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The salmonid industry faces challenges due to the susceptibility of fish to opportunistic pathogens, particularly in early developmental stages. Understanding the immunological capacity during these stages is crucial for developing effective disease control strategies. IL-8R, a member of the G-protein-coupled receptor family, acts as a receptor for Interleukin 8 (IL-8). The binding of IL-8 to IL-8R plays a major role in the pathophysiology of a wide spectrum of inflammatory conditions. This study focused on the immune response capacity of rainbow trout (Oncorhynchus mykiss) larvae by analyzing IL-8/CXCR1 response to lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Previous research demonstrated that LPS from P. aeruginosa acts as a potent immunostimulant in teleost, enhancing pro-inflammatory cytokines. The methodology included in silico analysis and the synthesis and characterization of an omCXCR1-derived epitope peptide, which was used to produce omCXCR1-specific anti98 serum in mice. The research revealed that rainbow trout larvae 19 days post-hatching (dph) exhibited pronounced immune responses post-stimulation with 1 µg/mL of LPS. This was evidenced by the upregulated protein expression of IL-8 and omCXCR1 in trout larvae 2 and 8 h after LPS challenge, as analyzed by ELISA and immunohistochemistry. Furthermore, fluorescence microscopy successfully revealed the colocalization of IL-8 and its receptor in cells from mucosal tissues after LPS challenge in larvae 19 dph. These findings underscore the efficacy of LPS immersion as a method to activate the innate immune system in trout larvae. Furthermore, we propose IL-8 and its receptor as molecular markers for evaluating immunostimulation in the early developmental stages of salmonids.

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Bacteriophages have been proposed as biological controllers to protect plants against different bacterial pathogens. In this scenario, one of the main challenges is the low viability of phages in plants and under adverse environmental conditions. This work explores the use of 12 compounds and 14 different formulations to increase the viability of a phage mixture that demonstrated biocontrol capacity against Pseudomonas syringae pv. actinidiae (Psa) in kiwi plants. The results showed that the viability of the phage mixture decreases at 44 °C, at a pH lower than 4, and under UV radiation. However, using excipients such as skim milk, casein, and glutamic acid can prevent the viability loss of the phages under these conditions. Likewise, it was demonstrated that the use of these compounds prolongs the presence of phages in kiwi plants from 48 h to at least 96 h. In addition, it was observed that phages remained stable for seven weeks when stored in powder with skim milk, casein, or sucrose after lyophilization and at 4 °C. Finally, the phages with glutamic acid, sucrose, or skim milk maintained their antimicrobial activity against Psa on kiwi leaves and persisted within kiwi plants when added through roots. This study contributes to overcoming the challenges associated with the use of phages as biological controllers in agriculture.

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Introduction: The chronic use of psychostimulants increases the risk of addiction and, there is no specific pharmacologic treatment for psychostimulant addiction. The vasopressin (AVP) system is a possible pharmacological target in drug addiction. Previous results obtained in our laboratory showed that amphetamine (AMPH) treatment decreases lateral septum (LS) AVP levels in male rats, and AVP microinjection in LS decreases addictive-like behavior. The aim of the present work was to investigate the effect of AMPH treatment on LS AVP levels and the effect of LS AVP administration on the expression of AMPH-conditioned place preference (CPP) in female rats. The secondary objectives were to study the effect of LS AVP administration on LS GABA and glutamate release in male and female rats and on nucleus accumbens (NAc) dopamine (DA) release in female rats. Methods: Female rats were conditioned with AMPH (1.5 mg/kg i.p.) or saline for 4 days. Results: Conditioning with AMPH did not change LS AVP content in females. However, AVP microinjection into the LS decreased the expression of conditioned place preference (CPP) to AMPH. Glutamate and GABA extracellular levels in the LS induced by AVP were studied in males and females. NAc GABA and DA extracellular levels induced by LS AVP microinjection in female rats were measured by microdialysis. In males, AVP perfusion produced a significant increase in LS GABA extracellular levels; however, a decrease in GABA extracellular levels was observed in females. Both in males and females, LS AVP perfusion did not produce changes in LS glutamate extracellular levels. Microinjection of AVP into the LS did not change GABA or DA extracellular levels in the NAc of females. Discussion: Therefore, AVP administration into the LS produces different LS-NAc neurochemical responses in females than males but decreases CPP to AMPH in both sexes. The behavioral response in males is due to a decrease in NAc DA levels, but in females, it could be due to a preventive increase in NAc DA levels. It is reasonable to postulate that, in females, the decrease in conditioning produced by AVP microinjection is influenced by other factors inherent to sex, and an effect on anxiety cannot be discarded.

