Journal articles on the topic 'Frogs defensive mechanisms'

AbstractAposematic prey are thought to move slowly and openly near predators, but exhibit reduced escape behaviour. We studied conspicuousness and escape by aposematic poison frogs (Dendrobates auratus and Oophaga pumilio). In circles of leaf litter, observers detected poison frogs quickly. Flight initiation distance (FID, predator-prey distance when escape begins) increases with approach speed in non-cryptic palatable prey, but not for frogs in clearings, which permitted close approach. On trails frogs moved slowly into forest and FID in D. auratus increased with approach speed. Distance from cover and handling exposing predators to distastefulness may account for greater reliance on aposematism in clearings. We observed responses to a simulated predator (stick with painted face) in three conditions: not approached, approached, and touched. Latency to hop and time to exit circles decreased and exit from circles was directed further away from the approach path in the order not approached, approached, touched. Oophaga pumilio changed directions less when approached than not; many exhibited no escape behaviour. Aposematic dendrobatids move slowly near predators, but retain risk-assessment mechanisms due to occasional predation. Differences in escape between dendrobatids and palatable Craugastor frogs suggest that dendrobatid defensive behavior may have been molded to maximize the effectiveness of aposematism.

Systematic characters, distribution, natural history, and hemipenial morphology are described for Dendrophidion brunneum (Günther), a poorly known snake from Ecuador and Peru. It has 17 midbody scale rows; 145–165 ventrals; 135–158 subcaudals; a tail 38–44% of total length; a relatively uniform green to brown dorsum; and a maximum length of 1370 mm (700–800 mm SVL). The species occurs from northern Ecuador to 8° S latitude in Peru, primarily on the Pacific slopes of the Andes but with a few documented lowland localities in Peru. Dendrophidion brunneum is diurnal, terrestrial, heliophilic, and oviparous. It occurs in humid montane forests or mixed puna grassland/highland forest. Recorded prey are small terrestrial frogs but a large individual possibly consumed a lizard. Defensive mechanisms include a pseudautotomic tail and behaviors such as striking, biting, and neck inflation. The hemipenis has a short, narrow base, a greatly expanded distal region, and a simple, centrolineal sulcus spermaticus. The expanded region is ornamented with spines, distal to which two flounces completely encircle the organ and another extends partially around one side. The flounces are unusual in being thin, membranous structures and in having mineralized rods (reduced spinules) completely embedded within the membranes. The apex is nude except for a few vestigial calyces. Unusual hemipenial characters are discussed.

Ecological corridors are an essential element in conserving the biodiversity and proper functioning of ecosystems. Without their connectivity, a very large number of species would not have access to all of the habitats needed for their life cycles. Although the concept of an ecological corridor has been discussed for many years, few studies on ecological corridors for frogs have been conducted. Frogs are often considered to be a keystone species. They are a good indicator of habitat health, and they are often the first to be harmed by pollution or ecosystem deterioration. However, there have been reports of frogs crossing ecological corridors and being attacked or consumed by natural enemies. It is vital to create ecological corridors for frogs that allow them to migrate quickly and safely. The purpose of this study was to propose a new ecological corridor design concept for frogs to address the limitations mentioned above. In this paper, grey system theory was employed to offer the necessary information for the frog ladder’s design. In addition, the frog’s high jump capacity and its defense mechanisms against natural enemies were used to determine the rest space and shelter.

Antimicrobial Peptides (AMPs) are one of the most common components of the innate immune system that protect multicellular organisms against microbial invasion. The vast majority of AMPs are isolated from the frog skin. Anuran (frogs and toads) skin contains abundant AMPs that can be developed therapeutically. Such peptides are a unique but diverse group of molecules. In general, more than 50% of the amino acid residues form the hydrophobic part of the molecule. Normally, there are no conserved structural motifs responsible for activity, although the vast majority of the AMPs are cationic due to the presence of multiple lysine residues; this cationicity has a close relationship with antibacterial activity. Notably, recent evidence suggests that synthesis of AMPs in frog skin may confer an advantage on a particular species, although they are not essential for survival. Frog skin AMPs exert potent activity against antibiotic-resistant bacteria, protozoa, yeasts, and fungi by permeating and destroying the plasma membrane and inactivating intracellular targets. Importantly, since they do not bind to a specific receptor, AMPs are less likely to induce resistance mechanisms. Currently, the best known amphibian AMPs are esculentins, brevinins, ranacyclins, ranatuerins, nigrocin-2, magainins, dermaseptins, bombinins, temporins, and japonicins-1 and -2, and palustrin-2. This review focuses on these frog skin AMPs and the mechanisms underlying their antimicrobial activity. We hope that this review will provide further information that will facilitate further study of AMPs and cast new light on novel and safer microbicides.

Cellular mechanisms underlying defense reactions induced by noxious heat and acids were studied in frogs ( Rana pipiens) by measuring whole cell membrane currents in cultured dorsal root ganglion (DRG) neurons. Seventy-eight of 82 DRG neurons exposed to 3-s ramps of increasing temperature to 48°C exhibited an inward current ( I HEAT) of 490 ± 70 pA at −70 mV. I HEAT exhibited reversal at ∼10 mV with a pronounced outward rectification, suggesting opening of nonselective cation channels. In frogs, in contrast to mammals, I HEAT was not influenced by capsaicin (5 μM), capsazepine (10 μM), or ruthenium red (10 μM). In a large proportion (∼80%) of heat-sensitive DRG neurons, acids produced a large slowly inactivating sodium carried current ( I ACID) with average pH50 5.7. I ACIDwas blocked by 1 mM amiloride (to 22%) and was absent if extracellular Na+ was substituted by Cs+. Elevating temperature to 38°C increased I ACID, whereas temperatures >40°C profoundly inhibited it (by 82 ± 2%; n = 42). The inhibition was long-lasting (>30 s) but fully reversible. Phorbol ester myristate acetate (PMA, 1 μM) and forskolin (1 μM) inhibited I ACID to 37 ± 5% ( n = 5) and 78 ± 8% ( n = 4), respectively. It is suggested that I HEAT in frog DRG neurons is carried through capsaicin-insensitive nonselective cation channels distinct from vanilloid receptor in mammals, whereas I ACID is carried through amiloride-sensitive sodium channels that are strongly inhibited by noxious heat, possibly due to activation of the intracellular messenger systems.

Frogs (Anura) are one of the most diverse vertebrate orders, comprising more than 7,000 species with a worldwide distribution and extensive ecological diversity. In contrast to other tetrapods, frogs have a highly derived body plan and simplified skull. In many lineages of anurans, increased mineralization has led to hyperossified skulls, but the function of this trait and its relationship with other aspects of head morphology are largely unexplored. Using three-dimensional morphological data from 158 species representing all frog families, we assessed wide-scale patterns of shape variation across all major lineages, reconstructed the evolutionary history of cranial hyperossification across the anuran phylogeny, and tested for relationships between ecology, skull shape, and hyperossification. Although many frogs share a conserved skull shape, several extreme forms have repeatedly evolved that commonly are associated with hyperossification, which has evolved independently more than 25 times. Variation in cranial shape is not explained by phylogenetic relatedness but is correlated with shifts in body size and ecology. The species with highly divergent, hyperossified skulls often have a specialized diet or a unique predator defense mechanism. Thus, the evolution of hyperossification has repeatedly facilitated the expansion of the head into multiple new shapes and functions.

