Frog Skin Research Articles

Modulation of platelet functions by European toad (Bufo Bufo) skin secretions components

Background: A growing number of reports indicate that amphibian skin secretions may have a remarkable medical importance; however, the effects of the components of some dermal secretions on blood platelets and hemostasis are inadequately recognized. Since our previous studies demonstrated that the general Bufo bufo skin secretions induced platelet aggregation in platelet-rich plasma, this work was designed to study the effects of the components of some fractions on platelet functions to comprehend its possible mechanism of action as platelet modulators. Methods: Chromatographic separation of B. bufo general skin secretions was carried out using size exclusion chromatography. Rabbit platelets were purified by column chromatography on Sepharose 4B. Various aspects of platelet function such as activation, aggregation, and adhesion were evaluated. Results: One fraction, out of 7, dose-dependently induced aggregation of isolated platelets and was used in further experiments. The studied fraction was shown to induce platelet adhesion onto fibrinogen-coated surface. Furthermore, the results demonstrated the effects of the fraction on some processes that involved in platelets activation: The fraction components facilitated (Ca2+) i mobilization and attenuated platelets Akt phosphorylation, but had no effect on platelet serotonin secretion. Membrane integrity was determined using lactate dehydrogenase (LDH) assay. No increased LDH release was recorded that means no platelet damage, which could lead to misinterpretation of the data, occurred. Conclusion: The results suggest that components of the B. bufo skin secretions may be a promising source of natural compounds which can modulate platelet functions.

Re-Examining Interaction between Antimicrobial Peptide Aurein 1.2 and Model Cell Membranes via SFG.

Aurein 1.2 (Aur), a highly efficient 13-residue antimicrobial peptide (AMP) with a broad-spectrum antibiotic activity originally derived from the Australian frog skin secretions, can nonspecifically disrupt bacterial membranes. To deeply understand the molecular-level detail of the antimicrobial mechanism, here, we artificially established comparative experimental models to investigate the interfacial interaction process between Aur and negatively charged model cell membranes via sum frequency generation vibrational spectroscopy. Sequencing the vibrational signals of phenyl, C-H, and amide groups from Aur has characteristically helped us differentiate between the initial adsorption and subsequent insertion steps upon mutual interaction between Aur and the charged lipids. The phenyl group at the terminal phenylalanine residue can act as an anchor in the adsorption process. The time-dependent signal intensity of α-helices showed a sharp rise once the Aur molecules came into contact with the negatively charged lipids, indicating that the adsorption process was ongoing. Insertion of Aur into the charged lipids then offered the detectable interfacial C-H signals from Aur. The achiral and chiral amide I signals suggest that Aur had formed β-folding-like aggregates after interacting with the charged lipids, along with the subsequent descending α-helical amide I signals. The above-mentioned experimental results provide the molecular-level detail on how the Aur molecules interact with the cell membranes, and such a mechanism study can offer the necessary support for the AMP design and later application.

Interspecific and intraspecific Taylor's laws for frog skin microbes

Amphibians are known to have an abundance of microorganisms colonizing their skin, and these symbionts often protect the host from disease. There are now many comprehensive studies on amphibian skin microbes, but the interspecific and intraspecific abundance distributions (or abundance heterogeneity) of amphibian skin microbes remain unclear. Furthermore, we have a very limited understanding of how the abundance and heterogeneity of microbial communities relate to the body size (or more specifically, skin surface area) of amphibian hosts. In this study, we evaluated the interspecific and intraspecific abundance distribution patterns of amphibian skin microbes and evaluated whether the symbiotic skin microbes of different anuran species share a fundamental heterogeneity scaling parameter. If scaling invariance exists, we hypothesize that a fundamental heterogeneity scaling value also exists. A total of 358 specimens of 10 amphibian host species were collected, and we used Type-I and III Taylor’s power law expansions (TPLE) to assess amphibian skin microbial heterogeneity at the community and mixed-species population levels, respectively. The obtained results showed that, at the community scale, a high aggregation of the microbial abundance distribution on the skin barely changed with host size. In a mixed-species population (i.e., a community context), the abundance distribution pattern of mixed microbial species populations also does not change with host size and always remains highly aggregated. These findings suggest that while amphibian skin microbiomes located in different hosts may have different environmental conditions, they share a fundamental heterogeneity scaling parameter, and thus, scale invariance exists. Finally, we found that microhabitat area provided by the host skin is vital to the stability of the symbiotic microbial community.