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Comprehending the immune defense mechanisms of new aquaculture species, such as the Chilean meagre (Cilus gilberti), is essential for sustaining large-scale production. Two bioassays were conducted to assess the impact of acute and intermittent hypoxia on the antibacterial activity of juvenile Chilean meagre epidermal mucus against the potential pathogens Vibrio anguillarum and Vibrio ordalii. Lysozyme and peroxidase activities were also measured. In general, fish exposed to hypoxia showed a 9-30% reduction in mucus antibacterial activity at the end of hypoxic periods and after stimulation with lipopolysaccharide. However, following water reoxygenation, the activity of non-stimulated fish was comparable to that of fish in normoxic conditions, inhibiting bacterial growth by 35-52%. In the case of fish exposed to chronic hypoxia, the response against V. anguillarum increased by an additional 19.8% after 6 days of control inoculation. Lysozyme exhibited a similar pattern, while no modulation of peroxidase activity was detected post-hypoxia. These results highlight the resilience of C. gilberti to dissolved oxygen fluctuations and contribute to understanding the potential of mucus in maintaining the health of cultured fish and the development of future control strategies.

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The HLA-B*35 alleles have been associated with a slow or rapid progression of HIV-1 infection. However, the mechanisms related to HIV-1 progression have yet to be entirely understood. Several reports indicate that the binding affinity between the HLA-I molecule and peptides could be associated with an increased CD8+ T-cell response. Novel HLA-B*35-restricted mutated variants have been described from HSNQVSQNY (HY9) and HPVHAGPIA (HA9) epitopes. Bioinformatic analysis has indicated that these mutated epitopes show low and high binding affinity towards HLA-B*35, respectively. However, the polyfunctionality of CD8+ T-cells stimulated with these mutated and wild-type epitopes has yet to be reported. The results suggest that the low-binding affinity H124 N/S125 N/N126S mutated peptide in the HY9 epitope induced a lower percentage of CD107a+CD8+ T-cells than the wild-type epitope. Instead, the high-binding affinity peptides I223V and I223A in the HA9 epitope induced a significantly higher frequency of polyfunctional CD8+ T-cells. Also, a higher proportion of CD8+ T-cells with two functions, with Granzyme B+ Perforin+ being the predominant profile, was observed after stimulation with mutated peptides associated with high binding affinity in the HA9 epitope. These results suggest that the high-affinity mutated peptides induced a more polyfunctional CD8+ T-cell response, which could be related to the control of viral replication.

