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Methyl Salicylate (MS), the principal constituent of Wintergreen oil (WO) was obtained from acetyl salicylic acid (ASA) by sequential transesterification-esterification reaction promoted by NbCl5for the first time. The reagents were added simultaneously, and the reaction process involved the transesterification and esterification reactions, which were accompanied by thin layer chromatography and gas chromatography. The conversion rate via GC was 100%, and the MS yield was 94%. A cytotoxicity of 50% and 64% for cultured S. aureus and metastatic melanoma cells, respectively, was observed for a concentration of 0.6 mg/mL, whereas no cytotoxicity for non-tumor cells was observed for this concentration, and it is considered to be the optimum concentration.

Ncediwe Tsolekile,1– 3 Sundararajan Parani,2,3 Erenilda Ferreira de Macedo,4 Thabang Calvin Lebepe,2,3 Rodney Maluleke,2,3 Vuyelwa Ncapayi,2,3 Mangaka Clara Matoetoe,1 Sandile Phinda Songca,5 Katia Conceição,6 Dayane Batista Tada,4 Oluwatobi Samuel Oluwafemi2,3 1Department of Chemistry, Cape Peninsula University of Technology, Cape Town, 2000, South Africa; 2Department of Chemical Sciences, University of Johannesburg, Johannesburg, 2028, South Africa; 3Centre for Nanomaterials Science Research, University of Johannesburg, Johannesburg, South Africa; 4Laboratory of Nanomaterials and Nanotoxicology, Institute of Science and Technology, Federal University of São Paulo, São Paulo, Brazil; 5Department of Chemistry, University of KwaZulu-Natal, Durban, 4000, South Africa; 6Laboratory of Peptide Biochemistry, Institute of Science and Technology, Federal University of São Paulo, São Paulo, BrazilCorrespondence: Oluwatobi Samuel Oluwafemi, Department of Chemical Sciences, University of Johannesburg, P. O. Box 17011, Doornfontein, Johannesburg, 2028, South Africa, Tel +27 011 559 9060, Email oluwafemi.oluwatobi@gmail.com Dayane Batista Tada, Laboratório de Nanomateriais e Nanotoxicologia, Universidade Federal de São Paulo, Talim, 330 - Vila Nair, São José dos Campos, São Paulo, 12231-280, Brazil, Email day.tada@gmail.comBackground: Photodynamic therapy (PDT) is a non-invasive treatment modality that destroys abnormally growing cells or microorganisms. Porphyrins are used as photosensitizers in PDT; however, their clinical application has been limited by their poor water solubility, resulting in aggregation and low quantum yields of reactive oxygen species (ROS).Methods: To overcome these limitations and improve PDT efficacy, we herein report the conjugation of ZnCuInS/ZnS (ZCIS/ZnS) quantum dots (QDs) to 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). The optimal conditions for QDs porphyrin conjugation formation were systematically evaluated.Discussion: This study further assessed the PDT efficacy and antibacterial potency of the synthesized ZCIS/ZnS-mTHPP conjugates. The PDT efficacy of the QDs, mTHPP, and conjugate was evaluated against the murine metastatic melanoma (B16 F10 Nex2) cell line. This was performed with and without LED irradiation.Results: The conjugate exhibited the highest reduction in cell viability following LED irradiation (72%) compared to the bare QDs (19%) and mTHPP (1%). Antimicrobial studies conducted on E. coli showed that the conjugation exhibits a higher antibacterial effect than the bare QDs, even without light.Conclusion: The results suggest that conjugate is a promising class of materials for anti-cancer and antimicrobial PDT.Keywords: Zn-Cu-In-S/ZnS QDs, 5,10,15,20-tetrakis(hydroxyphenyl)porphyrin, mTHPP, photodynamic therapy, murine metastatic melanoma (B16F10-Nex2), antibacterial activity

