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Listeria monocytogenes is a human opportunistic foodborne pathogen that produces life-threatening infections with a high mortality rate. The control of Listeria in the food production environment and effective clinical management of human listeriosis are challenging due to the emergence of antibiotic resistance. Hence we evaluate the in vitro anti-Listeria activity of two synthetic cruzioseptins reproducing their natural sequences CZS-9, and CZS-12, and one engineered sequence based on CZS-1, named [K4K15]CZS-1. The assessment of the in vitro potential of cruzioseptins, highlighted the promising antibacterial effect of [K4K15]CZS-1 in very low concentrations (0.91 μM) and its thermal stability at high-temperature conditions, is compatible with the food industry. Microscopic and metabolomic analyses suggest cruzioseptin induces anti-Listeria bioactivity through membrane disruption and changes in the intracellular metabolome. We also report that [K4K15]CZS-1 is not resistant to peptidases/proteases emphasizing a key advantage for their use as a food preservative. However, there is a need for further structural and functional optimisations for the potential clinical application as an antibiotic. In conclusion, [K4K15]CZS-1 stand out as membrane-active peptides with the ability to induce shifts in the bacteria metabolome and inspire the development of strategies for the prevention of L. monocytogenes emergence and dissemination., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Chagas disease, leishmaniasis, and malaria are major parasitic diseases disproportionately affecting the underprivileged population in developing nations. Finding new, alternative anti-parasitic compounds to treat these diseases is crucial because of the limited number of options currently available, the side effects they cause, the need for long treatment courses, and the emergence of drug-resistant parasites. Anti-microbial peptides (AMPs) derived from amphibian skin secretions are small bioactive molecules capable of lysing the cell membrane of pathogens while having low toxicity against human cells. Here, we report the anti-parasitic activity of five AMPs derived from skin secretions of three Ecuadorian frogs: cruzioseptin-1, cruzioseptin-4 (CZS-4), and cruzioseptin-16 from Cruziohyla calcarifer ; dermaseptin-SP2 from Agalychnis spurrelli ; and pictuseptin-1 from Boana picturata . These five AMPs were chemically synthesized. Initially, the hemolytic activity of CZS-4 and its minimal inhibitory concentration against Escherichia coli , Staphylococcus aureus , and Candida albicans were determined. Subsequently, the cytotoxicity of the synthetic AMPs against mammalian cells and their anti-parasitic activity against Leishmania mexicana promastigotes, erythrocytic stages of Plasmodium falciparum and mammalian stages of Trypanosoma cruzi were evaluated in vitro . The five AMPs displayed activity against the pathogens studied, with different levels of cytotoxicity against mammalian cells. In silico molecular docking analysis suggests this bioactivity may occur via pore formation in the plasma membrane, resulting in microbial lysis. CZS-4 displayed anti-bacterial, anti-fungal, and anti-parasitic activities with low cytotoxicity against mammalian cells. Further studies about this promising AMP are required to gain a better understanding of its activity.IMPORTANCEChagas disease, malaria, and leishmaniasis are major tropical diseases that cause extensive morbidity and mortality, for which available treatment options are unsatisfactory because of limited efficacy and side effects. Frog skin secretions contain molecules with anti-microbial properties known as anti-microbial peptides. We synthesized five peptides derived from the skin secretions of different species of tropical frogs and tested them against cultures of the causative agents of these three diseases, parasites known as Trypanosoma cruzi , Plasmodium falciparum , and Leishmania mexicana . All the different synthetic peptides studied showed activity against one of more of the parasites. Peptide cruzioseptin-4 is of special interest since it displayed intense activity against parasites while being innocuous against cultured mammalian cells, which indicates it does not simply hold general toxic properties; rather, its activity is specific against the parasites., Competing Interests: The authors declare no conflict of interest.

A computational study of the peptides Cruzioseptin-4 and Pictuseptin-1, identified in Cruziohyla calcarifer and Boana picturata respectively, has been carried out. The studies on Cruzioseptin-4 show that it is a cationic peptide with a chain of 23 amino acids that possess 52.17% of hydrophobic amino acids and a charge of + 1.2 at pH 7. Similarly, Pictuseptin-1 is a 22 amino acids peptide with a charge of + 3 at pH 7 and 45.45% of hydrophobic amino acids. Furthermore, the predominant secondary structure for both peptides is alpha-helical. The physicochemical properties were predicted using PepCalc and Bio-Synthesis; secondary structures using Jpred4 and PredictProtein; while molecular docking was performed using Autodock Vina. Geometry optimization of the peptides was done using the ONIOM hybrid method with the HF/6-31G basis set implemented in the Gaussian 09 program. Finally, the molecular docking study indicates that the viable mechanism of action for both peptides is through a targeted attack on the cell membrane of pathogens via electrostatic interactions with different membrane components, leading to cell lysis., (© 2024. The Author(s).)

