Strategy for the Identification of Host-Defense Peptides in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents.

Several amphibian peptides that were first identified on the basis of their antimicrobial or cytotoxic properties have subsequently shown potential for development into agents for the treatment of patients with Type 2 diabetes. A strategy is presented for the isolation and characterization of such peptides that are present in norepinephrine-stimulated skin secretions from a range of frog species. The methodology involves (1) fractionation of the secretions by reversed-phase HPLC, (2) identification of fractions containing components that stimulate the rate of release of insulin from BRIN-BD11 clonal ß-cells without simultaneously stimulating the release of lactate dehydrogenase, (3) identification of active peptides in the fractions in the mass range 1-6 kDa by MALDI-ToF mass spectrometry, (4) purification of the peptides to near homogeneity by further reversed-phase HPLC on various column matrices, and (5) structural characterization by automated Edman degradation. The effect of synthetic replicates of the active peptides on glucose homeostasis in vivo may be evaluated in appropriate animal models of Type 2 diabetes such as db/db mice and mice fed a high fat diet to produce obesity, glucose intolerance, and insulin resistance.

Early life nutrient restriction affects hypothalamic-pituitary-interrenal axis gene expression in a diet type-specific manner.

Stressful experiences in early life can alter phenotypic expression later in life. For instance, in vertebrates, early life nutrient restriction can modify later life activity of the hypothalamic-pituitary-adrenal/interrenal axis (the HPI in amphibians), including the up- and downstream regulatory components of glucocorticoid signaling. Early life nutrient restriction can also influence later life behavior and metabolism (e.g., fat accumulation). Yet, less is known about whether nutrient stress-induced carryover effects on HPA/HPI axis regulation can vary across environmental contexts, such as the type of diet on which nutrient restriction occurs. Here, we experimentally address this question using the plains spadefoot toad (Spea bombifrons), whose larvae develop in ephemeral habitats that impose intense competition over access to two qualitatively distinct diet types: detritus and live shrimp prey. Consistent with diet type-specific carryover effects of early life nutrient restriction on later life HPI axis regulation, we found that temporary nutrient restriction at the larval stage reduced juvenile (i.e., post-metamorphic) brain gene expression of an upstream glucocorticoid regulator (corticotropin-releasing hormone) and two downstream regulators (glucocorticoid and mineralocorticoid receptors) only on the shrimp diet. These patterns are consistent with known diet type-specific effects of larval nutrient restriction on juvenile corticosterone and behavior. Additionally, larval nutrient restriction increased juvenile body fat levels. Our study indicates that HPA/HPI axis regulatory responses to nutrient restriction can vary remarkably across diet types. Such diet type-specific regulation of the HPA/HPI axis might provide a basis for developmental or evolutionary decoupling of stress-induced carryover effects.

Dramatic genome-wide reprogramming of mRNA in hypometabolic muscle.

In response to seasonal droughts, the green striped burrowing frog Cyclorana alboguttata enters a reversible hypometabolic state called aestivation where heart rate and oxygen consumption can be reduced despite warm (>25C°) ambient temperatures. With a view to understanding molecular mechanisms we profiled aestivating versus control gastrocnemius muscle using mRNA sequencing. This indicated an extensive metabolic reprogramming, with nearly a quarter of the entire transcriptome (3996 of 16,960 mRNA) exhibiting a nominal >2-fold change. Consistent with a physiological adaptation to spare carbohydrate reserves, carbohydrate catabolism was systemically downregulated. A 630-fold downregulation of ENO3 encoding the enolase enzyme was most striking. The 590 frog orthologs of mRNA encoding the mitoproteome were, viewed as a population, significantly downregulated during aestivation, although not to the same extent as mRNA encoding carbohydrate catabolism. Prominent examples include members of the TCA cycle (IDH2), electron transport chain (NDUFA6), the ATP synthase complex (ATP5F1B) and ADP/ATP intracellular transport (SLC25A4). Moreover, mRNA derived from the mt genome itself (e.g. mt-ND1) were also downregulated. Most prominent among the upregulated mRNA are those encoding aspects of regulated proteolysis including the proteosome (e.g. PSME4L), peptidases (USP25), atrogins (FBXO32) and ubiquitination (VCP). Finally, we note the ∼5-fold upregulation of the mRNA EIFG3 that encodes part of the EIF4F complex. This possesses global control of protein synthesis. Given protein synthesis is repressed in aestivating frogs this indicates the skeletal musculature is poised for accelerated translation of mRNA upon emergence, supporting a strategy to rapidly restore function when the summer rains come.