Available treatments for invasive fungal infections have limitations, including toxicity and the emergence of resistant strains. Therefore, there is an urgent need for alternative solutions. Because of their unique mode of action and high selectivity, plant defensins (PDs) are worthy therapeutic candidates. Chemical synthesis remains a preferred method for the production of many peptide-based therapeutics. Given the relatively long sequence of PDs, as well as their complicated posttranslational modifications, the synthetic route can be considered challenging. Here, we describe a total synthesis of PvD1, the defensin from the common bean Phaseolus vulgaris. Analytical, structural, and functional characterization revealed that both natural and synthetic peptides fold into a canonical CSαβ motif stabilized by conserved disulfide bonds. Moreover, synthetic PvD1 retained the biological activity against four different Candida species and showed no toxicity in vivo. Adding the high resistance of synthetic PvD1 to proteolytic degradation, we claim that conditions are now met to consider PDs druggable biologicals.

Background: Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.

Natterin is an aerolysin-like pore-forming toxin responsible for the toxic effects of the venom of the medically significant fish Thalassophryne nattereri. Using a combination of pharmacologic and genetic loss-of-function approaches we conduct a systematic investigation of the regulatory mechanisms that control Natterin-induced neutrophilic inflammation in the peritonitis model. Our data confirmed the capacity of Natterin to induce a strong and sustained neutrophilic inflammation leading to systemic inflammatory lung infiltration and revealed overlapping regulatory paths in its control. We found that Natterin induced the extracellular release of mature IL-1β and the sustained production of IL-33 by bronchial epithelial cells. We confirmed the dependence of both ST2/IL-33 and IL-17A/IL-17RA signaling on the local and systemic neutrophils migration, as well as the crucial role of IL-1α, caspase-1 and caspase-11 for neutrophilic inflammation. The inflammation triggered by Natterin was a gasdermin-D-dependent inflammasome process, despite the cells did not die by pyroptosis. Finally, neutrophilic inflammation was mediated by non-canonical NLRP6 and NLRC4 adaptors through ASC interaction, independent of NLRP3. Our data highlight that the inflammatory process dependent on non-canonical inflammasome activation can be a target for pharmacological intervention in accidents by T. nattereri, which does not have adequate specific therapy.

SummaryEstablishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus.

Available treatments for invasive fungal infections have limitations, including toxicity and the emergence of resistant strains. Therefore, there is an urgent need for alternative solutions. Because of their unique mode of action and high selectivity, plant defensins (PDs) are worthy therapeutic candidates. Chemical synthesis remains a preferred method for the production of many peptide-based therapeutics. Given the relatively long sequence of PDs, as well as their complicated posttranslational modifications, the synthetic route can be considered challenging. Here, we describe a total synthesis of

Made available in DSpace on 2021-06-25T10:13:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In order to understand events and mechanisms present in the pathophysiology of tilapia's chronic inflammation and based on the immunomodulatory activity attributed to cyclophosphamide which is widely used to suppress immune responses in human medicine, the present study investigated the effects of cyclophosphamide (CYP) treatment on the modulation of foreign body inflammatory reaction in Nile tilapia (Oreochromis niloticus) with round glass coverslip implanted in the subcutaneous tissue (9 mm of diameter). Forty tilapia (151 ± 10,2 g) were randomly distributed in 5 aquariums (n = 8) with a capacity of 250 L of water each, to compose two treatments (sampled 3 and 6 days post-implantation): implanted/untreated (control) and implanted/treated with 200 mg of CYP kg−1 of b.w., through i.p. route. A fifth group (n = 8) was sampled without any stimulus (naive) to obtain reference values. CYP-treated tilapia showed decrease in macrophage accumulation, giant cell formation and Langhans cells on the glass coverslip when compared to control fish. The treatment with CYP resulted in decrease of leukocyte and thrombocyte counts. Decrease in alpha-2-macroglobulin, ceruloplasmin, albumin and transferrin levels, as well as increase in haptoglobin, complement C3 and apolipoprotein A1 were observed in tilapias during foreign body inflammation. Blood levels of complement C3, alpha-2-macroglobulin, ceruloplasmin and transferrin were modulated by treatment with CYP. Therefore, the treatment with 200 mg of CYP kg−1 of b.w. in tilapia resulted in an anti-inflammatory effect by suppressing the dynamics between leukocytes in the bloodstream and macrophage accumulation with giant cell formation in the inflamed focus, as well as by modulating APPs during foreign body reaction. Department of Pharmacology Institute of Biomedical Science University of São Paulo (USP) Department of Preventive Veterinary Medicine São Paulo State University (UNESP) Veterinary College Federal University of Rondonia (UNIR) Department of Graduate Program in Health Sciences Federal University of Roraima (UFRR) Laboratório de Bioquímica de Peptídeos UNIFESP Laboratory of Animal Pharmacology and Toxicology Brasil University Department of Preventive Veterinary Medicine São Paulo State University (UNESP) FAPESP: 2012/12256-7 FAPESP: 2013/11675-9 CNPq: 432830/2016-2 CNPq: 477816/2013-4