Molecular Cloning and Characterization of a Novel Antimicrobial Peptide from the Skin of Kaloula pulchra.

Bacterial resistance to all currently available conventional antibiotics has caused a global public health crisis and led to an imperative search for new agents. Antimicrobial peptides (AMPs) are essential components of host innate immune defense against microbial invasions. 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. 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. The cytotoxicity of brevinin-2KP was evaluated by the 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 morphological change in the 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. The precursor of brevinin-2KP contains 72 amino acid residues, including a conserved signal peptide, acidic propeptide with KR residues, and mature peptide with a 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 the water and an organized α-helical conformation in SDS solution. Further, this secondary structure is stable under high salt and high-temperature conditions. Brevinin-2KP is weakly active towards the tested Gram-positive and Gram-negative bacteria as well as fungi due to its membranolytic action. Moreover, brevinin-2KP inhibits the proliferation of several mammal cells with IC50 values ranging 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 the inflammatory response. This is the first report on 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.

Cinobufacini retards progression of pancreatic ductal adenocarcinoma through targeting YEATS2/TAK1/NF-κB axis

BackgroundCinobufacini, a sterilized hot water extract of dried toad skin, had significant effect against several human cancers. However, there are few studies reporting the effect of cinobufacini on pancreatic cancer. PurposeTo investigate the effects of cinobufacini on the progress of pancreatic ductal adenocarcinoma and the underlying mechanisms. MethodsCell counting, EdU incorporation and flow Cytometry were performed to evaluate the effect of cinobufacini on cell cycle and growth. MIA-PaCa2 cells were implanted into the nude mice to determine whether cinobufacini represses PDAC progression in vivo. Luciferase reporter assay, western blotting and qPCR were carried out to measure the activity of NF-κB pathway and the alteration of YEATS2 and TAK1. Ectopic gene expression introduced by plasmids was used to verify the molecular mechanism. ResultsOur results showed that cinobufacini induced cell cycle arrest and inhibited the growth of PDAC cell in vitro, and repressed MIA-derived PDAC in vivo. Cinobufacini inhibited the phosphorylation of IKK, IκB and NF-κB p65 in PDAC cells. Furthermore, cinobufacini decreased the abundance of intracellular YEATS2 and total TAK1 protein in a time- and dose dependent manner. Ectopic expression of YEATS2 re-elevated the level of TAK1 and phosphorylated IKKα/β, IκBα and p65 after cinobufacini treatment in PANC-1 cells. ConclusionCinobufacini retards the growth and progression of PDAC in vitro and in vivo through YEATS2/TAK1/NF-κB axis.

Widely quasi-quantitative analysis of both metabolites and tryptic peptides in animal-originated medicinal materials: Bufonis Venenum as a case

It is challenging to achieve in-depth quality analysis for animal-originated medicinal materials (AMMs) owing to containing abundant metabolites and proteins. RPLC–MS/MS intrinsically bears the ability to simultaneously monitor metabolites and peptides. Hence, its potential towards merging quasi-quantitative metabolomics and tryptic proteomics characterization is therefore assessed in current study, and a well-known AMM namely Bufonis Venenum (BV, Chinese name: Chansu) was employed as a proof-of-concept. Qualitative information of metabolites was acquired by RPLC–Qtof-MS and translated to plausible structures through careful “spectrum-to-structure” analysis. Bottom-up proteomics was conducted to characterize the tryptic peptidome using nanoLC–QExactive HF orbitrap-MS. Quantitative MS/MS parameters of either metabolites (72 ones) or tryptic peptides (28 unique peptides) were optimized using online energy-resolved MS and applied to configure RPLC–selected-reaction monitoring (RPLC–SRM) program. Ultimately, SRM response of each analyte was converted to quasi-content by serially diluting a so-called universal metabolome standard (UMS) solution and building regressive calibration curve set to achieve widely quasi-quantitative metabolomics and proteomics. Although being sourced from identical species, significant differences occurred among the metabolite and protein profiles between BV and toad skins, and bufadienolides (i.e., 3-(N-adipoyl-argininyl)-gamabufotalin/isomer) along with several tryptic peptides (i.e., ISGLIYEETR sourced from Histone H4) served as the primary differential variables. Above all, RPLC–SRM is a promising analytical tool for in-depth quality evaluation of AMMs, and more importantly, the workflow described here is a fit-for-purpose pipeline to merge quasi-quantitative metabolomics and bottom-up proteomics.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

Genomic characterization of antifungal Acinetobacter bacteria isolated from the skin of the frogs Agalychnis callidryas and Craugastor fitzingeri.

