The effects of preformed vitamin A and provitamin A carotenoid supplementation on tadpoles of the poison frog Phyllobates vittatus.

Understanding the nutritional requirements of captive animals is necessary for proper animal husbandry, however, the specific dietary requirements for many amphibian species commonly kept in captivity are unknown. Like most vertebrates, frogs cannot synthesize carotenoids and must therefore obtain these essential nutrients through diet. It is unclear if amphibians can cleave provitamin A carotenoids to form vitamin A metabolically within the body, so common practice is to supplement their captive diets with both preformed vitamin A and provitamin A carotenoids. We carried out a feeding experiment in tadpoles of Phyllobates vittatus, a commonly kept poison frog species, to test the effects of supplementing a fish flake diet with a provitamin A carotenoid (2.5 mg/g ß-carotene) and vitamin A (0.033-0.066 µg/mL retinyl acetate), both individually and in combination. Contrary to our expectations, supplementation had either no effect or adverse effects on tadpole growth and survivorship. Tadpoles reared under supplemented diets with vitamin A showed higher mortality rates, coupled with symptoms of hypervitaminosis A. Survivors had a smaller body size and mass at metamorphosis. ß-carotene supplementation alone had no detectable effect. The vitamin A and ß-carotene levels in our supplemented diet have been shown to be harmless or benefit tadpoles of other species, yet our results indicate that adding these amounts to what is found in a generalist fish flake mix can have detrimental effects on P. vittatus tadpoles. More broadly, this study highlights the importance of creating husbandry guidelines based on the specific physiological needs of the species (or species groups) being kept in captivity, rather than general ones for all amphibians, as is often done.

Dietary carotenoid supplementation has long-term and community-wide effects on the amphibian skin microbiome.

The amphibian skin microbiome plays a crucial role in host immunity and pathogen defence, yet we know little about the environmental drivers of skin microbial variation across host individuals. Inter-individual variation in the availability of micro-nutrients such as dietary carotenoids, which are involved in amphibian immunity, may be one factor that influences skin microbial assembly across different life history stages. We compared the effect of four carotenoid supplementation regimes during different life stages on the adult skin microbiome using a captive population of the critically endangered southern corroboree frog, Pseudophryne corroboree. We applied 16S rRNA sequencing paired with joint-species distribution models to examine the effect of supplementation on taxon abundances. We found that carotenoid supplementation had subtle yet taxonomically widespread effects on the skin microbiome, even 4.5 years post supplementation. Supplementation during any life-history stage tended to have a positive effect on the number of bacterial taxa detected, although explanatory power was low. Some genera were sensitive to supplementation pre-metamorphosis, but most demonstrated either additive or dominant effects, whereby supplementation during one life history stage had intermediate or similar effects, respectively, to supplementation across life. Carotenoid supplementation increased abundances of taxa belonging to lactic acid bacteria, including Lactococcus and Enterococcus, a group of bacteria that have previously been linked to protection against the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). While the fitness benefits of these microbial shifts require further study, these results suggest a fundamental relationship between nutrition and the amphibian skin microbiome which may be critical to amphibian health and the development of novel conservation strategies.

Lack of vitamin B12 impairs innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles.

Vitamin B12 is a micronutrient required by a variety of organisms for healthy cellular functioning. Despite the systemic effects observed in cases of B12 deficiency, relatively little is known about how vitamin B12 affects immune health, especially in amphibians, which are declining at unprecedented rates. In this study, we tested how supplementing an algae diet with B12 affects the innate and adaptive immunity of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We found that innate immunity, as measured by a bacterial killing assay, was significantly more robust in B12-supplemented tadpoles than control tadpoles, but no significant differences were found in natural antibody production or hematocrit between groups. Adaptive immunity, as measured by Aeromonas hydrophila-specific IgY antibodies, was significantly greater in tadpoles challenged with A. hydrophila and supplemented with B12 than in control tadpoles, those only challenged with A. hydrophila, and those only given B12. Our results suggest that vitamin B12 is an important factor in maintaining a functional immune system in tadpoles, which may also be true for all vertebrates.

Bioecological representations and social characteristics of students influence their attitudes toward wild vertebrates.