Amphibian host-defense peptides with potential for Type 2 diabetes therapy - an updated review.

Investigations conducted since 2018 have identified several host-defense peptides present in frog skin secretions whose properties suggest the possibility of their development into a new class of agent for Type 2 diabetes (T2D) therapy. Studies in vitro have described peptides that (a) stimulate insulin release from BRIN-BD11 clonal ß-cells and isolated mouse islets, (b) display ß-cell proliferative activity and protect against cytokine-mediated apoptosis and (c) stimulate production of the anti-inflammatory cytokine IL-10 and inhibit production of the pro-inflammatory cytokines TNF-α and IL-1ß. Rhinophrynin-27, phylloseptin-3.2TR and temporin F are peptides with therapeutic potential. Studies in vivo carried out in db/db and high fat-fed mice have shown that twice-daily administration of [S4K]CPF-AM1 and [A14K]PGLa-AM1, analogs of peptides first isolated from the octoploid frog Xenopus amieti, over 28 days lowers circulating glucose and HbA1c concentrations, increases insulin sensitivity and improves glucose tolerance and lipid profile. Peptide treatment produced potentially beneficial changes in the expression of skeletal muscle genes involved in insulin signaling and islet genes involved in insulin secretion in these murine models of T2D. Lead compounds uncovered by the study of frog HDPs may provide a basis for the design of new types of agents that can be used, alone or in combination with existing therapies, for the treatment of T2D.

A Comprehensive Review of Patented Antimicrobial Peptides from Amphibian Anurans.

Since the 1980s, studies of antimicrobial peptides (AMPs) derived from anuran skin secretions have unveiled remarkable structural diversity and a wide range of activities. This study explores the potential of these peptides for drug development by examining granted patents, amino acid modifications related to patented peptides, and recent amphibians' taxonomic updates influencing AMP names. A total of 188 granted patents related to different anuran peptides were found, with Asia and North America being the predominant regions, contributing 65.4% and 15.4%, respectively. Conversely, although the Neotropical region is the world's most diversified region for amphibians, it holds only 3.7% of the identified patents. The antimicrobial activities of the peptides are claimed in 118 of these 188 patents. Additionally, for 160 of these peptides, 66 patents were registered for the natural sequence, 69 for both natural and derivative sequences, and 20 exclusively for sequence derivatives. Notably, common modifications include alterations in the side chains of amino acids and modifications to the peptides' N- and C-termini. This review underscores the biomedical potential of anuran-derived AMPs, emphasizing the need to bridge the gap between AMP description and practical drug development while highlighting the urgency of biodiversity conservation to facilitate biomedical discoveries.

Computational modelling of the antimicrobial peptides Cruzioseptin-4 extracted from the frog Cruziohyla calcarifer and Pictuseptin-1 extracted from the frog Boana picturata.