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. A total of five isolates belonged to Acinetobacter pittii,Acinetobacter radioresistens, or Acinetobactermodestus, 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.

ЦИСТАМИН И ЦИСТИН ПОДАВЛЯЮТ ТРАНСПОРТ Na+ В ЭПИТЕЛИИ КОЖИ ЛЯГУШКИ

Amphibian skin and other isolated epithelial systems are classical model objects for studying the mechanisms of transepithelial ion transport. Na+ transport in osmoregulatory epithelium is a complex, multicomponent system, which involves Na+ transporting proteins and signaling cascades localized in various cell membranes. The protein components of this system may be a target for oxidative stress. At the same time, the effect of oxidizing and reducing agents on Na+ transport in native epithelial systems, such as frog skin epithelium, practically has not been studied. Using voltage-clamp technique the effect of disulfide-containing oxidizing agents cystine and cystamine on Na+ transport in frog skin was investigated. It has been shown for the first time that the treatment of the skin of the Rana temporaria frog with cystamine or cystine at a concentration of 10 μg/ml suppresses the transepithelial Na+ transport. It was also found that the preincubation with dithiothreitol, which reduces disulfide bonds in proteins, prevents the inhibitory effect of cystamine and cystine. The obtained results indicate that Na+ transport in the frog skin is sensitive to oxidative stress and is modulated by disulfide-containing oxidizing agents, such as cystamine or cystine, and also that the effect of cystamine and cystine on Na+ transport in the frog skin epithelium is mediated by their interaction with functionally important cysteine residues of Na+-transporting proteins.

Variation of Protein Band Pattern on the Skin Secretions of Chalcorana chalconota (Schlegel, 1837) Complex.

<b>Background and Objective:</b> <i> Chalcorana chalconota </i>is a complex frog species in West Sumatra which has been revised and designated as <i>Chalcorana parvaccola</i> and <i>Chalcorana rufipes</i> based on several studies such as morphology and genetics. Other studies such as protein band patterns can be a marker to differentiate species. This research was done to determine and prove the variations of protein band patterns found in skin secretions of the <i>C. chalconota </i>species complex. <b>Materials and Methods:</b> Frog samples were collected in the Pasia Laweh area, Pesisir Selatan, West Sumatra. The standard length measurement of the frog was carried out to determine the voltage that will be applied to the frog using an electric shock device (TAS/transcutaneous amphibian stimulator) in the process of removing the frog's skin secretions. Frog skin secretions were taken and used to see the pattern of protein bands using the SDS-PAGE method. <b>Results:</b> The protein band patterns of skin secretions of <i>C. chalconota</i> species complex were different between <i>C. parvaccola</i> and <i>C. rufipes</i>. In the skin secretions of <i>C. parvaccola</i>, there were eight protein bands with a molecular weight ranging between 12-103 kDa while for <i>C. rufipes</i> there were seven protein bands with a molecular weight ranging between 12-102 kDa. There were six protein bands shared by these two species. Two bands were only found in the skin secretions of <i>C. parvaccola</i> and one band was only found in <i>C. rufipes</i>. <b>Conclusion:</b> Pattern and molecular weight of protein in <i>C. parvaccola</i> and <i>C. rufipes</i> skin secretions can be used as protein markers to distinguish the two species.