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The skin of fish is a physicochemical barrier that is characterized by being formed by cells that secrete molecules responsible for the first defense against pathogenic organisms. In this study, the biological activity of peptides from mucus of Seriola lalandi and Seriolella violacea were identified and characterized. To this purpose, peptide extraction was carried out from epidermal mucus samples of juveniles of both species, using chromatographic strategies for purification. Then, the peptide extracts were characterized to obtain the amino acid sequence by mass spectrometry. Using bioinformatics tools for predicting antimicrobial and antioxidant activity, 12 peptides were selected that were chemically produced by simultaneous synthesis using the Fmoc-Tbu strategy. The results revealed that the synthetic peptides presented a random coil or extended secondary structure. The analysis of antimicrobial activity allowed it to be discriminated that four peptides, named by their synthesis code 5065, 5069, 5070, and 5076, had the ability to inhibit the growth of Vibrio anguillarum and affected the copepodite stage of C. rogercresseyi. On the other hand, peptides 5066, 5067, 5070, and 5077 had the highest antioxidant capacity. Finally, peptides 5067, 5069, 5070, and 5076 were the most effective for inducing respiratory burst in fish leukocytes. The analysis of association between composition and biological function revealed that the antimicrobial activity depended on the presence of basic and aromatic amino acids, while the presence of cysteine residues increased the antioxidant activity of the peptides. Additionally, it was observed that those peptides that presented the highest antimicrobial capacity were those that also stimulated respiratory burst in leukocytes. This is the first work that demonstrates the presence of functional peptides in the epidermal mucus of Chilean marine fish, which provide different biological properties when the fish face opportunistic pathogens.

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The meat industry uses phosphates to improve the water-holding capacity (WHC) of meat products, although excess phosphates can be harmful to human health. In this sense, protein hydrolysates offer an alternative with scientific evidence of improved WHCs. Salmon frames, a byproduct rich in protein, must be processed for recovery. Enzymatic technology allows these proteins to be extracted from muscle, and the sequential batch strategy significantly increases protein nitrogen extraction. This study focused on evaluating the WHC of protein hydrolysates from salmon frames obtained through double- and triple-sequential batches compared to conventional hydrolysis. Hydrolysis was carried out for 3 h at 55 °C with 13 mAU of subtilisin per gram of salmon frames. The WHC of each hydrolysate was measured as the cooking loss using concentrations that varied from 0 to 5% (w/w) in the meat matrix. Compared with those obtained through conventional hydrolysis, the hydrolysates obtained through the strategy of double- and triple-sequence batches demonstrated a 55% and 51% reduction in cooking loss, respectively, when they were applied from 1% by weight in the meat matrix. It is essential to highlight that all hydrolysates had a significantly lower cooking loss (p ≤ 0.05) than that of the positive control (sodium tripolyphosphate [STPP]) at its maximum allowable limit when applied at a concentration of 5% in the meat matrix. These results suggest that the sequential batch strategy represents a promising alternative for further improving the WHC of hydrolysates compared to conventional hydrolysis. It may serve as a viable substitute for polyphosphates.

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Currently, one of the primary challenges in salmon farming is caligidosis, caused by the copepod ectoparasites Caligus spp. The infection process is determined by the copepod's ability to adhere to the fish skin through the insertion of its chitin-composed filament. In this study, we examined several antimicrobial peptides previously identified in salmonid mucosal secretions, with a primary focus on their potential to bind to chitin as an initial step. The binding capacity to chitin was tested, with hepcidin and piscidin showing positive results. Further assessments involving cytotoxicity in salmonid cells RTgill-W1, SHK-1, RTS-11, and RT-gut indicated that the peptides did not adversely affect cell viability. However, hemolysis assays unveiled the hemolytic capacity of piscidin at lower concentrations, leading to the selection of hepcidin for antiparasitic assays. The results demonstrated that the nauplius II stage of C. rogercresseyi exhibited higher susceptibility to hepcidin treatments, achieving a 50% reduction in parasitic involvement at 50 µM. Utilizing fluorescence and scanning electron microscopy, we observed the localization of hepcidin on the surface of the parasite, inducing significant spherical protuberances along the exoskeleton of C. rogercresseyi. These findings suggest that cysteine-rich AMPs derived from fish mucosa possess the capability to alter the development of the chitin exoskeleton in copepod ectoparasites, making them therapeutic targets to combat recurrent parasitic diseases in salmon farming.

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One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron-Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-ßAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-ßAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-ßAla-OH, were synthesized by both strategies with high yield and purity.

