (Ailuropoda melanoleuca) by using a head turning paradigm and twenty-eight acoustic stimuli with different emotional valences which included twenty-four conspecific and four non-conspecific acoustic stimuli (white noise, thunder, and vocalization of a predator). There was no significant difference in auditory laterality in responses to conspecific or non-conspecific sounds. However, the left cerebral hemisphere processed the positive stimuli, whereas neither of the two hemispheres exhibited a preference for processing the negative stimuli. Furthermore, the right hemisphere was faster than the left hemisphere in processing emotional stimuli and conspecific stimuli. These findings demonstrate that giant pandas exhibit lateralization in response to different acoustic stimuli, which provides evidence of hemispheric asymmetry in this species.

Multisensory modalities increase working memory for mating signals in a treefrog.

Animal choruses, such as those found in insects and frogs, are often intermittent. Thus, females sampling males in the chorus might have to remember the location of the potential mates' calls during periods of silence. Although a number of studies have shown that frogs use and prefer multimodal mating signals, usually acoustic plus visual, it is not clear why they do so. Here we tested the hypothesis that preference for multimodal signals over unimodal signals might be due to multimodal signals instantiating longer memories than unimodal signals, particularly during the inter-chorus intervals. We tested this hypothesis in serrate-legged small treefrogs Kurixalus odontotarsus whose males produce advertisement calls accompanied by conspicuous vocal sac inflation. Females were tested with acoustic and acoustic + visual (video of inflating-deflating vocal sac) mating calls. We found that females prefer multimodal calls over unimodal, audio-only calls. Furthermore, multimodal calls are still preferred after a silent period of up to 30 s, a time that spans the average silent period of the chorus. This was not true of unimodal calls. Our results demonstrate that a multimodal signal can engage longer working memory than a unimodal signal, and thus female memory might favour the evolution of multimodal signals in males through sexual selection. Selection might also favour female preference for multimodal signals if longer memory facilitates mate searching and assessment. Our study does not allow us to elucidate the sequence of evolution of this trait and preference.

Subdivisions of the mesencephalon and isthmus in the lizard Gekko gecko as revealed by ChAT immunohistochemistry.

The distribution of cholinergic cell bodies and fibers was examined in the mesencephalon and isthmus of Gekko gecko. Distinct groups with prominent labeled cells were observed in the cranial nerve motor nuclei and isthmic nuclei, and weak labeled cell bodies and fibers were observed in the mesencephalic nucleus of the trigeminal nerve and the central nucleus of the torus semicircularis. After discussing the topological relationships within the tectum and isthmus, we unify the nomenclature of the caudal deep mesencephalic nucleus in lizards and the rostral magnocellular nucleus isthmi in turtles that is similar in terms of the preisthmic position, nontopographic connections with the tectum, and the same midbrain origin to the magnocellular preisthmic nucleus in birds, and may be homologous to the superficial cuneiform nucleus in mammals. None of them belong to the cholinergic nucleus isthmi, as the latter has isthmus origin and topographic reciprocal connections with the tectum. We also discuss the origin and intrinsic function of the inner longitudinal tract of the thick ChAT-ir fibers that course through the mesencephalon and diencephalon. We review the subdivisions of the mesencephalon and isthmus of Gekko gecko as revealed by ChAT immunohistochemistry, as well as the limits of the diencephalo-mesencephalic, mesencephalic-isthmo, and isthmo-rhombocephalic by the ChAT-ir cell- and fiber-poor distribution, and discuss the caudal limit of the isthmus. Our research on the subdivisions of the mesencephalon and isthmus in G. gecko as revealed by ChAT immunohistochemistry will serve as the neuroanatomical basis for subsequent relevant studies of Gekko gecko.

A new pit viper of the genus Trimeresurus (Lacépède, 1804) (Squamata: Viperidae) from Southwest China.

The white-lipped tree viper, Trimeresurus albolabris Gray, is one of the most common species of the genus Trimeresurus with a wide distribution from India eastwards to China and southwards to Indonesia. However, this species was previously split into five geographical clades with significant genetic divergence. Recent surveys in Yingjiang County, Yunnan Province, China resulted in the discovery of one cryptic species of the subgenus Trimeresurus. Combining molecular, morphological and ecological data, we describe it as a new species Trimeresurus caudornatus sp. nov. The new species differs from other Trimeresurus species by a combination of the following characteristics: (1) Head and body generally dark green, postocular stripes absent in both genders, upper labials light green; (2) ventrolateral stripe faint green yellow, present on the first row of DSR in both genders; (3) iris golden yellow in both genders; (4) dorsal tail mostly dark red, lateral and ventral green; an orange red stripe along the ventral part of the tail; (5) DSR 21/22 -21-15, VEN 161-163, SC 52-72; (6) first upper labial partially fused to the nasal. The new species was shown to be a strongly supported monophyletic group (BPP 100%) and sister to T. septentrionalis. The uncorrected pairwise distances of mitochondrial gene Cytb between the new species and other species of the subgenus Trimeresurus, ranging from 0.059 (T. septentrionalis) to 0.134 (T. kanburiensis).

Author Correction: Locally-adapted reproductive photoperiodism determines population vulnerability to climate change in burying beetles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Locally-adapted reproductive photoperiodism determines population vulnerability to climate change in burying beetles.

Understanding how phenotypic traits vary among populations inhabiting different environments is critical for predicting a species' vulnerability to climate change. Yet, little is known about the key functional traits that determine the distribution of populations and the main mechanisms-phenotypic plasticity vs. local adaptation-underlying intraspecific functional trait variation. Using the Asian burying beetle Nicrophorus nepalensis, we demonstrate that mountain ranges differing in elevation and latitude offer unique thermal environments in which two functional traits-thermal tolerance and reproductive photoperiodism-interact to shape breeding phenology. We show that populations on different mountain ranges maintain similar thermal tolerances, but differ in reproductive photoperiodism. Through common garden and reciprocal transplant experiments, we confirm that reproductive photoperiodism is locally adapted and not phenotypically plastic. Accordingly, year-round breeding populations on mountains of intermediate elevation are likely to be most susceptible to future warming because maladaptation occurs when beetles try to breed at warmer temperatures.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Sex differences in vocalization are reflected by event-related potential components in the music frog.

Sex differences in vocalization have been commonly found in vocal animals. It remains unclear, however, how animals perceive and discriminate these differences. The amplitudes and latencies of event-related potentials (ERP) components can reflect the auditory processing efficiency and time course. We investigated the neural mechanisms of auditory processing in the Emei music frog (Nidirana daunchina) using an Oddball paradigm with ERP. We recorded and analyzed eletroencephalogram (EEG) signals from the forebrain and midbrain when the subjects listened to white noise (WN) and conspecific sex-specific vocalizations. We found that (1) both amplitudes and latencies of some ERP components evoked by conspecific calls were significantly higher than those by WN, suggesting the music frogs can discriminate conspecific vocalizations from background noise; (2) both amplitudes and latencies of most ERP components evoked by female calls were significantly higher or longer than those by male calls, implying that the ERP components can reflect sex differences in vocalization; and (3) there were significant differences in ERP amplitudes between male and female subjects, suggesting a sexual dimorphism in auditory perception. Together, the present results indicate that the music frog could discriminate conspecific calls from noise, male's calls from female's ones, and sexual dimorphism of auditory perception existed in this species.