Status of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in liver and skin of the freeze tolerant wood frog

The wood frog (Rana sylvatica) has adapted full-body freezing and thawing as a means of sub-zero winter survival and early-breeding in ephemeral pools. One such protective process implicated recently in freeze-thaw tolerance is that of anti-apoptotic signaling, which has been proposed to play a cytoprotective role by modulating stress-induced death signals. This study employed the use of immunoblotting to examine response of a potent cell cycle and apoptosis regulator, known as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, to freezing and thawing in the liver and skin of the wood frog. This pathway demonstrably exhibits factor- and tissue-specific changes between non-frozen, 24 h-frozen, and 8 h-thawed conditions. There were few changes in JAK-STAT proteins in frozen frogs, but protective changes were observed upon thaw: Elevated levels of pJAK3 and nuclear localization of pSTAT3 and pSTAT5 suggested an increase in anti-apoptotic signaling after thaw. By contrast, both STAT1 and STAT3 signaling appeared to increase in frozen skin, suggesting frogs use homeostatic regulation of apoptotic- and anti-apoptotic signals, in an antagonistic and compensatory manner. As such, these findings support that JAK-STAT pathway signaling modulation is a plausible adaptation that contributes to fast and reversible manipulation of anti-apoptotic signals, thus assisting in freeze survival of the wood frog.

Multiple Mechanistic Action of Brevinin-1FL Peptide against Oxidative Stress Effects in an Acute Inflammatory Model of Carrageenan-Induced Damage.

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.

Temporal variation in skin microbiota of cohabitating amphibians.

Temporal changes and transmission patterns in host-associated microbial communities have important implications for host health. The diversity of amphibian skin microbial communities is associated with disease outcome in amphibians exposed to the fungal pathogen Batrachochytrium dendrobatidis (Bd). To successfully develop conservation strategies against Bd, we need a comprehensive understanding of how skin microbes are maintained and transmitted over time within populations. We used 16S rRNA sequence analysis to compare Epipedobates anthonyi frogs housed with one conspecific to frogs housed singly at four time points over the course of 1 year. We found that both α and β diversity of frog skin bacterial communities changed significantly over the course of the experiment. Specifically, we found that bacterial communities of cohabitating frogs became more similar over time. We also observed that some bacterial taxa were differentially abundant between frogs housed singly and frogs housed with a conspecific. These results suggest that conspecific contact may play a role in mediating amphibian skin microbial diversity and that turnover of skin microbial communities can occur across time. Our findings provide rationale for future studies exploring horizontal transmission as a potential mechanism of host-associated microbial maintenance in amphibians.

A pore-forming protein drives macropinocytosis to facilitate toad water maintaining

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water import and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporin, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress.

Design and modification of frog skin peptide brevinin-1GHa with enhanced antimicrobial activity on Gram-positive bacterial strains.

Naturally occurring frog skin peptides are one of largest sources of antimicrobial peptides that have many advantages including high potency, broad spectrum of targets and low susceptibility to multiple drug-resistance bacteria. However, they also have disadvantages such as hemolytic activity, low stability and high production costs. For these reasons, various strategies have been applied to overcome these drawbacks restricting their use in clinical trials. Previously reported brevinin-1GHa (BR-1GHa) is a 24 amino acid long antimicrobial peptide isolated from Hylarana guentheri with hemolytic activity. To enhance the antimicrobial activity of this peptide and to reduce its hemolytic activity, we designed five new temporin like analogues and examined their bioactivities. Temporins are another class of frog skin peptides without hemolytic activity and shorter than brevinins. When the antimicrobial activities of new analogues were examined against a panel of microorganisms, BR-1GHa-3, in which two alanine residues in the truncated version of BR-1GHa were replaced with leucine, exhibited significantly improved antimicrobial activity against Gram-positive bacterial strains (e.g., S. aureus ATCC 29213 and E. casseliflavus ATCC 700327) with lower hemolytic activity compared to the BR-1GHa peptide. Furthermore, BR-1GHa-4 analogue, in which Gly3 was replaced with Pro, did not show any hemolytic activity except for highest (128µM) concentration tested and have a strong antimicrobial effect on Gram-positive bacteria (e.g., E. faecalis ATCC 51299 and B. cereus ATCC 13061).