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The impact of salivary alterations on chickpea protein structure in the elderly has not been well documented. This study aimed to understand the role of simulated salivary alterations in the conformational properties and secondary structure of the chickpea protein isolate (CPI). Whey protein isolate (WPI) was used as the reference. Protein dispersions (10%) were subjected to in vitro oral processing under simulated salivary conditions in both the elderly and adult subjects. Proteins and their oral counterparts were characterized in terms of their composition, charge, size, solubility, water absorption, molecular weight (MW), and secondary structure (Circular Dichroism and Raman spectroscopy). Under condition of simulated oral digestion in the elderly population, the ordered secondary protein structure was significantly affected, decreasing α-helix by ~36% and ~29% in CPI and WPI compared to the control (adult) population, respectively. An increase in the unordered random coil state was observed. These results could be attributed to an increase in electrolytes in the salivary composition. The structure of CPI is more stable than that of WPI because of its higher MW, more rigid structure, less charged surface, and different amino acid compositions. This study is meaningful in understanding how alterations in the elderly oral system affect protein conformation and is expected to improve the understanding of plant-based protein digestibility.

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Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.

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Southern King Crab (SKC) represents an important fishery resource that has the potential to be a natural source of chitosan (CS) production. In tissue engineering, CS is very useful to generate biomaterials. However, CS has a lack of signaling molecules that facilitate cell-substrate interaction. Therefore, RGD (arginine-glycine-aspartic acid) peptides corresponding to the main integrin recognition site in extracellular matrix proteins have been used to improve the CS surface. The aim of this study was to evaluate in vitro cell adhesion and proliferation of CS films synthesized from SKC shell wastes functionalized with RGD peptides. The FTIR spectrum of CS isolated from SKC shells (SKC-CS) was comparable to commercial CS. Thermal properties of films showed similar endothermic peaks at 53.4 and 53.0 °C in commercial CS and SKC-CS, respectively. The purification and molecular masses of the synthesized RGD peptides were confirmed using HPLC and ESI-MS mass spectrometry, respectively. Mouse embryonic fibroblast cells showed higher adhesion on SKC-CS (1% w/v) film when it was functionalized with linear RGD peptides. In contrast, a cyclic RGD peptide showed similar adhesion to control peptide (RDG), but the highest cell proliferation was after 48 h of culture. This study shows that functionalization of SKC-CS films with linear or cyclic RGD peptides are useful to improve effects on cell adhesion or cell proliferation. Furthermore, our work contributes to knowledge of a new source of CS to synthesize constructs for tissue engineering applications.

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Growing consumer awareness of the potential negative health effects of synthetic antibiotics has prompted the search for more natural preservatives that can improve the safety and quality of food. In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Ile-Gln (Z-IQ) which is the precursor of Ile-Gln (IQ), a new antibacterial dipeptide, using an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate in 0.1 M Tris - HCl buffer pH 8. A partially purified proteolytic extract from the fruits of Solanum granuloso leprosum, named granulosain, proved to be a robust biocatalyst for the synthesis of Z-IQ, eliciting 71 ± 0.10% maximal peptide yield in the above described conditions. After cleaving and purifying IQ dipeptide, antimicrobial activity was assayed against Staphylococcus aureus ATCC 25923, Staphylococcus hominis A17771, and Staphylococcus aureus C00195, and MIC values between 118 ± 0.01 µg/mL and 133.7 ± 0.05 µg/mL were obtained. In addition, IQ showed MIC of 82.4 ± 0.01 µg/mL and 85.0 ± 0.00 µg/mL against Escherichia coli ATCC 25922 and Escherichia coli A17683, respectively. IQ did not show inhibitory activity against single-drug resistance (SDR) strains, such as Klebsiella oxytoca A19438 (SDR) and Pseudomonas aeruginosa C00213 (SDR), and against multidrug-resistant Enterococcus faecalis I00125 (MDR). IQ also caused growth inhibition of Helicobacter pylori NCTC 11638 and three wild-type H. pylori strains, which are sensitive to AML, MTZ, LEV and CLA (H. pylori 659), resistant to LEV (H. pylori 661 SDR), and resistant to MTZ (H. pylori 662 SDR). Finally, this study contributes with a new dipeptide (IQ) that can be used as an antimicrobial agent for food preservation or as a safe ingredient of functional foods.