Aposematic organisms couple conspicuous warning signals with a secondary defense to deter predators from attacking. Novel signals of aposematic prey are expected to be selected against due to positive frequency-dependent selection. How, then, can novel phenotypes persist after they arise, and why do so many aposematic species exhibit intrapopulation signal variability? Using a polytypic poison frog (Dendrobates tinctorius), we explored the forces of selection on variable aposematic signals using 2 phenotypically distinct (white, yellow) populations. Contrary to expectations, local phenotype was not always better protected compared to novel phenotypes in either population; in the white population, the novel phenotype evoked greater avoidance in natural predators. Despite having a lower quantity of alkaloids, the skin extracts from yellow frogs provoked higher aversive reactions by birds than white frogs in the laboratory, although both populations differed from controls. Similarly, predators learned to avoid the yellow signal faster than the white signal, and generalized their learned avoidance of yellow but not white. We propose that signals that are easily learned and broadly generalized can protect rare, novel signals, and weak warning signals (i.e., signals with poor efficacy and/or poor defense) can persist when gene flow among populations, as in this case, is limited. This provides a mechanism for the persistence of intrapopulation aposematic variation, a likely precursor to polytypism and driver of speciation.

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

Anurans may be brightly colored or completely cryptic. Generally, in the former situation, we are dealing with aposematism, and the latter is an example of camouflage. However, these are only simple views of what such colorations really mean and which defensive strategy is implied. For instance, a brightly colored frog may be part of a mimicry ring, which could be either Batesian, Müllerian, or Browerian. These are only examples of the diversity of color-usage systems as defensive strategies. Unfortunately, reports on the use of colors as defensive mechanisms are widespread in the available literature, and the possible functions are rarely mentioned. Therefore, we reviewed the literature and added new data to this subject. Then, we the use of colors (as defensive mechanism) into categories. Mimicry was divided into the subcategories camouflage, homotypy, and nondeceitful homotypy, and these groups were also subcategorized. Dissuasive coloration was divided into behavioral display of colors, polymorphism, and polyphenism. Aposematism was treated apart, but aposematic colorations may be present in other defensive strategies. Finally, we propose functions and forms of evolution for some color systems in post-metamorphic anurans and hope that this review can be the basis for future research, even on other animal groups.

This article describes how nature-inspired algorithms (NIAs) have evolved as efficient approaches for addressing the complexities inherent in the optimization of real-world applications. These algorithms are designed to imitate processes in nature that provide some ways of problem solving. Although various nature-inspired algorithms have been proposed by various researchers in the past, a robust and computationally simple NIA is still missing. A novel nature-inspired algorithm that adapts to the anti-predatory behavior of the frog is proposed. The algorithm mimics the self defense mechanism of a frog. Frogs use their reflexes as a means of protecting themselves from the predators. A mathematical formulation of these reflexes forms the core of the proposed approach. The robustness of the proposed algorithm is verified through performance evaluation on sixteen different unconstrained mathematical benchmark functions based on best and worst values as well as mean and standard deviation of the computed results. These functions are representative of different properties and characteristics of the problem domain. The strength and robustness of the proposed algorithm is established through a comparative result analysis with six well-known optimization algorithms, namely: genetic, particle swarm, differential evolution, artificial bee colony, teacher learning and Jaya. The Friedman rank test and the Holm-Sidak test have been used for statistical analysis of obtained results. The proposed algorithm ranks first in the case of mean result and scores second rank in the case of “standard deviation”. This proves the significance of the proposed algorithm.

The skin secretion of tree frogs contains a vast array of bioactive chemicals for repelling predators, but their structural and functional diversity is not fully understood. Paxilline (PAX), a compound synthesized by Penicillium paxilli, has been known as a specific antagonist of large conductance Ca2+-activated K+ Channels (BKCa). Here, we report the presence of PAX in the secretions of tree frogs (Hyla japonica) and that this compound has a novel function of inhibiting the potassium channel subfamily K member 18 (KCNK18) channels of their predators. The PAX-induced KCNK18 inhibition is sufficient to evoke Ca2+ influx in charybdotoxin-insensitive DRG neurons of rats. By forming π-π stacking interactions, four phenylalanines located in the central pore of KCNK18 stabilize PAX to block the ion permeation. For PAX-mediated toxicity, our results from animal assays suggest that the inhibition of KCNK18 likely acts synergistically with that of BKCa to elicit tingling and buzzing sensations in predators or competitors. These results not only show the molecular mechanism of PAX-KCNK18 interaction, but also provide insights into the defensive effects of the enriched PAX.

Social monogamy, typically characterized by the formation of a pair bond, increased territorial defense, and often biparental care, has independently evolved multiple times in animals. Despite the independent evolutionary origins of monogamous mating systems, several homologous brain regions and neuropeptides and their receptors have been shown to play a conserved role in regulating social affiliation and parental care, but little is known about the neuromolecular mechanisms underlying monogamy on a genomic scale. Here, we compare neural transcriptomes of reproductive males in monogamous and nonmonogamous species pairs ofPeromyscusmice,Microtusvoles, parid songbirds, dendrobatid frogs, andXenotilapiaspecies of cichlid fishes. We find that, while evolutionary divergence time between species or clades did not explain gene expression similarity, characteristics of the mating system correlated with neural gene expression patterns, and neural gene expression varied concordantly across vertebrates when species transition to monogamy. Our study provides evidence of a universal transcriptomic mechanism underlying the evolution of monogamy in vertebrates.

Toxins are the poisonous products of organisms. Toxins serve vital defensive and offensive functions for those that harbor them: stinging scorpions, pesticidal plants, sanguinary snakes, fearless frogs, sliming snails, noxious newts, and smarting spiders. For physiologists, toxins are integral chemical tools that hijack life’s fundamental processes with remarkable molecular specificity. Our understanding of electrophysiological phenomena has been transformed time and time again with the help of some terrifying toxins. For this reason, studies of toxin mechanism are an important and enduring facet of The Journal of General Physiology (JGP). This Milestone in Physiology reflects on toxins studied in JGP over its first 100 years, what they have taught us, and what they have yet to reveal.

Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD+ kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca2+/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

Abstract In a series of papers starting in the early 1980s, Toft proposed a general scenario to explain dietary evolution in leaf litter anurans in which species would “form a continuum from those that specialize on ants and mites, through generalists, to species that avoid ants and mites”, and these differences would in turn correlate with foraging strategies, morphology, and defense mechanisms. In this study, we reassess this hypothesis using a global dataset on the dietary composition of 120 anuran species. Surprisingly, we found that the relative contribution of ants and mites in anuran diets were largely orthogonal to one another. Moreover, we did not find evidence for the continuum of dietary composition envisioned by Toft. These results suggest that, although ants and mites have played a major role in the evolution of aposematic species, the trends found in those species might not be directly extrapolated to all leaf litter anurans.

SUMMARY For most vertebrates, cutting off the oxygen supply to the brain results in a rapid (within minutes) loss of ATP, the failure of ATP-dependent ion-transport process, subsequent anoxic depolarization of neuronal membrane potential and consequential neuronal death. The few species that survive brain anoxia for days or months, such as the freshwater turtle Trachemys scripta, avoid anoxic depolarization and maintain brain ATP levels through a coordinated downregulation of brain energy demand processes. The frog Rana pipiens represents an intermediate in anoxia-tolerance, being able to survive brain anoxia for hours. However, the anoxic frog brain does not defend its energy stores. Instead, anoxia-tolerance appears to be related to a retarded rate of ATP depletion. To investigate the relationship between this slow ATP depletion and the loss of ionic homeostasis, cerebral extracellular K+ concentrations were monitored and ATP levels measured during anoxia, during the initial phase of anoxic depolarization and during complete anoxic depolarization. Extracellular K+ levels were maintained at normoxic levels for at least 3 h of anoxia, while ATP content decreased by 35 %. When ATP levels reached 0.33±0.06 mmol l–1 (mean ± s.e.m., N=5), extracellular K+ levels slowly started to increase. This value is thought to represent a critical ATP concentration for the maintenance of ion homeostasis. When extracellular [K+] reached an inflection value of 4.77±0.84 mmol l–1 (mean ± s.e.m., N=5), approximately 1 h later, the brain quickly depolarized. Part of the reduction in ATP demand was attributable to an approximately 50 % decrease in the rate of K+ efflux from the anoxic frog brain, which would also contribute to the retarded rate of increase in extracellular [K+] during the initial phase of anoxic depolarization. However, unlike the anoxia-tolerant turtle brain, adenosine did not appear to be involved in the downregulation of K+ leakage in the frog brain. The increased anoxia-tolerance of the frog brain is thought to be a matter more of slow death than of enhanced protective mechanisms.