© King Abdulaziz City for Science and Technology 2020, The emergence of bacterial resistance due to the indiscriminate use of antibiotics warrants the need for developing new bioactive agents. In this context, antimicrobial peptides are highly useful for managing resistant microbial strains. In this study, we report the isolation and characterization of peptides obtained from the venom of the toadfish Thalassophryne nattereri. These peptides were active against Gram-positive and Gram-negative bacteria and fungi. The primary amino acid sequences showed similarity to Cocaine and Amphetamine Regulated Transcript peptides, and two peptide analogs—Tn CRT2 and Tn CRT3—were designed using the AMPA algorithm based on these sequences. The analogs were subjected to physicochemical analysis and antimicrobial screening and were biologically active at concentrations ranging from 2.1 to 13 μM. Zeta potential analysis showed that the peptide analogs increased the positive charge on the cell surface of Grampositive and Gram-negative bacteria. The toxicity of Tn CRT2 and Tn CRT3 were analyzed in vitro using a hemolytic assay and tetrazolium salt reduction in fibroblasts and was found to be significant only at high concentrations (up to 40 μM). These results suggest that this methodological approach is appropriate to design novel antimicrobial peptides to fight bacterial infections and represents a new and promising discovery in fish venom., This research was supported by FAPESP grant No. 2017/00032-0.

Recently, fish byproducts have been appraised as a source of structurally diverse bioactive compounds. In this context, fish mucus has revealed a myriad of pharmacological activities. This study describes a new bioactive molecule release system composed of protein nanoparticles from pacama ( Lophiosilurus alexandri ) mucus, an endemic fish to the Sao Francisco River (Brazil). The process of obtaining and applying these nanoparticles, composed of proteins present in the pacama mucus, as biocompatible carriers of bioactive molecules is also addressed herein. Therefore, the external mucus of the fish was collected, lyophilized and hydrated in deionized water containing the template molecule. After pH adjustment to 7.5, followed by sonication and centrifugation, the formed nanoparticles were collected in the supernatant. Additionally, the mucus and the particles were characterized, and pharmacological effects were evaluated regarding their antifungal, antibacterial, anticancer, anti or pro-inflammatory and antinociceptive proprieties. NP-Mucus did not exhibit antibacterial activity against Pseudomonas aeruginosa or Staphylococcus aureus . However, it showed potential effects against Candida albicans with a minimum inhibitory concentration (MIC) of 1 µg/mL, and significant pro-inflammatory role, reflected by cellular recruitment activity and healing effects. Therefore, future studies are now needed in order to identify specific compounds in NP-mucus responsible for the observed effects in order to provide new pharmacological and therapeutic strategies.