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The SARS-CoV-2 worldwide outbreak prompted the development of several tools to detect and treat the disease. Among the new detection proposals, the use of peptides mimetics has surged as an alternative to avoid the use of antibodies, of which there has been a shortage during the COVID-19 pandemic. However, the use of peptides in detection systems still presents some questions to be answered, mainly referring to their stability under different environmental conditions. In this work, we synthesized an ACE2 peptide mimic and evaluated its stability in different pH, salinity, polarity, and temperature conditions. Further, the same conditions were assessed when using the ability of the peptide mimic to detect the recombinant SARS-CoV-2 spike protein in a biotin-streptavidin-enzyme-linked assay. Finally, we also tested the capacity of the peptide to detect SARS-CoV-2 from patients' samples. The results indicate that the peptide is structurally sensitive to the medium conditions, with relevance to the pH, where basic pH favored its performance when used as a SARS-CoV-2 detector. Further, the proposed peptide mimic was able to detect SARS-CoV-2 comparably to RT-qPCR results. Therefore, the present study promotes knowledge advancement, particularly in terms of stability considerations, in the application of peptide mimics as a replacement for antibodies in detection systems.

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Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is a prototype of the "Repeat in Toxins" (RTX) family. It was demonstrated that HlyA-cholesterol interaction facilitates the insertion of the toxin into membranes. Putative cholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC), and CARC (analogous to CRAC but with the opposite orientation) were identified in the HlyA sequence. In this context, two peptides were synthesized, one derived from a CARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other one from a CRAC site from the domain between the acylated lysines (residues 639-644) (PEP 2), to study their role in the interaction of HlyA with membranes. The interaction of peptides with membranes of different lipid compositions (pure POPC and POPC/Cho of 4:1 and 2:1 molar ratios) was analyzed by surface plasmon resonance and molecular dynamics simulations. Results demonstrate that both peptides interact preferentially with Cho-containing membranes, although PEP 2 presents a lower KD than PEP 1. Molecular dynamics simulation results indicate that the insertion and interaction of PEP 2 with Cho-containing membranes are more prominent than those caused by PEP 1. The hemolytic activity of HlyA in the presence of peptides indicates that PEP 2 was the only one that inhibits HlyA activity, interfering in the binding between the toxin and cholesterol.

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Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by "reader" proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.

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Shrimp antilipopolysaccharide factors (ALFs) form a multifunctional and diverse family of antimicrobial host defense peptides (AMPs) composed of seven members (groups A to G), which differ in terms of their primary structure and biochemical properties. They are amphipathic peptides with two conserved cysteine residues stabilizing a central ß-hairpin that is understood to be the core region for their biological activities. In this study, we synthetized three linear (cysteine-free) peptides based on the amino acid sequence of the central ß-hairpin of the newly identified shrimp (Litopenaeus vannamei) ALFs from groups E to G. Unlike whole mature ALFs, the ALF-derived peptides exhibited an α-helix secondary structure. In vitro assays revealed that the synthetic peptides display a broad spectrum of activity against both Gram-positive and Gram-negative bacteria and fungi but not against the protozoan parasites Trypanosoma cruzi and Leishmania (L.) infantum. Remarkably, they displayed synergistic effects and showed the ability to permeabilize bacterial membranes, a mechanism of action of classical AMPs. Having shown low cytotoxicity to THP-1 human cells and being active against clinical multiresistant bacterial isolates, these nature-inspired peptides represent an interesting class of bioactive molecules with biotechnological potential for the development of novel therapeutics in medical sciences.