Host defense peptides (HDPs) are a group of antimicrobial peptides (AMPs) that are crucial components of the innate immune system of many different organisms. These small peptides actively kill microbes and prevent infection. Despite the presence of AMPs in the amphibian immune system, populations of these organisms are in decline globally. Magainin is an AMP derived from the African clawed frog (Xenopus laevis) and has displayed potent antimicrobial effects against a wide variety of microbes. Included in this group of microbes are known pathogens of the African clawed frog and other amphibian species. Arguably, the most deleterious amphibious pathogen is Batrachochytrium dendrobatidis, a chytrid fungus. Investigating the mechanism of action of magainin can help understand how to effectively fight off infection. By understanding amphibian AMPs’ role in the frog, a potential conservation strategy can be developed for other species of amphibians that are susceptible to infections, such as the North American green frog (Rana clamitans). Considering that population declines of these organisms are occurring globally, this effort is crucial to protect not only these organisms but the ecosystems they inhabit as well.

Magainins, antimicrobial peptides secreted by granular glands of frog skin, may be related to the high resistance to infections of this epithelial surface. The oral mucosa of healthy individuals is another tissue in which infection is not frequent, probably owing to the activity of potent salivary and mucosal defense mechanisms. To investigate if magainin-like factors are a component of these oral defense mechanisms, human and animal minor (mucosal) and major salivary glands were examined by immunohistochemistry, using a polyclonal rabbit anti-magainin antibody. Cryostat sections of (para) formaldehyde-fixed tissues were incubated with the antibody and then stained with fluorescein-complexed anti-rabbit IgG. Specific staining was observed in the apical portion of the cytoplasm of ductal epithelial cells of human submandibular and labial salivary glands. Diffuse staining was present in submandibular acinar cells. Bovine, rat, hamster, and mouse tissues were unreactive. The presence of magainin-like substances in human salivary gland duct cells is consistent with reports of the occurrence of other biologically active substances in salivary gland ducts.

Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 µM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC).

Nature-Inspired Algorithms (NIAs) are one of the most efficient methods to solve the optimization problems. A recently proposed NIA is the anti-predatory NIA, which is based on the anti-predatory behavior of frogs. This algorithm uses five different types of self-defense mechanisms in order to improve its anti-predatory strength. This paper demonstrates the computation steps of anti-predatory for solving the Rastrigin function and attempts to solve 20 unconstrained minimization problems using anti-predatory NIA. The performance of anti-predatory NIA is compared with the six competing meta-heuristic algorithms. A comparative study reveals that the anti-predatory NIA is a more promising than the other algorithms. To quantify the performance comparison between the algorithms, Friedman rank test and Holm-Sidak test are used as statistical analysis methods. Anti-predatory NIA ranks first in both cases of “Mean Result” and “Standard Deviation.” Result measures the robustness and correctness of the anti-predatory NIA. This signifies the worth of anti-predatory NIA in the domain of mathematical optimization.

Cytotoxic peptides are relatively small cationic molecules such as those found 1) in venoms, e.g., melittin in bee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, and lycotoxin I and II in wolf spider; 2) in skin secretions (e.g., magainin I and II from Xenopus laevis, dermaseptin from frog, antimicrobials from carp) and cells of the immune system (e.g., insect, scorpion, and mammalian defensins and cryptdins); 3) as autocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic β-cells, prion peptide fragment 106–126 [PrP-(106–126)], and amyloid β-protein (AβP) cytotoxic to neurons; and 4) as designed synthetic peptides based on the sequences and properties of naturally occurring cytotoxic peptides. The small cytotoxic peptides are composed of β-sheets, e.g., mammalian defensins, AβP, amylin, and PrP-(106–126), whereas the larger cytotoxic peptides have several domains composed of both α-helices and β-sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modify cell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These two nonexclusive mechanisms of action lead to changes in second messenger systems that further augment the abnormal electrical activity and distortion of the signal transduction causing cell death.

Amphibian skin secretions are remarkable sources of novel bioactive peptides. Among these, antimicrobial peptides have demonstrated an outstanding efficacy in killing microorganisms via a general membranolytic mechanism, which may offer the prospect of solving specific target-driven antibiotic resistance. Here, the discovery of a novel defensive peptide is described from the skin secretion of the African frog, Kassina senegalensis. Named kassinatuerin-3, it was identified through a combination of “shot-gun” cloning and MS/MS fragmentation sequencing. Subsequently, a synthetic replicate was subjected to biofunctional evaluation. The results indicated that kassinatuerin-3 possessed antimicrobial activity against Gram-positive bacteria but no effect against Gram-negative bacteria. Additionally, it was active in biofilm eradication on S. aureus and MRSA and in the antiproliferation of selected cancer cell lines. Moreover, it had a very mild hemolytic effect, which demonstrated a high therapeutic index for kassinatuerin-3. Collectively, although kassinatuerin-3 did not demonstrate remarkable bioactivities compared with other natural or synthetic antimicrobial peptides (AMPs), it offered a new insight into the design of antimicrobial derivatives.

Recently, bioactive peptides have attracted attention for their therapeutic applications in the pharmaceutical industry. Among them, antimicrobial peptides are candidates for new antibiotic drugs. Since pseudin-2 (Ps), isolated from the skin of the paradoxical frog Pseudis paradoxa, shows broad-spectrum antibacterial activity with high cytotoxicity, we previously designed Ps-K18 with a Lys substitution for Leu18 in Ps, which showed high antibacterial activity and low toxicity. Here, we examined the potency of Ps-K18, aiming to develop antibiotics derived from bioactive peptides for the treatment of Gram-negative sepsis. We first investigated the antibacterial mechanism of Ps-K18 based on confocal micrographs and field emission scanning electron microscopy, confirming that Ps-K18 targets the bacterial membrane. Anti-inflammatory mechanism of Ps-K18 was investigated by secreted alkaline phosphatase reporter gene assays and RT-PCR, which revealed that Ps-K18 activates innate defense via Toll-like receptor 4-mediated nuclear factor-kappa B signaling pathways. Moreover, we investigated the antiseptic effect of Ps-K18 using a lipopolysaccharide or Escherichia coli K1-induced septic shock mouse model. Ps-K18 significantly reduced bacterial growth and inflammatory responses in the septic shock model. Ps-K18 showed low renal and liver toxicity and attenuated lung damage effectively. This study suggests that Ps-K18 is a potent peptide antibiotic that could be applied therapeutically to Gram-negative sepsis.

Abstract The frog skin host-defense peptide tigerinin-1R stimulates insulin release in vitro and improves glucose tolerance and insulin sensitivity in animal models of type 2 diabetes. This study extends these observations by investigating the molecular mechanisms of action underlying the beneficial metabolic effects of the analogue [Arg4]tigerinin-1R in mice with diet-induced obesity, glucose intolerance and insulin resistance. The study also investigates the electrophysiological effects of the peptide on KATP and L-type Ca2+ channels in BRIN-BD11 clonal β cells. Non-fasting plasma glucose and glucagon concentrations were significantly (p<0.05) decreased and plasma insulin increased by twice daily treatment with [Arg4]tigerinin-1R (75 nmol/kg body weight) for 28 days. Oral and intraperitoneal glucose tolerance were significantly (p<0.05) improved accompanied by enhanced secretion and action of insulin. The peptide blocked KATP channels and, consistent with this, improved beta cell responses of isolated islets to a range of secretagogues. Peptide administration resulted in up-regulation of key functional genes in islets involved insulin secretion (Abcc8, Kcnj11, Cacna1c and Slc2a2) and in skeletal muscle involved with insulin action (Insr, Irs1, Pdk1, Pik3ca, and Slc2a4). These observations encourage further development of tigerinin-1R analogues for the treatment of patients with type 2 diabetes.