A computational study of the peptides Cruzioseptin-4 and Pictuseptin-1, identified in Cruziohyla calcarifer and Boana picturata respectively, has been carried out. The studies on Cruzioseptin-4 show that it is a cationic peptide with a chain of 23 amino acids that possess 52.17% of hydrophobic amino acids and a charge of + 1.2 at pH 7. Similarly, Pictuseptin-1 is a 22 amino acids peptide with a charge of + 3 at pH 7 and 45.45% of hydrophobic amino acids. Furthermore, the predominant secondary structure for both peptides is alpha-helical. The physicochemical properties were predicted using PepCalc and Bio-Synthesis; secondary structures using Jpred4 and PredictProtein; while molecular docking was performed using Autodock Vina. Geometry optimization of the peptides was done using the ONIOM hybrid method with the HF/6-31G basis set implemented in the Gaussian 09 program. Finally, the molecular docking study indicates that the viable mechanism of action for both peptides is through a targeted attack on the cell membrane of pathogens via electrostatic interactions with different membrane components, leading to cell lysis.

Metabolomic response of striped marsh frog (Limnodynastes peronii) tadpoles exposed to the fire retardant Phos-Chek LC95W.

Climate change and other factors have contributed to an increased frequency and intensity of global wildfires in recent years. Ammonium-based fire retardants are widely used to suppress or delay the spread of fire and have generally been regarded as presenting a low risk of acute toxicity to fauna. However, studies have raised concerns about their potential to cause indirect or sub-lethal effects, and toxicity information regarding the potential for such impacts in aquatic species is limited. To address these knowledge gaps, we used an untargeted metabolomics approach to evaluate the sub-lethal physiological and metabolic responses of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a concentration gradient of the ammonium polyphosphate (APP)-based fire retardant Phos-Chek LC95W (PC). Acute exposure (96 h) to PC significantly altered the relative abundance of 14 metabolites in whole tadpoles. The overall metabolic response pattern was consistent with effects reported for ammonia toxicity and also suggestive of energy dysregulation and osmotic stress associated with alterations to physicochemical water quality parameters in the PC treatments. Results suggest that run-off or accidental application of this formulation into waterways can have significant sub-lethal consequences on the biochemical profiles (i.e., the metabolome) of aquatic organisms and may be a concern for frog species that breed and develop in small, often ephemeral, waterbodies. Our study highlights the benefits of integrating untargeted metabolomics with other ecological and toxicological endpoints to provide a more holistic characterisation of the sub-lethal impacts associated with bushfire-fighting chemicals and with environmental contaminants more broadly.

Bioactive alkaloids from the venom of Dendrobatoidea Cope, 1865: a scoping review.

Bioactive compounds derived from secondary metabolism in animals have refined selectivity and potency for certain biological targets. The superfamily Dendrobatoidea is adapted to the dietary sequestration and secretion of toxic alkaloids, which play a role in several biological activities, and thus serve as a potential source for pharmacological and biotechnological applications. This article constitutes a scoping review to understand the trends in experimental research involving bioactive alkaloids derived from Dendrobatoidea based upon scientometric approaches. Forty-eight (48) publications were found in 30 journals in the period of 60 years, between 1962 and 2022. More than 23 structural classes of alkaloids were cited, with 27.63% for batrachotoxins, 13.64% for pyridinics, with an emphasis on epibatidine, 16.36% for pumiliotoxins, and 11.82% for histrionicotoxins. These tests included in vivo (54.9%), in vitro (39.4%), and in silico simulations (5.6%). Most compounds (54.8%) were isolated from skin extracts, whereas the remainder were obtained through molecular synthesis. Thirteen main biological activities were identified, including acetylcholinesterase inhibitors (27.59%), sodium channel inhibitors (12.07%), cardiac (12.07%), analgesic (8.62%), and neuromuscular effects (8.62%). The substances were cited as being of natural origin in the "Dendrobatidae" family, genus "Phyllobates," "Dendrobates," and seven species: Epipedobates tricolor, Phyllobates aurotaenia, Oophaga histrionica, Oophaga pumilio, Phyllobates terribilis, Epipedobates anthonyi, and Ameerega flavopicta. To date, only a few biological activities have been experimentally tested; hence, further studies on the bioprospecting of animal compounds and ecological approaches are needed.