BACKGROUND: The origin of different human emotions directed towards animals (whether in the utilitarian, affective, conflictual, or cosmological context) is strongly influenced by sociocultural factors, although our genetic predispositions also play an important role in the origin of these emotions. Such emotions guide people's representations of different species, which in turn affect their attitudes toward them. For this reason, understanding the factors that guide such attitudes becomes a key element in making conservationist decisions. In this sense, the main objective of this study was to analyze how sociocultural characteristics and bioecological representations can influence students' attitudes of empathy or antipathy towards vertebrate species; as well as which classes and species are related to greater and lesser support in people for their conservation. METHODS: To do so, 667 interviews were conducted with students from urban (n = 1) and rural (n = 2) schools in the Brazilian semi-arid region. We used mixed generalized linear models (GLMM) to examine the effect of social factors and bioecological representations on empathy and antipathy attitudes and multiple factor analysis (MFA) to examine the relationship between the biological characteristics of the animals (positive or negative) and the attitudes toward them (antipathetic or empathetic). RESULTS: Through GLMM, we found that students from the urban area and from lower school levels are more extreme in their responses, more frequently expressing both empathy and antipathy towards wild animals. Regarding gender, women had a higher frequency of responses associated with aversion than men for species perceived as dangerous and poisonous (p < 0.001). Through the MFA, we found greater support (empathy) for the conservation of fish species (31.56%), birds (29.37%) and mammals (25.94%), with emphasis on the Red-cowled cardinal (Paroaria dominicana) and clownfish (Amphiprion ocellaris) species, and less support (antipathy) for reptile and amphibian species such as rattlesnakes (Crotalus durissus) and horned frogs (Ceratophrys joazeirensis). CONCLUSIONS: The attitudinal ambivalence reflected by varying empathy for certain species and antipathy to others has important implications for wildlife conservation. Understanding the socioeconomic factors and emotions that influence attitudes towards animals can enable integrating educational strategies for the conservation of species, especially those which are culturally important.

Proliferative parathyroid lesions in captive-bred American bullfrogs (Lithobates catesbeianus) with metabolic bone disease.

Parathyroid gland lesions in anurans are infrequently reported and most often occur secondary to experimental interventions. Husbandry-related parathyroid changes have not been documented in this order of Amphibia. Three American bullfrogs (Lithobates catesbeianus) living in a captive colony were euthanized due to clinical concern for metabolic bone disease secondary to lack of consistent dietary supplementation with vitamin D3. Necropsy revealed cystic dilation and variable proliferation of unidentified structures within the cranial coelom corresponding to the anatomical location of anuran parathyroid glands. Histologically, the structures consisted of sheets and whorls of elongated cells. Immunohistochemistry for pan-cytokeratin revealed strong cytoplasmic staining and Grimelius staining identified neuroendocrine granules in the elongated cells of these structures, supportive of a parathyroid origin.

Comparative transcriptomes of nine tissues for the Heilongjiang brown frog (Rana amurensis).

The Heilongjiang brown frog (Rana amurensis) is widely used in traditional Chinese medicine. In particular, the oviduct and skin have been developed into various health products. However, limited numbers of complete genomes of amphibian species have been reported, excluding the Heilongjiang brown frog. Here, the transcriptomes of 45 samples from the liver, spleen, heart, ovaries, thigh muscles, skin, oviduct, stomach and intestine of five Heilongjiang brown frog were reassembled and analyzed. A total of 1,085,532 unigenes with an average length of 676.6 bp and N50 of 722 bp were obtained. Comparative transcriptomics of different tissues detected tissue-specific expression. There were 3248 differentially expressed genes (DEGs) in the ovary, and the number of unique DEGs between the ovary and spleen was the largest. The results of DEGs enrichment showed there were many pathways and items related to protein synthesis and metabolism in the oviduct. The DEGs of the skin were enriched with many bacterial defense items, indicating that there were a large number of antimicrobial peptides in the skin. Thus, these were suitable as biological sources for the development and extraction of antimicrobial peptides. Through the assembly of transcriptome sequencing data and functional annotation of the Heilongjiang brown frog genome, this study provides reference materials for further exploring and utilizing functional gene resources of frogs and lays a foundation for medical research and the development of new products.

Moisture content monitoring in withering leaves during black tea processing based on electronic eye and near infrared spectroscopy.