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

Whole genome sequencing (WGS) is a very valuable resource to understand the evolutionary history of poorly known species. However, in organisms with large genomes, as most amphibians, WGS is still excessively challenging and transcriptome sequencing (RNA-seq) represents a cost-effective tool to explore genome-wide variability. Non-model organisms do not usually have a reference genome and the transcriptome must be assembledde-novo. We used RNA-seq to obtain the transcriptomic profile forOreobates cruralis, a poorly known South American direct-developing frog. In total, 550,871 transcripts were assembled, corresponding to 422,999 putative genes. Of those, we identified 23,500, 37,349, 38,120 and 45,885 genes present in the Pfam, EggNOG, KEGG and GO databases, respectively. Interestingly, our results suggested that genes related to immune system and defense mechanisms are abundant in the transcriptome ofO. cruralis. We also present a pipeline to assist with pre-processing, assembling, evaluating and functionally annotating ade-novotranscriptome from RNA-seq data of non-model organisms. Our pipeline guides the inexperienced user in an intuitive way through all the necessary steps to buildde-novotranscriptome assemblies using readily available software and is freely available at:https://github.com/biomendi/TRANSCRIPTOME-ASSEMBLY-PIPELINE/wiki.

Freeze tolerance is an amazing winter survival strategy used by various amphibians and reptiles living in seasonally cold environments. These animals may spend weeks or months with up to ∼65% of their total body water frozen as extracellular ice and no physiological vital signs, and yet after thawing they return to normal life within a few hours. Two main principles of animal freeze tolerance have received much attention: the production of high concentrations of organic osmolytes (glucose, glycerol, urea among amphibians) that protect the intracellular environment, and the control of ice within the body (the first putative ice-binding protein in a frog was recently identified), but many other strategies of biochemical adaptation also contribute to freezing survival. Discussed herein are recent advances in our understanding of amphibian and reptile freeze tolerance with a focus on cell preservation strategies (chaperones, antioxidants, damage defense mechanisms), membrane transporters for water and cryoprotectants, energy metabolism, gene/protein adaptations, and the regulatory control of freeze-responsive hypometabolism at multiple levels (epigenetic regulation of DNA, microRNA action, cell signaling and transcription factor regulation, cell cycle control, and anti-apoptosis). All are providing a much more complete picture of life in the frozen state.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.

Amphibian skin is acknowledged to contain an antioxidant system composed of various gene-encoded antioxidant peptides, which exert significant effects on host defense. Nevertheless, recognition of such peptides is in its infancy so far. Here, we reported the antioxidant properties and underlying mechanism of a new antioxidant peptide, brevinin-1FL, identified from Fejervarya limnocharis frog skin. The cDNA sequence encoding brevinin-1FL was successfully cloned from the total cDNA of F. limnocharis and showed to contain 222 bp. The deduced mature peptide sequence of brevinin-1FL was FWERCSRWLLN. Functional analysis revealed that brevinin-1FL could concentration-dependently scavenge ABTS+, DPPH, NO, and hydroxyl radicals and alleviate iron oxidation. Besides, brevinin-1FL was found to show neuroprotective activity by reducing contents of MDA and ROS plus mitochondrial membrane potential, increasing endogenous antioxidant enzyme activity, and suppressing H2O2-induced death, apoptosis, and cycle arrest in PC12 cells which were associated with its regulation of AKT/MAPK/NF-κB signal pathways. Moreover, brevinin-1FL relieved paw edema, decreased the levels of TNF-α, IL-1β, IL-6, MPO, and malondialdehyde (MDA), and restored catalase (CAT) and superoxide dismutase (SOD) activity plus glutathione (GSH) contents in the mouse injected by carrageenan. Together, these findings indicate that brevinin-1FL as an antioxidant has potent therapeutic potential for the diseases induced by oxidative damage. Meanwhile, this study will help us further comprehend the biological functions of amphibian skin and the mechanism by which antioxidants protect cells from oxidative stress.

ABSTRACTHost defense antimicrobial peptides (AMPs) are a promising source of antibiotics for the treatment of multiple-drug-resistant pathogens. Lipopolysaccharide (LPS), the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, functions as a permeability barrier against a variety of molecules, including AMPs. Further, LPS or endotoxin is the causative agent of sepsis killing 100,000 people per year in the United States alone. LPS can restrict the activity of AMPs inducing aggregations at the outer membrane, as observed for frog AMPs, temporins, and also in model AMPs. Aggregated AMPs, “trapped” by the outer membrane, are unable to traverse the cell wall, causing their inactivation. In this work, we show that these inactive AMPs can overcome LPS-induced aggregations while conjugated with a short LPS binding β-boomerang peptide motif and become highly bactericidal. The generated hybrid peptides exhibit activity against Gram-negative and Gram-positive bacteria in high-salt conditions and detoxify endotoxin. Structural and biophysical studies establish the mechanism of action of these peptides in LPS outer membrane. Most importantly, this study provides a new concept for the development of a potent broad-spectrum antibiotic with efficient outer membrane disruption as the mode of action.