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Mate calling alters expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain of male frog Microhyla nilphamariensis.

Croaking is a unique component of reproductive behaviour in amphibians which plays a key role in intraspecies communication and mate evaluation. While gonadal hormones are known to induce croaking, central regulation of sound production is less studied. Croaking is a dramatic, transient activity that sets apart an animal from non-croaking individuals. Herein, we aim at examining the profile of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in actively croaking and non-croaking frog Microhyla nilphamariensis. In anurans, this peptide is widely expressed in the areas inclusive of acoustical nuclei as well as areas relevant to reproduction. CART immunoreactivity was far more in the preoptic area (POA), anteroventral tegmentum (AV), ventral hypothalamus (vHy), pineal (P) and pituitary gland of croaking frog compared to non-croaking animals. On similar lines, tissue fragments collected from the mid region of the brain inclusive of POA, vHy, AV, pineal and pituitary gland of croaking frog showed upregulation of CART mRNA. However, CART immunoreactivity in the neuronal perikarya of raphe (Ra) was completely abolished during croaking activity. The data suggest that CART signaling in the brain may be an important player in mediating croaking behaviour in the frog.

Early onset of urea synthesis and ammonia detoxification pathways in three terrestrially developing frogs.

Frogs evolved terrestrial development multiple times, necessitating mechanisms to avoid ammonia toxicity at early stages. Urea synthesis from ammonia is a key adaptation that reduces water dependence after metamorphosis. We tested for early expression and plasticity of enzymatic mechanisms of ammonia detoxification in three terrestrial-breeding frogs: foam-nest-dwelling larvae of Leptodactylus fragilis (Lf) and arboreal embryos of Hyalinobatrachium fleischmanni (Hf) and Agalychnis callidryas (Ac). Activity of two ornithine-urea cycle (OUC) enzymes, arginase and CPSase, and levels of their products urea and CP in tissues were high in Lf regardless of nest hydration, but reduced in experimental low- vs. high-ammonia environments. High OUC activity in wet and dry nests, comparable to that under experimental high ammonia, suggests terrestrial Lf larvae maintain high capacity for urea excretion regardless of their immediate risk of ammonia toxicity. This may aid survival through unpredictably long waiting periods before rain enables their transition to water. Moderate levels of urea and CP were present in Hf and Ac tissues and enzymatic activities were lower than in Lf. In both species, embryos in drying clutches can hatch and enter the water early, behaviorally avoiding ammonia toxicity. Moreover, glutamine synthetase was active in early stages of all three species, condensing ammonia and glutamate to glutamine as another mechanism of detoxification. Enzyme activity appeared highest in Lf, although substrate and product levels were higher in Ac and Lf. Our results reveal that multiple biochemical mechanisms of ammonia detoxification occur in early life stages of anuran lineages that evolved terrestrial development.

Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

The Significance of Hypervariability and Conserved Motifs in Antimicrobial Peptides from Tree Frogs.

Amphibians' skin is a rich source of natural antimicrobial peptides (AMPs). These AMPs exhibit marked inter- and intraspecific sequence divergence linked to the arms race between host and pathogens. Here, we combine peptidomics, molecular modeling, and phylogenetic analyses to understand the evolution of AMPs in Cophomantini, a diverse clade of neotropical tree frogs, and to investigate their interaction with bacterial membranes. Consistent with results in other amphibians, all species of Cophomantini secrete a mixture of peptides. We selected the hylin peptide family to survey sequence variability and the presence of common amino acid motifs. We found that most species secrete a unique set of hylins that, though variable, share the conserved motif Gly-X-X-X-Pro-Ala-X-X-Gly, with Gly and Pro colocalizing with charged or polar residues. Our modeling revealed that Pro curves the peptide through a hinge, facilitating its insertion into the bacterial membrane and, once inserted, contributes to stabilizing the pore structure. The phylogenetic inference using hylid prepro-peptides showed the need to classify new AMPs using the full-length sequence of the prepro-peptide region and highlighted the complex relationships between peptide families. Our findings revealed that conserved motifs occurred independently in distinct AMP families, suggesting a convergent evolution and a significant role in peptide-membrane interactions.