Seasonality of host immunity in a tropical disease system

AbstractInfectious disease systems frequently exhibit strong seasonal patterns, yet the mechanisms that underpin intra‐annual cycles are unclear, particularly in tropical regions. We hypothesized that host immune function fluctuates seasonally, contributing to oscillations in infection patterns in a tropical disease system. To test this hypothesis, we investigated a key host defense of amphibians against a lethal fungal pathogen, Batrachochytrium dendrobatidis (Bd). We integrated two field experiments in which we perturbed amphibian skin secretions, a critical host immune mechanism, in Panamanian rocket frogs (Colostethus panamansis). We found that this immunosuppressive technique of reducing skin secretions in wild frog populations increased Bd prevalence and infection intensity, indicating that this immune defense contributes to resistance to Bd in wild frog populations. We also found that the chemical composition and anti‐Bd effectiveness of frog skin secretions varied across seasons, with greater pathogen inhibition during the dry season relative to the wet season. These results suggest that the effectiveness of this host defense mechanism shifts across seasons, likely contributing to seasonal infection patterns in a lethal disease system. More broadly, our findings indicate that host immune defenses can fluctuate across seasons, even in tropical regions where temperatures are relatively stable, which advances our understanding of intra‐annual cycles of infectious disease dynamics.

Identification of peptides of cinobufacini by gel filter chromatography and peptidomics.

Aqueous extract of toad skin (named as Cinobufacini or Huachansu) provides plentiful sources of bioactive peptides that remain undetected and unidentified. High-resolution mass spectrometry-based peptidomics platforms have developed into a major approach to the discovery of natural peptides, with data-dependent acquisition modes providing a wealth of peptide profiling information. In this study, we used a gel- and HLB (a solid phase extraction cartridge)-based two-dimensional separation and purification system and nano-liquid chromatography-tandem mass spectrometry-based peptidomic studies with homology matching for the identification of peptides from Cinobufacini. We evaluated 232 multi-charged peptides and found several specific peptides, some of which were validated by target parallel reaction monitoring mode. These peptides are the first to be identified in Cinobufacini and are completely different from ones identified in toad venom. So, this mapping provides key peptide information for the quality control of Bufo bufo gargarizans skin and its preparation.

Antimicrobial Activity of Fejervarya Skin Secretions (Anura: Dicroglossidae) in West Sumatra, Indonesia

Research on the antimicrobial test of Fejervarya frog skin secretions (Anura: Dicroglossidae) in West Sumatra. This study aimed to analyze the ability of compounds secreted from the skin of F. cancrivora and F. limnocharis in West Sumatra to inhibit the growth of Gram Negative bacteria, Gram Positive bacteria, antibiotics-resistant bacteria and fungi. This study used the diffusion method with paper discs for antimicrobial test of frog skin secretions. Result from this study showed that the skin secretions of F. cancrivora and F. limnocharis in West Sumatra, Indonesia did not show any antimicrobial properties.

Cinobufagin inhibits tumor progression and reduces doxorubicin resistance by enhancing FOXO1-mediated transcription of FCGBP in osteosarcoma

Ethnopharmacological relevanceCinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma. Aim of the studyThe study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance. Materials and methodsCell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments. ResultsCinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts. ConclusionCinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

Antifungal Activity of the Frog Skin Peptide Temporin G and Its Effect on Candida albicans Virulence Factors.

The increasing resistance to conventional antifungal drugs is a widespread concern, and a search for new compounds, active against different species of fungi, is demanded. Antimicrobial peptides (AMPs) hold promises in this context. Here we investigated the activity of the frog skin AMP Temporin G (TG) against a panel of fungal strains, by following the Clinical and Laboratory Standards Institute protocols. TG resulted to be active against (i) Candida species and Cryptococcus neoformans, with MIC50 between 4 µM and 64 µM after 24 h of incubation; (ii) dermatophytes with MIC80 ranging from 4 to 32 µM, and (iii) Aspergillus strains with MIC80 of 128 µM. In addition, our tests revealed that TG reduced the metabolic activity of Candida albicans cells, with moderate membrane perturbation, as proven by XTT and Sytox Green assays, respectively. Furthermore, TG was found to be effective against some C. albicans virulence factors; indeed, at 64 µM it was able to inhibit ~90% of yeast–mycelial switching, strongly prevented biofilm formation, and led to a 50% reduction of metabolic activity in mature biofilm cells, and ~30–35% eradication of mature biofilm biomass. Even though further studies are needed to deepen our knowledge of the mechanisms of TG antifungal activity, our results suggest this AMP as an attractive lead compound for treatment of fungal diseases.