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Infectious diseases account for nine percent of annual human deaths, and the widespread emergence of antimicrobial resistances threatens to significantly increase this number in the coming decades. The prospect of antimicrobial peptides (AMPs) derived from venomous animals presents an interesting alternative for developing novel active pharmaceutical ingredients (APIs). Small, cationic and amphiphilic peptides were predicted from the venom gland transcriptome of Pamphobeteus verdolaga using a custom database of the arthropod's AMPs. Ninety-four candidates were chemically synthesized and screened against ATCC® strains of Escherichia coli and Staphylococcus aureus. Among them, one AMP, named PvAMP66, showed broad-spectrum antimicrobial properties with selectivity towards Gram-negative bacteria. It also exhibited activity against Pseudomonas aeruginosa, as well as both an ATCC® and a clinically isolated multidrug-resistant (MDR) strain of K. pneumoniae. The scanning electron microscopy analysis revealed that PvAMP66 induced morphological changes of the MDR K. pneumoniae strain suggesting a potential "carpet model" mechanism of action. The isobologram analysis showed an additive interaction between PvAMP66 and gentamicin in inhibiting the growth of MDR K. pneumoniae, leading to a ten-fold reduction in gentamicin's effective concentration. A cytotoxicity against erythrocytes or peripheral blood mononuclear cells was observed at concentrations three to thirteen-fold higher than those exhibited against the evaluated bacterial strains. This evidence suggests that PvAMP66 can serve as a template for the development of AMPs with enhanced activity and deserves further pre-clinical studies as an API in combination therapy.

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Cell-penetrating peptides rich in arginine are good candidates to be considered as antibacterial compounds, since peptides have a lower chance of generating resistance than commonly used antibiotics. Model homopeptides are a useful tool in the study of activity and its correlation with a secondary structure, constituting an initial step in the construction of functional heteropeptides. In this report, the 11-residue arginine homopeptide (R11) was used to determine its antimicrobial activity against Staphylococcus aureus and Escherichia coli and the effect on the secondary structure, caused by the substitution of the arginine residue by the amino acids Ala, Pro, Leu and Trp, using the scanning technique. As a result, most of the substitutions improved the antibacterial activity, and nine peptides were significantly more active than R11 against the two tested bacteria. The cell-penetrating characteristic of the peptides was verified by SYTOX green assay, with no disruption to the bacterial membranes. Regarding the secondary structure in four different media-PBS, TFE, E. coli membrane extracts and DMPG vesicles-the polyproline II structure, the one of the parent R11, was not altered by unique substitutions, although the secondary structure of the peptides was best defined in E. coli membrane extract. This work aimed to shed light on the behavior of the interaction model of penetrating peptides and bacterial membranes to enhance the development of functional heteropeptides.

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The discovery and improvements of antimicrobial peptides (AMPs) have become an alternative to conventional antibiotics. They are usually small and heat-stable peptides, exhibiting inhibitory activity against Gram-negative and Gram-positive bacteria. In this way, studies on broad-spectrum AMPs found in amphibians with the remarkable capability to regenerate a wide array of tissues are of particular interest in the search for new strategies to treat multidrug-resistant bacterial strains. In this work, the use of bioinformatic approaches such as sequence alignment with Fasta36 and prediction of antimicrobial activity allowed the identification of the Ramosin peptide from the de novo assembled transcriptome of the plethodontid salamander Bolitoglossa ramosi obtained from post-amputation of the upper limb tissue, heart, and intestine samples. BLAST analysis revealed that the Ramosin peptide sequence is unique in Bolitoglossa ramosi. The peptide was chemically synthesized, and physicochemical properties were characterized. Furthermore, the in vitro antimicrobial activity against relevant Gram-positive and Gram-negative human pathogenic bacteria was demonstrated. Finally, no effect against eukaryotic cells or human red blood cells was evidenced. This is the first antibacterial peptide identified from a Colombian endemic salamander with interesting antimicrobial properties and no hemolytic activity.