Introduction Such symptoms as hard, complex, bodily or mental feelings, that turn our everyday life into a hell, at first, lead us to a doctor, and then - to a psychotherapist. A sick man is keen to get rid of a symptom. A doctor prescribes medication, that is ought to eliminate a symptom. A psychotherapist searches for a reason of the problem that needs to be removed. There is such an idea that a neurotic symptom, in particular, an anxiety - is a pathological (spare or extra) response of a body. It is generally believed that such anxiety doesn’t have some real, objective reasons and that it is the result of a nervous system disorder, or some disruption of a cognitive sphere etc. Meanwhile, it is known that in the majority of cases, medical examinations of anxious people show that they don’t have any organic damages, including nervous system. It often happens that patients even wish doctors have found at least any pathology and have begun its treatment. And yet - there is no pathology. All examinations indicate a high level of functionality of a body and great performance of the brain's work. Doctors throw their hands up, as they can't cure healthy people. One of my clients told me her story of such medical examinations (which I’ll tell you with her permission). She said that it was more than 10 years ago. So, when she told her doctor all of her symptoms - he seemed very interested in it. He placed a helmet with electrodes on her head and wore some special glasses, when, according to her words, he created some kind of stressful situation for her brain, as she was seeing some flashings of bright pictures in her eyes. She said that he had been bothered with her for quite a long time, and at the end of it he had told her that her brain had been performing the best results in all respects. He noted that he’d rarely got patients with such great health indicators. My client asked the doctor how rare that was. And he answered: “one client in two or three months.” At that moment my client didn’t know whether to be relieved, flattered or sad. But since then, when someone told her that anxiety was a certain sign of mental problems, or problems with the nervous system, or with a body in general, she answered that people who had anxiety usually had already got all the required medical examinations sufficiently, and gave them the advice to go through medical screening by themselves before saying something like that. Therefore, we see a paradoxical situation, when some experts point to a neurotic anxiety as if it is a kind of pathology, in other words - some result of a nervous system disorder. Other specialists in the same situation talk about cognitive impairments. And some, after all the examinations, are ready to send such patients into space Main text I don’t agree with the statement that any neurotic anxiety that happens is excessive and unfounded. It often happens that there is objective, specific and real causes for appearance of anxiety conditions. And these causes require solutions. And it’s not about some organic damages of the brain or nervous system. The precondition that may give a rise to anxiety disorder is the development of such a life story that at some stage becomes too toxic - when, on the one hand, a person interacts with the outside world in a way that destroys his or her personality, and, on the other hand, this person uses repression and accepts such situation as common and normal. Repression - is an essential condition for the development of a neurotic symptom. Sigmund Freud was the first who pointed this out. Repression is such a defense mechanism that helps people separate themselves from some unpleasant feelings of discomfort (pain) while having (external or internal) irritations. It is the situation when, despite the presence of irritations and painful feelings, a person, however, doesn't feel any of it and is not aware of them in his or her conscious mind. Repression creates the situation of so-called emotional anesthesia. As a result, a displacement takes place, so a body starts to signal about the existing toxic life situation via a symptom. Anxiety disorder is usually an appropriate response (symptom) of a healthy body to an unhealthy life situation, which is seen by a person as normal. And it’s common when such a person is surrounded by others (close people), who tend to benefit from such situation, and so they actively maintain this state of affairs, whether it is conscious for them or not. At the beginning of a psychotherapy almost all clients insist that everything is good in their lives, even great, as it is like in everyone else’s life. They say that they have only one problem, which is that goddamn symptom. So they focus all of their attention on that symptom. They are not interested in all the other aspects of their life, and they show their irritation when it comes to talking about it. People want to get rid of it, whatever it takes, but they often tend to keep their lives the way that it was. In such cases a psychotherapist is dealing with the resistance of clients, trying to turn their attention from a symptom to their everyday situation that includes their way of thinking, interactions with themselves and with others and with the external world in order to have the opportunity to see the real problem, to live it through, to rethink and to change the story of their lives. For better understanding about how it works I want to tell you three allegorical tales. The name of the first tale is “A frog in boiling water”. There is one scientific anecdote and an assumption (however, it is noted that such experiments were held in 19 century), that if we put a frog in a pot with warm water and start to slowly heat the water, then this frog get used to the temperature rise and stays in a hot water, the frog doesn’t fight the situation, slowly begins to lose its energy and at the last moment it couldn’t find enough strength and energy to get out of that pot. But if we throw a frog abruptly in hot water - it jumps out very quickly. It is likely that a frog, that is seating in boiling water, will have some responses of the body (symptoms). For example, the temperature of its body will rise, the same as the color of it, etc., that is an absolutely normal body response to the existing situation. But let us keep fantasizing further. Imagine a cartoon where such a frog is the magical cartoon hero, that comes to some magical cartoon doctor, shows its skin, that has changed the color, to the doctor, and asks to change the situation by removing this unpleasant symptom. So the doctor prescribes some medication to return the natural green color of the frog’s skin back. The frog gets back in its hot water. For some period of time this medication helps. But then, after a while, the frog’s body gets over the situation, and the redness of the frog's skin gets back. And the magical cartoon doctor states that the resistance of the body to this medication has increased, and each time prescribes some more and more strong drugs. In this example with the frog it is perfectly clear that the true solution of the problem requires the reduction of the water temperature in that pot. We could propose that magical cartoon frog to think and try to realize that: 1) the water in that pot is hot, and that is the reason why the skin is red; 2) the frog got used to this situation and that is why it is so unnoticeably for this frog; 3) if the temperature of the water in the pot still stay so hot, without any temperature drop, then all the medication works only temporarily; 4) if we lower the temperature in that pot - the redness disappears on its own, automatically and without any medication. Also this cartoon frog, that will go after the doctor to some cartoon physiotherapist, will face the necessity to give itself some answers for such questions as: 1) What is going on? Who has put this frog in that pot? Who is raising the temperature progressively? Who needs it? And what is the purpose or benefit for this person in that? Who benefits? 2) Why did the frog get into the pot? What are the benefits in it for the frog? Or why did the frog agree to that? 3) What does the frog lose when it gets out of this pot? What are the consequences of it for the frog? What does the frog have to face? What are the possible difficulties on the way? Who would be against the changes? With whom the frog may confront? 4) Is the frog ready to take control over its own pot in its own hands and start to regulate the temperature of the water by itself, so to make this temperature comfortable for itself? Is this frog ready to influence by itself on its own living space, to take the responsibility for it to itself? The example “A frog in boiling water” is often used as a metaphorical portrayal of the inability of people to respond (or fight back) to significant changes that slowly happen in their lives. Also this tale shows that a body, while trying to adjust to unfavorable living conditions, will react with a symptom. And it is very important to understand this symptom. Symptom - is the response of a body, it’s a way a body adjusts to some unfriendly environment. Symptom, on the one hand, informs about the existence of a problem, and from the other hand - tries to regulate this problem, at least in some way (like, to remove or reduce), at the level on which it can do it. The process is similar to those when, for example, in a body, while it suffers from some infectious disease, the temperature rises. Thus, on the one hand, the temperature informs about the existence of some infection. On the other hand, the temperature increase creates in a body the situation that is damaging for the infection. So, it would be good to think about in what way does an anxiety symptom help a body that is surrounded by some toxic life situation. And this is a good topic for another article. Here I want to emphasize that all the attempts to remove a symptom without a removal of a problem, without changing the everyday life story, may lead to strengthening of the symptom in the body. Even though the removal of a symptom without elimination of its cause has shown success, it only means that the situation was changed into the condition of asymptomatic existence of a problem. And it is, in its essence, a worse situation. For example, it can cause an occurrence of cancer. The tale “A frog in boiling water” is about the tendency of people to treat a symptom, instead of seeing their real problems, as its cause, and trying to solve it. People don’t want to see their problems, but it doesn’t mean that the problem doesn’t exist. The problem does exist and it continues to destroy a person, unnoticeably for him or her. A person with panic disorder could show us anxiety that is out of control (fear, panic), which, by its essence, seems to exist without any logical reason. Meanwhile the body of such a person could be in such processes that are similar to those that occur in the conditions of some real dangers, when the instinct for self-preservation is triggered and an automatic response of a body to fight or flight implements for its full potential. We can see or feel signs of this response, for example, in cases when some person tries to avoid some real or imaginary danger via attempts to escape (the feeling of fear), or tries to handle the situation by some attempts to fight (the feeling of anger). As I mentioned before, many doctors believe that such fear is pathological, as there is no real reason for such intense anxiety. They may see the cause of the problem in worrisome temper, so they try to remove specifically anxiety rather than help such patients to understand specific reason of their anxiety, they use special psychotherapeutic methods that are designed to help clients to develop logical thinking, so it must help them to realize the groundlessness of their anxiety. In my point of view, such anxiety often has specific, real reasons, when this response of a body, fight or flight, is absolutely appropriate, but not excessive or pathological. Inadequacy, in fact, is in the unconsciousness, but not in the reactions of a body. For a better understanding of the role of anxiety in some toxic environment, that isn’t realized, I want to tell you another allegorical tale called “The wolf and the hare”. Let us imagine that two cages were brought together in one room. The wolf was inside one cage and the hare was in another. The cages were divided by some kind of curtain that makes it impossible for them to see each other. At this point a question arises whether the animals react to each other in some way in such a situation, or not? I think that yes, they will. Since there are a lot of other receptors that participate in the receiving and processing of the sensory information. As well as sight and hearing, we have of course a range of other senses. For example, animals have a strong sense of smell. It is well known that people, along with verbal methods of communicating