Antimicrobial peptides have been developed based on plant-derived molecular scaffolds for the treatment of infectious diseases. Chenopodin is an abundant seed storage protein in quinoa, an Andean plant with high nutritional and therapeutic properties. Here, we used computer- and physicochemical-based strategies and designed four peptides derived from the primary structure of Chenopodin. Two peptides reproduce natural fragments of 14 amino acids from Chenopodin, named Chen1 and Chen2, and two engineered peptides of the same length were designed based on the Chen1 sequence. The two amino acids of Chen1 containing amide side chains were replaced by arginine (ChenR) or tryptophan (ChenW) to generate engineered cationic and hydrophobic peptides. The evaluation of these 14-mer peptides on Staphylococcus aureus and Escherichia coli showed that Chen1 does not have antibacterial activity up to 512 µM against these strains, while other peptides exhibited antibacterial effects at lower concentrations. The chemical substitutions of glutamine and asparagine by amino acids with cationic or aromatic side chains significantly favoured their antibacterial effects. These peptides did not show significant hemolytic activity. The fluorescence microscopy analysis highlighted the membranolytic nature of Chenopodin-derived peptides. Using molecular dynamic simulations, we found that a pore is formed when multiple peptides are assembled in the membrane. Whereas, some of them form secondary structures when interacting with the membrane, allowing water translocations during the simulations. Finally, Chen2 and ChenR significantly reduced SARS-CoV-2 infection. These findings demonstrate that Chenopodin is a highly useful template for the design, engineering, and manufacturing of non-toxic, antibacterial, and antiviral peptides.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Peptide engineering has gained attraction as a source of new cationicity-enhanced analogues with high potential for the design of next-generation antibiotics. In this context, cruzioseptin-1 (CZS-1), a peptide identified from Cruziohyla calcarifer, is recognized for its antimicrobial potency. However, this amidated-peptide is moderately hemolytic. In order to reduce toxicity and increase antimicrobial potency, 3 peptide analogues based on cruzioseptin-1 were designed and evaluated. [K4K15]CZS-1, an analogue with increased cationicity and reduced hydrophobicity, showed antibacterial, antifungal and antiproliferative properties. In addition, [K4K15]CZS-1 is less hemolytic than CZS-1. The in silico and scanning electron microscopy analysis reveal that [K4K15]CZS-1 induces a membranolytic effect on bacteria. Overall, these results confirm the potential of CZS-1 as source of inspiration for design new selective antimicrobial analogues useful for development of new therapeutic agents., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The Imbabura treefrog (Boana picturata) is an underexplored source of bioactive peptides. The combination of molecular cloning and mass spectrometry allowed us to identify three new peptide families, named "Picturins" (PTR), "Pictuseptins" (PTS), and "Boanins" (BNS). PTR is composed of three 25-mer peptides, characterized by the N-terminal sequence: GVFKDALKQ and the C-terminal sequence: AANALKPK. The sequences of PTR-1, -2 and - 3 are highly conserved only showing two divergent sites: (L/F) in position 10 and (K/Q) in position 17. PTS gathers six peptides. PTS -1, -2 and - 4 have 22 amino acid residues in length, while PTS -3, -5 and - 6 are composed of 26 residues. Whereas BNS are four 28-37 mer peptides, showing two conserved regions: the N-terminal sequence FLGAL and the C-terminal sequence KALNP. PTR-1 to 3 and PTS -1 to -3 were chemically synthetized and their antimicrobial and haemolytic activity was assessed. PTR displayed moderate activity against Escherichia coli (MIC 24.80 to 48.95 μM), while PTS showed a broad antimicrobial and antifungal effect. PTS-1 was the most active peptide against E. coli (6.8 μM) followed by PTS-3 (11.7 μM) and PTS-2 (14.24 μM). These peptides also showed low haemolytic activity, pointing to a favorable selectivity. Overall, new unique non-hemolytic and cationic peptide sequences were characterized that could be valuable for the next-generation of anti-infective drugs. Future functional studies should explore the pharmacological potential of Boanins to include them as antimicrobial scaffolds. BIOLOGICAL SIGNIFICANCE: Nature-inspired solutions have shown their importance mainly for the development of the pharmaceutical industry. Frog skin peptides are excellent examples of the biomedical potential of naturally evolved molecules for specific targets, including multi-resistant bacteria. The characterization of new chemical entities from poorly studied skin secretions of Ecuadorian biodiversity, such as B. picturata, represents an unprecedented opportunity to identify candidates to tackle global concerns, for instance, antibiotic resistance., (Copyright © 2022. Published by Elsevier B.V.)