IgG Fc-binding protein positively regulates the assembly of pore-forming protein complex ßγ-CAT evolved to drive cell vesicular delivery and transport.

Cell membranes form barriers for molecule exchange between the cytosol and the extracellular environments. ßγ-CAT, a complex of pore-forming protein BmALP1 (two ßγ-crystallin domains with an aerolysin pore-forming domain) and the trefoil factor BmTFF3, has been identified in toad Bombina maxima. It plays pivotal roles, via inducing channel formation in various intracellular or extracellular vesicles, as well as in nutrient acquisition, maintaining water balance, and antigen presentation. Thus, such a protein machine should be tightly regulated. Indeed, BmALP3 (a paralog of BmALP1) oxidizes BmALP1 to form a water-soluble polymer, leading to dissociation of the ßγ-CAT complex and loss of biological activity. Here, we found that the B. maxima IgG Fc-binding protein (FCGBP), a well-conserved vertebrate mucin-like protein with unknown functions, acted as a positive regulator for ßγ-CAT complex assembly. The interactions among FCGBP, BmALP1, and BmTFF3 were revealed by co-immunoprecipitation assays. Interestingly, FCGBP reversed the inhibitory effect of BmALP3 on the ßγ-CAT complex. Furthermore, FCGBP reduced BmALP1 polymers and facilitated the assembly of ßγ-CAT with the biological pore-forming activity in the presence of BmTFF3. Our findings define the role of FCGBP in mediating the assembly of a pore-forming protein machine evolved to drive cell vesicular delivery and transport.

A Pore Forming Toxin-like Protein Derived from Chinese Red Belly Toad Bombina maxima Triggers the Pyroptosis of Hippomal Neural Cells and Impairs the Cognitive Ability of Mice.

Toxin-like proteins and peptides of skin secretions from amphibians play important physiological and pathological roles in amphibians. ßγ-CAT is a Chinese red-belly toad-derived pore-forming toxin-like protein complex that consists of aerolysin domain, crystalline domain, and trefoil factor domain and induces various toxic effects via its membrane perforation process, including membrane binding, oligomerization, and endocytosis. Here, we observed the death of mouse hippocampal neuronal cells induced by ßγ-CAT at a concentration of 5 nM. Subsequent studies showed that the death of hippocampal neuronal cells was accompanied by the activation of Gasdermin E and caspase-1, suggesting that ßγ-CAT induces the pyroptosis of hippocampal neuronal cells. Further molecular mechanism studies revealed that the pyroptosis induced by ßγ-CAT is dependent on the oligomerization and endocytosis of ßγ-CAT. It is well known that the damage of hippocampal neuronal cells leads to the cognitive attenuation of animals. The impaired cognitive ability of mice was observed after intraperitoneal injection with 10 µg/kg ßγ-CAT in a water maze assay. Taken together, these findings reveal a previously unknown toxicological function of a vertebrate-derived pore-forming toxin-like protein in the nerve system, which triggers the pyroptosis of hippocampal neuronal cells, ultimately leading to hippocampal cognitive attenuation.

Identification of thyroid hormone response genes in the remodeling of dorsal muscle during Microhyla fissipes metamorphosis.