information, like language and speaking, also have other means of transmitting information - non-verbal, such as tone of voice, intonation, look, gestures, body language, facial expressions etc., that gives us the opportunity to receive additional information from each other. The lie detector works by using this principle: due to detecting non-verbal signals, it distinguishes the level of the accuracy of information that is transmitted. It is assumed, that about 30% of information, that we receive from the environment, comes through words, vision, hearing, touches etc. This is the information that we are aware of in our consciousness, so we could consciously (logically) use it to be guided by. And approximately 70% of everyday information about the reality around us we receive non-verbally, and this information in the majority of cases could remain in us without any recognition. It is the situation when we’ve already known something, and we even have already started to respond to it via our body, but we still don’t know logically and consciously that we know it. We can observe the responses of our own body without understanding what are the reasons for such responses. We can recognize this unconscious information through certain pictures, associations, dreams, or with the help of psychoanalysis. Psychoanalysis is a great tool that can help to recognize the information from the unconscious mind, so that it can be logically processed further on, in other words, a person then receives the opportunity to indicate the real problems and to make right decisions. But let us return to the tale where the hare and the wolf stay in one room and don’t see each other, and, maybe, don’t hear, though - feel. These feelings (in other words - non-verbal information that the hare receives) activate a certain response in the hare’s body. And it reacts properly and adequately to the situation, for instance, the body starts to produce adrenaline and runs the response “fight or flight”. So the hare starts to behave accordingly and we could see the following symptoms: the hare is running around his cage, fussing, having some tremor and an increased heart rate, etc.. And now let us imagine this tale in some cartoon. The hare stays in its house, and the wolf wanders about this house. But the hare doesn’t see the wolf. Though the body of the hare gives some appropriate responses. And then that cartoon hare goes to a cartoon doctor and asks that doctor to give it some pill from its tremor and the increased heart rate. And in general asks to treat in some way this incomprehensible, confusing, totally unreasonable severe anxiety. If we try to replace the situation from this fairy-tale to a life story, we could see that it fits well to the script of interdependent relationships, where there are a couple “a victim and an aggressor”, and where such common for our traditional families’ occurrences as a domestic family violence, psychological and physical abuse take place. Only in 2019 a law was passed that follows the European norms and gives a legislative definition of such concepts as psychological domestic abuse, sexual abuse, physical abuse, bullying, that criminalizes all of these occurrences, establishes the punishment and directly points to people that could be a potential abuser. Among them are: a husband towards his wife, parents towards their children, a wife towards her husband, a superior towards a subordinate, a teacher towards his or her students, children towards each other etc.. When it comes to recognition of something as unacceptable, it seems more easy to put to that category such occurrences as physical and sexual abuse, as we could see here some obvious events. For example, beating or sexual harassment. Our society is ready to respond to these incidents in more or less adequate way, and to recognize them as a crime. But it is harder to deal with the recognition of psychological abuse as an offence. Psychological abuse in our families is common. Psychological abuse occurs through such situations, when one person, while using different psychological manipulations, such as violation of psychological borders, imposition of feeling of guilty or shame, etc., force another person to give up his or her needs and desires, and so in such a way make this person live another’s life. Such actions have an extremely negative effect on the mental health of these people, just as much as physical abuse. It can destroy a person from the inside, ruin self-esteem and a feeling of self-worth, create the situation of absolute dependence such victim from an abuser, including financial dependence etc.. It often happens that psychological abuse takes place against the backdrop of demonstrations of care and love. So you've got this story about the wolf and the hare, that are right next to each other, and the shield between two of them is a repression - a psychological defense mechanism, when a person turns a blind eye to such offences, that take place in his or her own life and towards him or her. And this person considers this as normal, doesn't realize, doesn't have a resource to realize, that it is a crime. Most importantly - doesn’t feel anything, as a repression takes place. But a body responds in a right way - from a certain point of the existence of such a toxic situation the response “fight or flight” is launched in a body at full, in other words - the fear and anxiety with the associated symptoms. The third allegorical tale I called “Defective suit”, which I read in the book of Clarissa Pinkola Estés with the name “Running With the Wolves". “Once one man came to a tailor and started to try on a suit. When he was standing in front of a mirror, he saw that the costume had uneven edges. - Don’t worry, - said the tailor. - If you hold the short edge of the suit by your left hand - nobody notices it. But then the man saw that a lapel of a jacket folded up a little bit. - It's nothing. You only need to turn your head and to nail it by your chin. The customer obeyed, but when he put on trousers, he saw that they were pulling. - All right, so just hold your trousers like this by your right hand - and everything will be fine, - the tailor comforts him. The client agreed with him and took the suit. The next day he put on his new suit and went for a walk, while doing everything exactly in the way that the tailor told him to. He waddled in a park, while holding the lapel by his chin, and holding the short edge of the suit by his left hand, and holding his trousers by his right hand. Two old men, who were playing checkers, left the game and started to watch him. - Oh, God! - said one of them. - Look at that poor cripple. - Oh, yes - the limp - is a disaster. But I'm wondering, where did he get such a nice suit?” Clarissa wrote: “The commentary of the second old man reflects the common response of the society to a woman, who built a great reputation for herself, but turned into a cripple, while trying to save it. “Yes, she is a cripple, but look how great her life is and how lovely she looks.” When the “skin” that we put on ourselves towards society is small, we become cripples, but try to hide it. While fading away, we try to waddle perky, so everyone could see that we are doing really well, everything is great, everything is fine”. As for me, this tale is also about the process of forming a symptom in a situation when one person tries very hard to match to another one, whether it is a husband, a wife or parents. It’s about a situation when such a person always tries to support the other one, while giving up his or her own needs and causing oneself harm in such a way by feeling a tension every day, that becomes an inner normality. And so this person doesn’t give oneself a possibility to relax, to be herself (or himself), to be spontaneous, free. As a result, in this situation the person, who was supported, looks perfect from the outside, but those who tried to match, arises some visible defect, like a limp - a symptom. And so this person lives like a cripple, under everyday stress and tension, trying to handle it, while sacrificing herself (or himself) and trying to maintain this situation, so not to lose the general picture of a beautiful family and to avoid shame. The tailor, who made this defective suit and tells how to wear the suit properly, in order to keep things going as they are going, often is a mother who raised a problematic child and then tells another person how to deal with her child in the right way. It is the situation when a mother-in-law tells her daughter-in-law how to treat her son properly. In other words, how to support him, when to keep silent, to handle, how to fit in, so that her problematic son and this relationship in general looks perfect. Or vice versa, when a mother-in-law tells her son-in-law how to support her problematic daughter, how to fit in etc.. When, for example, a woman acts like this in her marriage and with her husband, with these excessive efforts to fit in - then after a while everybody will talk like: “Look at this lovely man: he lives with his sick wife, and their family seems perfect!”. But when such a woman becomes brave enough to relax and to just let the whole thing go, everybody will see that the relationship in her marriage isn’t perfect, and it is the other one who has problems. Each time when someone tries excessively to match up to another one, while turning oneself in some kind of a cripple, - he or she, on the one hand, supports the comfort of that person, to whom he or she tries to match up, and on the other hand - such a situation always arises in that person such conditions as a continuous tension, anxiety, fear to act spontaneously. A symptom - is like a visible defect, that shows itself through the body (and may look like some kind of injury). It is the result of a hidden inner prison. As a result of evolution, a pain tells us about a problem that is needed to be solved. When we repress our pain we can’t see our needs and our problems at full. And then a body starts to talk to us via a symptom. Psychotherapy aims for providing a movement from a symptom to a resumption of sensitivity to feelings, a resumption of the ability to feel your psychological pain, so you can realize your own toxic story. In this perspective another fairy-tale looks interesting to analyze - it is Andersen's fairytale “Princess and the Pea”. In the tale a prince wanted to find a princess to marry. There was one requirement for women candidates, so the prince could select her among commoner - high level of sensitivity, as the real princess would feel a pea through the mountain of mattresses, and so she could have the ability to feel discomfort, to be in a good contact with her body, to tell about her discomfort without such feeling as shame and guilt, and to refuse that discomfort, so to have the readiness to solve her problems and to demand from others the respect for her needs. It is common for our culture that the expression “a princess on a pea” very often uses for a negative meaning. So people who are in good contact with their body and who can demand comfort for themselves are often called capricious. At the same time the heroes who are ready to suffer and to tolerate their pain, who are able to repress (stop to feel) their pain represents a good example to be followed in our society. So, we may see the next algorithm in cases of various anxiety disorders: the existence of some toxic situation that brings some danger to a person. And we need not to be confused: a danger exists not for a body, but for a personality. A toxic live situation as well as having a panic attack is not a threat for the health of a body (that is what medical examinations show), and vice versa - it’s like every day intensive sport training, that could be good for your health only to some degree. A toxic situation destroys a person as a personality, who longs for one self’s expression; the existence of such a defense mechanism as repression - it’s a life with closed eyes, in pink glasses, when there is inability (or the absence of the desire) to see its own toxic story; 3.the presence of a symptom - a healthy response of a body “fight or flight” to some toxic situation; displacement - it’s replacement of the attention from the situation to a symptom, when a person starts to see and search for the problem in some other place, not where it really is. A symptom takes as some spare, pathological reaction that we need to get rid of. The readiness to fight the symptom arises, and that is the goal of such methods of therapy as pharmacological therapy, CBT and many others; the absence of adequate actions that are directed towards the change of a toxic situation itself. The absence of the readiness to show aggression when it comes to protect its space. All of it is a mechanism of formation of primary anxiety and preparation for launch of secondary anxiety. A complete anxiety disorder is the interaction between a primary and a secondary anxiety.