Despite the potential antimicrobial activity of metallic nanoparticles, the increasing concerns about nanosafety have been holding back the use of these materials in therapeutics and biomedical devices. In the last years, several studies called attention to metallic nanoparticles toxicity. In the most part of in vitro studies performed with mammalian cells, metallic NPs reduced cell viability and induced genotoxicity and inflammatory responses. Bimetallic NPs have attracted great attention because they present distinct and even more advanced characteristics when compared to nanoparticles formed by a single metal. Recently, bimetallic NPs have emerged as an alternative to improve the antimicrobial activity of metallic nanoparticles, aiming at the broadening of the action spectrum and the reduction of the toxicity. However, the biocompatibility of bimetallic nanoparticles has been demonstrated only by in vitro studies. In the present work, the toxicity of AuPt nanoparticles was addressed both in vitro and in vivo. In addition, the antimicrobial activity of AuPt bimetallic nanoparticles has been evaluated in comparison with Au and Ag nanoparticles. The nanoparticles were characterized by ultraviolet-visible spectroscopy, dynamic light scattering, transmission electron microscopy, inductively coupled plasma optical emission spectroscopy, and X-ray diffraction. The antimicrobial activity was studied against Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. The toxicity of nanoparticles was evaluated in vitro by analyzing their toxicity against human fibroblast cells (HS68 cell line) and in vivo by embryonic toxicity test in zebrafish (Danio rerio). The results confirmed the intrinsic antimicrobial activity of the three types of nanoparticles but different toxicity. Bimetallic nanoparticles showed enhanced antimicrobial activity in comparison with Au nanoparticles but lower antimicrobial activity compared with Ag nanoparticles. However, AuPt nanoparticles showed great advantage over Ag nanoparticles due to the absence of cytotoxicity and lower toxicity in vivo.

Made available in DSpace on 2019-10-06T16:39:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-15 Nanoparticles to delivery drugs and immunomodulatory substances in fish have been widely studied by several research groups. In general, these nanoparticles are polymers, ceramic, fullerenes and metal derivates which can be toxic to fish and other aquatic organisms. We reported for the first time a nanostructured system composed of proteins obtained from pacamã fish (Lophiosilurus alexandri) mucus. We identified and characterized the formation of nanoparticles with 210.8 nm and zeta potential of −13.4 (mV). These characteristics suggest a possible application of this material as a biocompatible coating. These nanoparticles are biodegradable, metabolizable and can be easily manipulated; as well as they can easily undergo surface modification to transport biomolecules, thus exhibiting greater versatility. Our study suggests that these nanoparticles from fish mucus could be used to delivery drugs and to modulate the immune system, since they demonstrated to be biocompatible coating. Universidade Brasil Campus Itaquera Faculdade de Medicina São Leopoldo Mandic Departamento de Morfologia Universidade Federal de Minas Gerais Universidade Estadual Paulista Instituto de Ciência e Tecnologia de Sorocaba Laboratório de Bioquímica de Peptídeos Universidade Federal de São Paulo Laboratório de Farmacologia e Toxicologia Animal Descalvado/SP Universidade Brasil Departamento de Medicina Veterinária Preventiva Universidade Estadual Paulista- Unesp Jaboticabal Departamento de Zootecnia Universidade Federal de Minas Gerais Escola de Veterinária Laboratório de Aquacultura Universidade Estadual Paulista Instituto de Ciência e Tecnologia de Sorocaba Laboratório de Farmacologia e Toxicologia Animal Descalvado/SP Universidade Brasil Departamento de Medicina Veterinária Preventiva Universidade Estadual Paulista- Unesp Jaboticabal

Made available in DSpace on 2019-10-06T17:05:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Acute-phase protein (APPs) serum levels have been studied in many human diseases, and their components contribute to host defense during the evolution of infectious diseases by acting as part of the innate immune system. Based on the importance of establishing new experimental models, the present investigation evaluated the modulation of APPs following inflammatory stimulus by the inoculation of Aeromonas hydrophila in tilapias. Fish were sampled 6 and 24 hours post-infection. Tilapias presented increase of positive APPs such as ceruloplasmin, haptoglobin, alpha-2-macroglobulin and complement C3, as well as decrease of negative APPs such as albumin and transferrin. The protein response of tilapias during the course of bacterial infection showed correlation with the kinetics of cellular accumulation in the inflamed focus with significant increase of granulocytes, thrombocytes, lymphocytes and macrophages. However, granulocytes were the predominant cells, associated with increment in the reactive oxygen species (ROS) production. Showing responses similar to those observed in humans, the modulation of APPs and the kinetics of cellular accumulation in the exudate demonstrate the feasibility of this alternative experimental model for advances and studies to understand changes in pathophysiological mechanisms of acute inflammatory reaction due to bacterial infection. Department of Preventive Veterinary Medicine São Paulo State University (UNESP) Department of Pharmacology and Morphology ICB-UFMG Immunoregulation Unit of the Special Laboratory of Applied Toxicology (CEPID/FAPESP) Butantan Institute Laboratório de Bioquímica de Peptídeos UNIFESP Veterinary College Federal University of Rondonia Laboratory of Animal Pharmacology and Toxicology Brasil University Department of Preventive Veterinary Medicine São Paulo State University (UNESP)