Monitoring the moisture content of withering leaves in black tea manufacturing remains a difficult task because the external and internal information of withering leaves cannot be simultaneously obtained. In this study, the spectral data and the color/texture information of withering leaves were obtained using near infrared spectroscopy (NIRS) and electronic eye (E-eye), respectively, and then fused to predict the moisture content. Subsequently, the low- and middle-level fusion strategy combined with support vector regression (SVR) was applied to detect the moisture level of withering leaves. In the middle-level fusion strategy, the principal component analysis (PCA) and random frog (RF) were employed to compress the variables and select effective information, respectively. The middle-level-RF (cutoff line = 0.8) displayed the best performance because this model used fewer variables and still achieved a satisfactory result, with 0.9883 and 5.5596 for the correlation coefficient of the prediction set (Rp) and relative percent deviation (RPD), respectively. Hence, our study demonstrated that the proposed data fusion strategy could accurately predict the moisture content during the withering process.

The role of environmental calcium in the extreme acid tolerance of northern banjo frog (Limnodynastes terraereginae) larvae.

Many aquatically respiring animals acutely exposed to low pH waters suffer inhibition of ion uptake and loss of branchial (gill) epithelial integrity, culminating in a fatal loss of body Na+. Environmental calcium levels ([Ca2+]e) are pivotal in maintaining branchial junction integrity, with supplemental Ca2+ reversing the negative effects of low pH in some animals. Tolerance of some naturally acidic environments by aquatic animals is further complicated by low [Ca2+]e, yet many of these environments are surprisingly biodiverse. How animals overcome the damaging actions of low pH and low environmental Ca2+ remains unknown. We examined the effects of [Ca2+]e on the response to low pH in larvae of the highly acid-tolerant frog Limnodynastes terraereginae. Acute exposure to low pH water in the presence of low (5 µmol l-1) [Ca2+]e increased net Na+ efflux. Provision of additional [Ca2+]e reduced net Na+ efflux, but the effect was saturable. Acclimation to both low and high (250 µmol l-1) [Ca2+]e improved the resistance of larvae to Na+ efflux at low pH. Exposure to the Ca2+ channel inhibitor ruthenium red resulted in an abrupt loss of tolerance in low pH-acclimated larvae. Acclimation to acidic water increased branchial gene expression of the intracellular Ca2+ transport protein calbindin, consistent with a role for increased transcellular Ca2+ trafficking in the tolerance of acidic water. This study supports a role for [Ca2+]e in promoting branchial integrity and highlights a potential mechanism via the maintenance of transcellular Ca2+ uptake in the acid tolerance of L. terraereginae larvae.

Species perceived to be dangerous are more likely to have distinctive local names.

BACKGROUND: Species with direct uses, such as sources of food, shelter, building material and medicine tend to have more specific local names. But could the same apply for species that people fear? METHODS: To address this question, here we explore the behavior and perception of species diversity and dangerousness through a survey of 1037 households in nine villages in Cabo Delgado, northern Mozambique. We compare people's knowledge of snakes with that of lizards and amphibians. RESULTS: We find that northern Mozambicans know four to five times more local names for snakes than for lizards and frogs, despite the local species richness of snakes being comparable to the diversity of lizards and frogs. We further find that local knowledge was on par with the academic literature regarding snakebite symptoms. CONCLUSIONS: Our results suggest that fear can increase the level of specificity in naming species among indigenous communities, which could lead to biases in the mapping and protection of species that include data from citizen reports.

In Vitro and In Vivo Studies on the Antibacterial Activity and Safety of a New Antimicrobial Peptide Dermaseptin-AC.