ABSTRACT The phytotelm-dwelling frogs from the genus Phyllodytes Wagler, 1830 have been characterized as specialist frogs regarding their diet strategy which is mainly composed by colonial insects. Herein, we used two species of ants (Camponotus sp. and Gnamptogenys sp.) with distinct defensive mechanisms to test the predatory behavior of Phyllodytes luteolus Wied, 1824. The experiment was conducted with frogs inhabiting a patch of 20 bromeliads (Aechmea cf. blanchetiana). Ants were offered randomly to the frogs until we obtained ten observations of predation of each ant species. We observed and recorded the time that P. luteolus needed to keep each ant species inside its mouth before it could ingest it. Predatory behavior was highly distinct. While Camponotus were caught and swallowed within six seconds and without apparent discomfort, individuals of P. luteolus had more difficulty in swallowing Gnamptogenys individuals, the time of manipulation ranging from 57 to 177 seconds. The mean values of time of predation observed in each treatment was highly significant (p<0.001). We conclude that differences found in the time of manipulation are highly correlated with defense mechanisms of each species of ants.

Defense against ultraviolet (UV) radiation exposure is essential for survival, especially in high-elevation species. Although some specific genes involved in UV response have been reported, the full view of UV defense mechanisms remains largely unexplored. Herein, we used integrated approaches to analyze UV responses in the highest-elevation frog, Nanorana parkeri . We show less damage and more efficient antioxidant activity in skin of this frog than those of its lower-elevation relatives after UV exposure. We also reveal genes related to UV defense and a corresponding temporal expression pattern in N. parkeri . Genomic and metabolomic analysis along with large-scale transcriptomic profiling revealed a time-dependent coordinated defense mechanism in N. parkeri . We also identified several microRNAs that play important regulatory roles, especially in decreasing the expression levels of cell cycle genes. Moreover, multiple defense genes (i.e., TYR for melanogenesis) exhibit positive selection with function-enhancing substitutions. Thus, both expression shifts and gene mutations contribute to UV adaptation in N. parkeri . Our work demonstrates a genetic framework for evolution of UV defense in a natural environment.

Hyla annectans is a tree frog living in the southwestern plateau area of China where there is strong ultraviolet radiation and long duration of sunshine. So their naked skin may possess chemical defense components that protect it from acute photo-damage. However, no such peptide or components has been identified till to date. In the current work, two novel peptides (FW-1, FWPLI-NH2 and FW-2, FWPMI-NH2) were identified from the skin of the tree frog. Five copies of FW-1 and four copies of FW-2 are encoded by an identical gene and released from the same protein precursor, which possess 167 amino acid residues. FW-1 and -2 can exert significant anti-inflammatory functions by directly inhibiting Ultraviolet B irradiation (UVB)-induced secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). They may achieve this function by modulating the UV-induced stress signaling pathways such as Mitogen-activated protein kinases (MAPK) and Nuclear Factor Kappa B (NF-κB). Besides, FW-1 and -2 showed potential antioxidant effects on epidermis by attenuating the UVB-induced reactive oxygen species (ROS) production through an unknown mechanism. Considering small peptides’ easy production, storage, and potential photo-protective activity, FW-1/2 might be exciting leading compounds or templates for the development of novel pharmacological agents for the suppression of UVB-induced skin inflammation. Moreover, this study might expand our knowledge on skin defensive mechanism of tree frog upon UVB irradiation.

The roles of bactericidal cathelicidins against bacterial infection have been extensively studied. However, the antibacterial property and mechanism of action of non-bactericidal cathelicidins are rarely known. Herein, a novel naturally occurring cathelicidin (PopuCATH) from tree frog (Polypedates puerensis) did not show any direct anti-bacterial activity in vitro. Intriguingly, intraperitoneal injection of PopuCATH before bacterial inoculation significantly reduced the bacterial load in tree frogs and mice, and reduced the inflammatory response induced by bacterial inoculation in mice. PopuCATH pretreatment also increased the survival rates of septic mice induced by a lethal dose of bacterial inoculation or cecal ligation and puncture (CLP). Intraperitoneal injection of PopuCATH significantly drove the leukocyte influx in both frogs and mice. In mice, PopuCATH rapidly drove neutrophil, monocyte/macrophage influx in mouse abdominal cavity and peripheral blood with a negligible impact on T and B lymphocytes, and neutrophils, monocytes/macrophages, but not T and B lymphocytes, were required for the preventive efficacy of PopuCATH. PopuCATH did not directly act as chemoattractant for phagocytes, but PopuCATH obviously drove phagocyte migration when it was cultured with macrophages. PopuCATH significantly elicited chemokine/cytokine production in macrophages through activating p38/ERK mitogen-activated protein kinases (MAPKs) and NF-κB p65. PopuCATH markedly enhanced neutrophil phagocytosis via promoting the release of neutrophil extracellular traps (NETs). Additionally, PopuCATH showed low side effects both in vitro and in vivo. Collectively, PopuCATH acts as a host-based immune defense regulator that provides prophylactic efficacy against bacterial infection without direct antimicrobial effects. Our findings reveal a non-bactericidal cathelicidin which possesses unique anti-bacterial action, and highlight the potential of PopuCATH to prevent bacterial infection.

Abstract Chytridiomycosis, a lethal fungal disease caused by Batrachochytrium dendrobatidis (Bd), is responsible for population declines and extinctions of amphibians worldwide. However, not all amphibian species are equally susceptible to the disease; some species persist in Bd enzootic regions with no population reductions. Recently, it has been shown that the amphibian skin microbiome plays a crucial role in the defense against Bd. Numerous bacterial isolates with the capacity to inhibit the growth of Batrachochytrium fungi have been isolated from the skin of amphibians. Here, we characterized eight Acinetobacter bacteria isolated from the frogs Agalychnis callidryas and Craugastor fitzingeri at the genomic level. Five isolates belonged to A. pittii, A. radioresistens, or A. modestus, and three were not identified as any of the known species, suggesting they are members of new species. We showed that seven isolates inhibited the growth of Bd and that all eight isolates inhibited the growth of the phytopathogen fungus Botrytis cinerea. Finally, we identified the biosynthetic gene clusters that could be involved in the antifungal activity of these isolates. Our results suggest that the frog skin microbiome includes Acinetobacter isolates that are new to science and have broad antifungal functions, perhaps driven by distinct genetic mechanisms.