Laboratory-based practical classes are an essential component in teaching molecular biology for undergraduate students. Universidad Regional Amazonica Ikiam is a higher education institution located in the Ecuadorian Amazon rainforest, a high biodiversity place, including amphibians. Based on this, we have established a practical molecular biology program with eight sessions that contextualize the biodiverse surroundings of the University. This program stimulates synchronization of information between theory and practice and improves research skills. During these sessions, students are motivated to identify and characterize antimicrobial peptides from Ecuadorian frog skin secretions, using molecular biology techniques and biochemistry and microbiology knowledge. This practical course was held twice with a total of 56 students from the fifth semester of the biotechnology engineering. The evaluation of the practical program was carried out through a questionnaire applied to students using the Likert scale. Overall, this form of teaching had high receptivity and presented benefits for student learning. Interestingly, 80% of respondents strongly agreed that this course provided tools and knowledge for the development of their undergraduate dissertation. Therefore, practical courses tailored to the student's context can stimulate student learning and interest. Additionally, this experimental methodology is interdisciplinary and can be applied to other research fields and subjects., (© 2022 International Union of Biochemistry and Molecular Biology.)

Amphibians are in peril, given the ongoing sixth mass extinction of wildlife. Thus, Conservation Breeding Programs (CBPs) are attempting to breed some species under laboratory conditions. The incorporation of assisted reproduction technologies (ARTs), such as hormonal stimulation, sperm collection and cryopreservation, and in vitro fertilization is contributing to successful CBPs. The objective of this study was to apply ARTs in sexually mature individuals of an undescribed species of Atelopus (spumarius complex) (harlequin frog). Our procedure involves hormonal induction of gametogenesis in this species. We were able to induce gamete release through administration of human chorionic gonadotropin (hCG) in males, and in females this has been achieved through the sequential administration of hCG (priming doses), and combinations of hCG with gonadotropin releasing hormone analogue, GnRHa (ovulary dose). We standardized sperm cryopreservation by performing toxicity tests of cryoprotectants, fast/slow freezing and thawing, as well as supplementation of non-penetrating cryoprotectants (sugars). Next, we performed in vitro fertilization, evaluated the fertilization capacity of the cryopreserved sperm, and describe external features of fresh and cryopreserved sperm. We found that 10 IU/g hCG induced the release of the highest sperm concentrations between 3 and 5 h post-injection, while 2.5 IU/g hCG induced the release of eggs in most treated females. Under cryopreservation conditions, the highest recovery of forward progressive motility or FPM was 26.3 ± 3.5%, which was obtained in cryosuspensions prepared with the 5% DMF and 2.5% sucrose. Cryopreserved sperm showed narrower mitochondrial vesicles after thawing, while in frozen samples without cryodiluent showed 31% of spermatozoa lost their tails. In most cases, our attempts of in vitro fertilization were successful. However, only ∼10% of embryos were viable. Overall, our study demonstrates that the development of ARTs in individuals of Atelopus sp. (spumarius complex) bred in laboratory can be successful, which result in viable offspring through in vitro fertilization. Our study provides a baseline for assisted breeding protocols applicable to other harlequin frogs of the genus Atelopus., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Peptides have positively impacted the pharmaceutical industry as drugs, biomarkers, or diagnostic tools of high therapeutic value. However, only a handful have progressed to the market. Toxicity is one of the main obstacles to translating peptides into clinics. Hemolysis or hemotoxicity, the principal source of toxicity, is a natural or disease-induced event leading to the death of vital red blood cells. Initial screenings for toxicity have been widely evaluated using erythrocytes as the gold standard. More recently, many online databases filled with peptide sequences and their biological meta-data have paved the way toward hemolysis prediction using user-friendly, fast-access machine learning-driven programs. This review details the growing contributions of in silico approaches developed in the last decade for the large-scale prediction of erythrocyte lysis induced by peptides. After an overview of the pharmaceutical landscape of peptide therapeutics, we highlighted the relevance of early hemolysis studies in drug development. We emphasized the computational models and algorithms used to this end in light of historical and recent findings in this promising field. We benchmarked seven predictors using peptides from different data sets, having 7-35 amino acids in length. According to our predictions, the models have scored an accuracy over 50.42% and a minimal Matthew's correlation coefficient over 0.11. The maximum values for these statistical parameters achieved 100.0% and 1.00, respectively. Finally, strategies for optimizing peptide selectivity were described, as well as prospects for future investigations. The development of in silico predictive approaches to peptide toxicity has just started, but their important contributions clearly demonstrate their potential for peptide science and computer-aided drug design. Methodology refinement and increasing use will motivate the timely and accurate in silico identification of selective, non-toxic peptide therapeutics.