Antimicrobial resistance has been an increasing public health threat in recent years. Antimicrobial peptides are considered as potential drugs against drug-resistant bacteria because they are mainly broad-spectrum and are unlikely to cause resistance. In this study, a novel peptide was obtained from the skin secretion of Agalychnis callidryas using the "shotgun" cloning method. The amino acid sequence, molecular weight, and secondary structure of Dermaseptin-AC were determined. The in vitro antimicrobial activity, hemolysis, and cytotoxicity of Dermaseptin-AC were evaluated. MICs and minimum bactericidal concentrations (MBCs) of Dermaseptin-AC against seven different bacterial strains ranged between 2 ∼ 4 µM and 2 ∼ 8 µM. The HC50 (50% maximum hemolysis concentration) of Dermaseptin-AC against horse erythrocytes was 76.55 µM. The in vivo anti-MRSA effect was tested on immune-suppressed MRSA pneumonia in mice. Dermaseptin-AC showed anti-MRSA effects similar to the same dose of vancomycin (10 mg/kg body weight). Short-term (7 days of intraperitoneal injection, 10 mg/kg body weight) in vivo safety evaluation of Dermaseptin-AC was tested on mice. The survival rate during the 7-day injection was 80%. Dermaseptin-AC showed no obvious effect on the liver, heart, spleen, kidney, and blood, but did induce slight pulmonary congestion. The skin safety of Dermaseptin-AC was evaluated on wounds on the back skin of a rat, and no irritation was observed. IMPORTANCE In this study, we discovered a new antimicrobial peptide, Dermaseptin-AC, and studied its in vitro and in vivo antimicrobial activity. These studies provide some data for finding new antimicrobial peptides for overcoming antimicrobial resistance. Dermaseptin-AC showed strong broad-spectrum antibacterial activity and relatively low hemolysis, and was more cytotoxic to cancer cells than to normal cells. Dermaseptin-AC was active in vivo, and its anti-MRSA effect was similar to that of vancomycin when administered by intraperitoneal injection. Safety studies found that continuous injection of Dermaseptin-AC may cause mild pulmonary congestion, while there was no obvious irritation when it was applied to skin wounds. Chronic wounds are often accompanied by high bacterial burdens and, at the same time, antimicrobial resistance is more likely to occur during repeated infections and treatments. Therefore, developing Dermaseptin-AC to treat chronic wound infection may be an attractive choice.

Does dietary ß-carotene influence ontogenetic colour change in the southern corroboree frog?

Ontogenetic colour change occurs in a diversity of vertebrate taxa and may be closely linked to dietary changes throughout development. In various species, red, orange and yellow colouration can be enhanced by the consumption of carotenoids. However, a paucity of long-term dietary manipulation studies means that little is known of the role of individual carotenoid compounds in ontogenetic colour change. We know even less about the influence of individual compounds at different doses (dose effects). The present study aimed to use a large dietary manipulation experiment to investigate the effect of dietary ß-carotene supplementation on colouration in southern corroboree frogs (Pseudophryne corroboree) during early post-metamorphic development. Frogs were reared on four dietary treatments with four ß-carotene concentrations (0, 1, 2 and 3 mg g-1), with frog colour measured every 8 weeks for 32 weeks. ß-Carotene was not found to influence colouration at any dose. However, colouration was found to become more conspicuous over time, including in the control treatment. Moreover, all frogs expressed colour maximally at a similar point in development. These results imply that, for our study species, (1) ß-carotene may contribute little or nothing to colouration, (2) frogs can manufacture their own colour, (3) colour development is a continual process and (4) there may have been selection for synchronised development of colour expression. We discuss the potential adaptive benefit of ontogenetic colour change in P. corroboree. More broadly, we draw attention to the potential for adaptive developmental synchrony in the expression of colouration in aposematic species.

Peculiarities of Neurostimulation by Intense Nanosecond Pulsed Electric Fields: How to Avoid Firing in Peripheral Nerve Fibers.

Intense pulsed electric fields (PEF) are a novel modality for the efficient and targeted ablation of tumors by electroporation. The major adverse side effects of PEF therapies are strong involuntary muscle contractions and pain. Nanosecond-range PEF (nsPEF) are less efficient at neurostimulation and can be employed to minimize such side effects. We quantified the impact of the electrode configuration, PEF strength (up to 20 kV/cm), repetition rate (up to 3 MHz), bi- and triphasic pulse shapes, and pulse duration (down to 10 ns) on eliciting compound action potentials (CAPs) in nerve fibers. The excitation thresholds for single unipolar but not bipolar stimuli followed the classic strength-duration dependence. The addition of the opposite polarity phase for nsPEF increased the excitation threshold, with symmetrical bipolar nsPEF being the least efficient. Stimulation by nsPEF bursts decreased the excitation threshold as a power function above a critical duty cycle of 0.1%. The threshold reduction was much weaker for symmetrical bipolar nsPEF. Supramaximal stimulation by high-rate nsPEF bursts elicited only a single CAP as long as the burst duration did not exceed the nerve refractory period. Such brief bursts of bipolar nsPEF could be the best choice to minimize neuromuscular stimulation in ablation therapies.

Tree frog-inspired nanopillar arrays for enhancement of adhesion and friction.