© 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved., Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins—KTP-NH2, KTP–NH2–DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg—were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates—Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S—obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application., This research was supported by FAPESP (Grant No. 2017/00032-0). This article is also part of the Fungal Adaptation to Hostile Challenges special issue for the third International Symposium on Fungal Stress (ISFUS), which is supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant No. 2018/20571-6) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Grant No. 88881.289327/2018-01).

Conjugation of photosensitizers (PS) with nanoparticles has been largely used as a strategy to stabilize PS in the biological medium resulting in photosensitizing nanoparticles of enhanced photoactivity. Herein, (Meso-5, 10, 15, 20-tetrakis (3-hydroxyphenyl) phorphyryn (mTHPP) was conjugated with diamond nanoparticles (ND) by covalent bond. Nanoconjugate ND-mTHPP showed suitable stability in aqueous suspension with 58 nm of hydrodynamic diameter and Zeta potential of −23 mV. The antibacterial activity of ND-mTHPP was evaluated against Escherichia coli for different incubation times (0–24 h). The optimal activity was observed after 2 h of incubation and irradiation (660 nm, 51 J/cm2) performed right after the addition of ND-mTHPP (100 μg/mL) to the bacterial suspension. The inhibitory activity was 56% whereas ampicillin at the same conditions provided only 14% of bacterial growth inhibition. SEM images showed agglomerate of ND-mTHPP adsorbed on the bacterial cell wall, suggesting that the antimicrobial activity of ND-mTHPP was afforded by inducing membrane damage. Cytotoxicity against murine embryonic fibroblast cells (MEF) was also evaluated and ND-mTHPP was shown to be noncytotoxic since viability of cells cultured for 24 h in the presence of the nanoconjugate (100 μg/mL) was 78%. Considering the enhanced antibacterial activity and the absence of cytotoxic effect, it is possible to consider the ND-mTHPP nanoconjugate as promising platform for application in antimicrobial photodynamic therapy (aPDT).

Gender related variation in the molecular composition of venoms and secretions have been described for some animal species, and there are some evidences that the difference in the toxin (s) profile among males and females may be related to different physiopathological effects caused by the envenomation by either gender. In order to investigate whether this same phenomenon occurs to the toadfish Thalassophryne maculosa, we have compared some biological and biochemical properties of female and male venoms. Twenty females and males were collected in deep waters of the La Restinga lagoon (Venezuela) and, after protein concentration assessed, the induction of toxic activities in mice and the biochemical properties were analyzed. Protein content is higher in males than in females, which may be associated to a higher size and weight of the male body. In vivo studies showed that mice injected with male venoms presented higher nociception when compared to those injected with female venoms, and both venoms induced migration of macrophages into the paw of mice. On the other hand, mice injected with female venoms had more paw edema and extravasation of Evans blue in peritoneal cavity than mice injected with male venoms. We observed that the female venoms had more capacity for necrosis induction when compared with male venoms. The female samples present a higher proteolytic activity then the male venom when gelatin, casein and FRETs were used as substrates. Evaluation of the venoms of females and males by SDS-PAGE and chromatographic profile showed that, at least three components (present in two peaks) are only present in males. Although the severity of the lesion, characterized by necrosis development, is related with the poisoning by female specimens, the presence of exclusive toxins in the male venoms could be associated with the largest capacity of nociception induction by this sample.