Introduction: Extensive morphological, biochemical, and cellular changes occur during anuran metamorphosis, which is triggered by a single hormone, thyroid hormone (TH). The function of TH is mainly mediated through thyroid receptor (TR) by binding to the specific thyroid response elements (TREs) of direct response genes, in turn regulating the downstream genes in the cascade. The remodeling of dorsal skeletal muscle during anuran metamorphosis provides the perfect model to identify the immediate early and direct response genes that are important during apoptosis, proliferation, and differentiation of the muscle. Methods: In our current study, we performed Illumina sequencing combined with single-molecule real-time (SMRT) sequencing in the dorsal muscle of Microhyla fissipes after TH, cycloheximide (CHX), and TH_CHX treatment. Results and Discussion: We first identified 1,245 differentially expressed transcripts (DETs) after TH exposure, many of which were involved in DNA replication, protein processing in the endoplasmic reticulum, cell cycle, apoptosis, p53 signaling pathway, and protein digestion and absorption. In the comparison of the TH group vs. control group and TH_CHX group vs. CHX group overlapping gene, 39 upregulated and 6 downregulated genes were identified as the TH directly induced genes. Further analysis indicated that AGGTCAnnTnAGGTCA is the optimal target sequence of target genes for TR/RXR heterodimers in M. fissipes. Future investigations on the function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian dorsal muscle remodeling. These full-length and high-quality transcriptomes in this study also provide an important foundation for future studies in M. fissipes metamorphosis.

Evolution of bitter receptor genes and ontogenetic dietary shift in a frog.

Vertebrate Tas2r taste receptors detect bitter compounds that are potentially poisonous. Previous studies found substantial variation in the number of Tas2r genes across vertebrates, with some frog species carrying the largest number. Peculiar among vertebrates, frogs undergo metamorphosis, often associated with a dietary shift between tadpoles and adults. A possible explanation for the large size of frog Tas2r families could be that distinct sets of Tas2r genes are required for tadpoles and adults, suggesting differential expression of Tas2r genes between tadpoles and adults. To test this hypothesis, we first examined 20 amphibian genomes and found that amphibians generally possess more Tas2r genes than do other vertebrate clades. We next focused on the American bullfrog (Lithobates catesbeianus) to examine the expression of its Tas2r genes in herbivorous tadpoles and insectivorous adult frogs. We report that close to one fifth of its 180 Tas2r genes are differentially expressed (22 genes enriched in adults and 11 in tadpoles). Tuning properties were determined for a subset of differentially expressed genes by a cell-based functional assay, with the adult-enriched Tas2r gene set covering a larger range of ligands compared to the tadpole-enriched subset. These results suggest a role of Tas2r genes in the ontogenetic dietary shift of frogs and potentially initiate a new avenue of ontogenetic analysis of diet-related genes in the animal kingdom.

Artifical light at night triggers slight transcriptomic effects on melatonin signaling but not synthesis in tadpoles of two anuran species.

The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N-acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

MicroRNA transcriptomics in liver of the freeze-tolerant gray tree frog (Dryophytes versicolor) indicates suppression of energy-expensive pathways.

The rapid and reversible nature of microRNA (miRNA) transcriptional regulation is ideal for implementing global changes to cellular processes and metabolism, a necessary asset for the freeze-tolerant gray tree frog (Dryophytes versicolor). D. versicolor can freeze up to 42% of its total body water during the winter and then thaw completely upon more favorable conditions of spring. Herein, we examined the freeze-specific miRNA responses in the gray tree frog using RBiomirGS, a bioinformatic tool designed for the analysis of miRNA-seq transcriptomics in non-genome sequenced organisms. We identified 11 miRNAs differentially regulated during freezing (miR-140-3p, miR-181a-5p, miR-206-3p, miR-451a, miR-19a-3p, miR-101-3p, miR-30e-5p, miR-142-3p and -5p, miR-21-5p, and miR-34a-5p). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis suggests these miRNAs play roles in downregulating signaling pathways, apoptosis, and nuclear processes while enhancing ribosomal biogenesis. Overall, these findings point towards miRNA inducing a state of energy conservation by downregulating energy-expensive pathways, while ribosomal biogenesis may lead to prioritization of critical processes for freeze-tolerance survival.

Antimicrobial, cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs.

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 →Lys and the double substitution Asp4 →Lys and Ala18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.