Bioinspired structure adhesives have received increasing interest for many applications, such as climbing robots and medical devices. Inspired by the closely packed keratin nanopillars on the toe pads of tree frogs, tightly arranged polycaprolactone nanorod arrays are prepared by mold process and chemical modification. Nanorod arrays show enhanced adhesion and friction on both smooth and rough surfaces compared to the arrays with hexagonal micropillars. The bonding of nanorods results in a larger stiffness of the nanorod surface, contributing mainly to friction rather than adhesion. The results suggest the function of closely packed keratin nanopillars on the toe pad of tree frogs and offer a guiding principle for the designing of new structured adhesives with strong attaching abilities.

A novel peptide identified from skin secretions of Bombina maxima possesses LPS-neutralizing activity.

Lipopolysaccharide (LPS) is a major pathogenic factor in endotoxin shock or sepsis. Most antibiotics have little clinical anti-endotoxin activity, but some antimicrobial peptides (AMPs) have been shown to be effective in blocking LPS. We identified a novel peptide from the skin secretions of Bombina maxima (B. _maxima) by challenging the skin of frogs with an LPS solution. Peptide 2 has an amino acid sequence of LVGKLLKGAVGDVCGLLPIC. Peptide 2 possesses low hemolytic activity, low cytotoxicity against RAW 264.7 cells, and strong anti-inflammatory activity. Moreover, peptide 2 plays an anti-inflammatory role by inhibiting inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). A biolayer interferometry (BLI) assay indicated that peptide 2 binds to LPS with strong affinity and that this interaction has an affinity constant (KD) value of 1.05 × 10-9 M. A survival study showed that peptide 2 possesses potent LPS-neutralizing activity to protect LPS-treated mice from death. In conclusion, we have identified a potent peptide with LPS neutralizing activity, which lays a foundation for future research and development.

Kambô-Induced Systemic Inflammatory Response: A Case Report of Acute Disease Progression of Cholangiocarcinoma.

Kambô is a cleansing ritual involving the application of a toxin produced by the giant leaf frog (Phyllomedusa bicolor). The Kambô ritual has increasingly been adopted among cancer patients in Europe. Accumulating data indicate various adverse effects. We report another severe adverse reaction to Kambô, a systemic inflammatory response syndrome mimicking disease progression in a patient with cholangiocarcinoma. We describe a systemic reaction to Kambô, manifested as tachycardia, tachypnea, impaired liver cholestatic enzymes, and enlargement of lymphadenopathy mimicking disease progression. The clinical features and onset of symptoms, the rapid reaction, and the lack of other identified causes make the diagnosis of Kambô-induced SIRS highly probable. This case report calls for future studies examining standard oncological care such as chemotherapy, radiotherapy, and immunotherapy in conjunction with alternative therapy. Additionally, greater awareness and physician education should be promoted, encouraging inquiry of oncology patients' administration of alternative, complementary, and integrative medicine.

Connected to the spirit of the frog: An Internet-based survey on Kambô, the secretion of the Amazonian Giant Maki Frog (Phyllomedusa bicolor): Motivations for use, settings and subjective experiences.

BACKGROUND/AIM: Kambô is a name for the secretion of the Giant Maki Frog (Phyllomedusa bicolor), which has been used by indigenous cultures from the Amazonas basin and has recently become popular in alternative healing circles in Western countries, with a certain overlap with psychedelic self-exploration. METHODS: We carried out an online-based survey in English (54.92%) and German investigating motivations for using Kambô, settings in which rituals take place, and subjective experiences during and after the application. RESULTS: Participants (n = 386, mean age: 38.08 years, (standard deviation = 9.95)) were well-educated individuals with an increased lifetime prevalence of the use of ayahuasca (67.88%). A plethora of motivations for using Kambô was reported, including general healing, detoxification and spiritual growth. Acute effects included severe physical reactions and mild psychoactive effects, most surprisingly, the feeling of being connected to the frog's spirit (41.97%), whereas predominantly positive persisting psychological effects were reported. Few participants reported long-lasting physical (2.85%) or mental (1.81%) health problems which they attributed to Kambô. Of the participants, 87.31% reported an increase in personal well-being or life satisfaction, and 64.26% considered Kambô to have been at least of 'very much' spiritual significance for their lives. CONCLUSIONS: The majority of users claimed beneficial effects including more health-orientated behaviors, whereas only very few participants complained about new health problems which they ascribed to Kambô. In retrospect, Kambô was given a high personal and spiritual significance by many participants. Additional research is needed to determine in how far reported effects are modulated by setting and subjective expectations.