ABSTRACT Global amphibian declines due to the fungal pathogen Batrachochytrium dendrobatidis (Bd) have led to questions about how amphibians defend themselves against skin diseases. A total of two amphibian defense mechanisms are antimicrobial peptides (AMPs), a component of amphibian innate immune defense and symbiotic skin bacteria, which can act in synergy. We characterized components of these factors in four populations of Columbia spotted frogs (Rana luteiventris) to investigate their role in disease defense. We surveyed the ability of their AMPs to inhibit Bd, skin bacterial community composition, skin metabolite profiles and presence and intensity of Bd infection. We found that AMPs from R. luteiventris inhibited Bd in bioassays, but inhibition did not correlate with Bd intensity on frogs. R. luteiventris had two prevalent and abundant core bacteria: Rhizobacter and Chryseobacterium. Rhizobacter relative abundance was negatively correlated with AMP's ability to inhibit Bd, but was not associated with Bd status itself. There was no relationship between metabolites and Bd. Bacterial communities and Bd differ by location, which suggests a strong environmental influence. R. luteiventris are dominated by consistent core bacteria, but also house transient bacteria that are site specific. Our emergent hypothesis is that host control and environmental factors shape the microbiota on R. luteiventris.

A grafting techniques or using various synthetic and biological dressing also widely used to protect the wound area. There are 8 peptides with differential antimicrobial activities contained in Odorrana hosii’s skin secretion. However, to our best knowledge no study has been scientifically conducted to reveal the value off this species on wound healing. Primarily, the aim of this study was to look at the potential use of O. hosii’s skin as a biological dressing in wound healing management. This study assessed the wound healing in rat compared between wound grafted with O. hosii’s skin and wound treated with normal saline dressing. Histological examination was done to assess the wound healing activities after 14 days. The result shown, both wounds which were treated with O. hosii’s skin and untreated wound heal completely on day 14 as the epidermis and dermis completely close. Histologically, the percentage of neutrophils, macrophages and fibroblasts, were reduced on day 14. However, wounded skin, which was treated with O. hosii’s skin, had better healing quality as more new tissues and hair follicle regrowth compared with the untreated wound. It is suggested that poison gland in the O. hosii’s skin did not harm the wounded rat skin, instead, poison that act as defensive mechanism can help the species to fight the pathogen on the wound.

Abstract Background The skin microbiome serves as a first line defense against pathogens in vertebrates. In amphibians, it has the potential to protect against the chytrid fungus Batrachochytrium dendrobatis (Bd), a likely agent of amphibian declines. Alteration of the microbiome associated with unfavorable environmental changes produced by anthropogenic activities may make the host more susceptible to pathogens. Some amphibian species that were thought to be “extinct” have been rediscovered years after population declines in the late 1980s probably due to evolved Bd-resistance and are now threatened by anthropogenic land-use changes. Understanding the effects of habitat disturbance on the host skin microbiome is relevant for understanding the health of these species, along with its susceptibility to pathogens such as Bd. Here, we investigate the influence of habitat alteration on the skin bacterial communities as well as specifically the putative Bd-inhibitory bacterial communities of the montane frog Lithobates vibicarius. This species, after years of not being observed, was rediscovered in small populations inhabiting undisturbed and disturbed landscapes, and with continuous presence of Bd. Results We found that cutaneous bacterial communities of tadpoles and adults differed between undisturbed and disturbed habitats. The adults from disturbed habitats exhibited greater community dispersion than those from undisturbed habitats. We observed a higher richness of putative Bd-inhibitory bacterial strains in adults from disturbed habitats than in those from undisturbed habitats, as well as a greater number of these potential protective bacteria with a high relative abundance. Conclusions Our findings support the microbial “Anna Karenina principle”, in which disturbance is hypothesized to cause greater microbial dispersion in communities, a so-called dysbiosis, which is a response of animal microbiomes to stress factors that decrease the ability of the host or its microbiome to regulate community composition. On the positive side, the high richness and relative abundance of putative Bd-inhibitory bacteria may indicate the development of a defense mechanism that enhances Bd-protection, attributed to a co-occurrence of more than 30-years of host and pathogen in these disturbed habitats. Our results provide important insight into the influence of human-modified landscapes on the skin microbiome and health implications of Bd-survivor species.

Abstract Background Physiological processes, as immediate responses to the environment, are important mechanisms of phenotypic plasticity and can influence evolution at ecological time scales. In stressful environments, physiological stress responses of individuals are initiated and integrated via the release of hormones, such as corticosterone (CORT). In vertebrates, CORT influences energy metabolism and resource allocation to multiple fitness traits (e.g. growth and morphology) and can be an important mediator of rapid adaptation to environmental stress, such as acidification. The moor frog, Rana arvalis, shows adaptive divergence in larval life-histories and predator defense traits along an acidification gradient in Sweden. Here we take a first step to understanding the role of CORT in this adaptive divergence. We conducted a fully factorial laboratory experiment and reared tadpoles from three populations (one acidic, one neutral and one intermediate pH origin) in two pH treatments (Acid versus Neutral pH) from hatching to metamorphosis. We tested how the populations differ in tadpole CORT profiles and how CORT is associated with tadpole life-history and morphological traits. Results We found clear differences among the populations in CORT profiles across different developmental stages, but only weak effects of pH treatment on CORT. Tadpoles from the acid origin population had, on average, lower CORT levels than tadpoles from the neutral origin population, and the intermediate pH origin population had intermediate CORT levels. Overall, tadpoles with higher CORT levels developed faster and had shorter and shallower tails, as well as shallower tail muscles. Conclusions Our common garden results indicate among population divergence in CORT levels, likely reflecting acidification mediated divergent selection on tadpole physiology, concomitant to selection on larval life-histories and morphology. However, CORT levels were highly environmental context dependent. Jointly these results indicate a potential role for CORT as a mediator of multi-trait divergence along environmental stress gradients in natural populations. At the same time, the population level differences and high context dependency in CORT levels suggest that snapshot assessment of CORT in nature may not be reliable bioindicators of stress.

Background: Bacterial resistance to all currently available conventional antibiotics has caused a global public health crisis and an imperative search for new agents. Antimicrobial peptides (AMPs) are essential components of host innate immune defense against microbial invasions. Aims: We reported a novel AMP, brevinin-2KP and described structural and biological characterization. Objective: The objective of this study was to report a novel AMP, brevinin-2KP, from the skin of the black Kaloula pulchra frog and describe its structural and biological characterization. Materials and Methods: : The physical and chemical parameters of brevinin-2KP were predicted with the ExPASy Bioinformatics Resource Portal. The assembled sequences were aligned with ClustalW and the phylogenetic tree was constructed using Mega. Circular dichroism (CD) experiments were carried out to identify the secondary structure and the stability of peptide in different solvent environments. Cytotoxicity of brevinin-2KP was undertook by MTT test. To determine antibacterial activity of brevinin-2KP, a standard two-fold broth dilution method was used. SEM was carried out to observe the morphology change of bacterial treated by brevinin-2KP. The live/dead bacterial viability was measured with a LIVE/DEAD® BacLight kit. Histamine release and mast cell degranulation assays were performed. Results: The precursor of brevinin-2KP contains 72 amino acid residues, which includes a conserved signal peptide, acidic propeptide with KR residues and mature peptide with sequence of GVITDALKGAAKTVAAELLKKAHCKLTNSC. Phylogenetic analysis based on the amino acid sequences of 34 brevinin-2 peptides from 30 anuran species demonstrates that K. pulchra is genetically closely related to the genus Hylarana. The CD spectra analysis indicates that brevinin- 2KP adopts random coil in water and an organized α-helical conformation in SDS solution. Furtherly, this secondary structure is stable under high salt and high temperature conditions. Brevinin-2KP is weakly active towards the tested Gram-positive and -negative bacteria as well as fungi by its membranolytic action. Moreover, brevinin-2KP inhibits the proliferation of several mammal cells with IC50 values from 3.27 to 59.75 μM. In addition, brevinin-2KP promotes degranulation and histamine release of mast cells, indicating that it is involved in inflammatory response. Conclusion: This is the first report about AMP identified from the skin of K. pulchra. Brevinin-2KP adopts a typical amphipathic α-helix conformation in membrane mimic environment and shows antimicrobial and antitumor activities by potential membranolytic mechanism. In addition, brevinin- 2KP can promote degranulation and histamine release of mast cells. Brevinin-2KP is expected to become a good drug temple molecule.