Observations on spindly leg syndrome in a captive population of Andinobates geminisae.

Amphibian health problems of unknown cause limit the success of the growing number of captive breeding programs. Spindly leg syndrome (SLS) is one such disease, where affected individuals with underdeveloped limbs often require euthanization. We experimentally evaluated husbandry-related factors of SLS in a captive population of the critically endangered frog, Andinobates geminisae. SLS has been linked to tadpole nutrition, vitamin B deficiency, water filtration methods, and water quality, but few of these have been experimentally tested. We tested the effects of water filtration method and vitamin supplementation (2017) and the effects of tadpole husbandry protocol intensity (2018) on time to metamorphosis and the occurrence of SLS. We found that vitamin supplementation and reconstituted reverse osmosis filtration of tadpole rearing water significantly reduced SLS prevalence and that reduced tadpole husbandry delayed time to metamorphosis. A fortuitous accident in 2018 resulted in a decrease in the phosphate content of rearing water, which afforded us an additional opportunity to assess the influence of phosphate on calcium sequestration. We found that tadpoles that had more time to sequester calcium for ossification during development had decreased the prevalence of SLS. Taken together, our results suggest that the qualities of the water used to rear tadpoles plays an important role in the development of SLS. Specifically, filtration method, vitamin supplementation, and calcium availability of tadpole rearing water may play important roles. Focused experiments are still needed, but our findings provide important information for amphibian captive rearing programs affected by high SLS prevalence.

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis.

RATIONALE: Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides. METHODS: Manual interpretation of complementary collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation. RESULTS: Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28. CONCLUSIONS: Mirror-symmetrical peptides are sequenced using N-terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b-ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.

Identification and functional characterisation of Esculentin-2 HYba peptides and their C-terminally amidated analogs from the skin secretion of an endemic frog.

Here, we report the identification, functional characterisation, and the effect of C-terminal amidation on the activity profile of two novel Esculentin-2 peptides (Esculentin-2 HYba1 and Esculentin-2 HYba2). The parent peptides and their analogs exhibited potent activity against the tested Gram-positive and Gram-negative bacteria. The effect of amidation was evident in the activity profile of fish pathogens and killing kinetics. The analogs showed a 10-fold decrease in MIC, and the killing time was reduced to 10-15 minutes. The hemolytic potential was unaltered upon amidation. The selectivity index revealed that these peptides are more selective to bacteria than mammalian cells. Cytotoxicity against Hep3B cells reveals their potential to destroy cancer cells; they showed potential inhibition compared to anticancer drug silymarin. The study also highlights the need for further truncations and modifications of esculentin peptides for developing them as lead drug molecules.

Three auditory brainstem response (ABR) methods tested and compared in two anuran species.

Hearing sensitivity has been extensively investigated, often by measuring the auditory brainstem response (ABR). ABR measurements are relatively non-invasive, easy to reproduce, and allow the assessment of sensitivity when psychophysical data are difficult to obtain. However, the experimental methods differ greatly in respect to stimulation, which may result in different audiograms. We used three different methods in the same individual frogs: stimulating with brief tone bursts (tABR), long-duration tones (ltABR) and masked ABR (mABR), where transients are masked by a long-duration sinusoid, and the sensitivity is assessed by the difference between unmasked and masked ABR. We measured sensitivity in a range from 100 to 3500 Hz, and the resulting audiograms show two sensitivity peaks at 400-600 Hz and 1500-1600 Hz (both sensitive down to 30 dB re. 20 µPa). We found similar results below 1000 Hz, but when stimulating with long-duration tones, the sensitivity decreased more rapidly above this frequency. We showed that the frequency specificity of tone bursts becomes poorly defined with shorter duration at low frequencies. Comparisons between subjectively (visual inspection by researchers) and objectively (thresholds defined by signal-to-noise ratio) defined audiograms showed very little variation. In conclusion, the mABR method gave the most sensitive audiograms. The tABR method showed a similar audiogram when using relatively long-duration tone bursts (25 ms). The ltABR method is not a good choice for studying hearing thresholds above 1000 Hz because of the bias introduced by spike rate saturation in the nerve fibers and their inability to phase lock.