Fish, like other aquatic organisms, are a potential source of structurally diverse bioactive compounds. Literature data analysis confirms that fish may be used not only for nutritional purposes but also as sources of unique peptides with a broad spectrum of biological activities. Since peptides are mostly involved in cell signaling, they are capable of playing roles as agonists, antagonists, modulators, mediators, hormones, effectors, cofactors, activators, stimulators, and so on. Studies about the pharmacological effects of fish peptides have revealed antihypertensive, immunomodulatory, antioxidant, antitumor and antimicrobial activities. The development of new peptide therapies is still a crucial task for several pharmaceutical companies in the design of new drugs; several approaches are now emerging for the development of new peptides against pathologies. In this scenario, fish bioactive peptides emerge as a promising group of compounds with potential application to human health, diagnosis and therapy. This work analyzed systematically the currently existing data about fish bioactive peptides from the year 1986 to till date 2019. The peptides identified were grouped according to their pre-established families and had their discovery and activity discussed. Most of identified molecules are of antimicrobial peptides, followed by vasoactive and inflammatory activity. The importance to continue investigating fish venoms and poisons and exploring their potential as new drugs and physiological tools is clear. We believe this review can serve as a guide for the development of new projects, revealing new directions for future research, and compiling information about the exploration methods that attained success in the field.

Toadfish are fish from the family Batrachoididae that are found in marine and brackish environment around the world. Among the toadfish, Porichthys genus is very common, where Porichthys porosissimus, also called Atlantic Midshipman is found in Southwest Atlantic, from Rio de Janeiro, Brazil to eastern Argentina. There was no consensus about the classification of the genus Porichthys as venomous fish because so far there are no published studies regarding human envenomations and/or toxic activities induced in animal models. Herein, we report two conclusive envenoming in human beings caused by P porosissimus spines, with clear signs and symptoms that were very important for the development of our experimental studies. We demonstrated that the P. porosissimus spine extract, now venom, can induce nociceptive and edematogenic responses in mice as well an induction of an inflammatory response elicited by intravital microscopy and leukocyte migration. Finally, we identified in the P. porosissimus spine extract, through analysis by mass spectrometry, the presence of proteins previously detected in the venoms of other fish species and other venomous animals. We believe that based on our studies we will dismiss the non-venomous nature of this fish and clarify this issue.

The subfamily Phyllomedusinae has attracted a great interest of many researchers mainly due to the high diversity of these frog species and plethora of pharmacological activities frequently observed for their skin secretions. Despite of this fact, mainly for new species, limited information is available regarding the molecular composition of these skin secretions and the cellular components involved in their production. Phyllomedusa nordestina is a recently described Brazilian frog species also popularly known as ‘tree-frogs’. Aiming at contributing to the biological knowledge of this species, we show here the gene expression profile of this frog skin secretion using a global ESTs analysis of a cDNA library. The marked aspect of this analysis revealed a significant higher transcriptional level of the opioid peptide dermorphins in P. nordestina skin secretion than in Phyllomedusa hypochondrialis , which is its closest related species, belonging both to the same phylogenetic group. Precursors of bioactive peptides as dermaseptins, phylloseptins, tryptophyllins, and bradykinin-like peptideswere also found in this library. Transcripts encoding proteins related to ordinary cellular functions and pathways were also described. Some of them are chiefly involved in the production of the skin secretion. Taken together, the data reported here constitute a contribution to the characterization of the molecular diversity of gene-encoded polypeptides with potential possibility of pharmacological exploitation. The transcriptional composition of the skin secretion may also help to give the necessary support for the definition of P. nordestina as a new species, which actually relies basically on frog morphological characteristics and geographical distribution.

© 2016 American Chemical Society, Kyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (l-Tyr-l-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of d amino acid residues. Intravital microscopy data show that KTP-NH2 having a d-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 μM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 μM increase leukocyte rolling up to nearly 5-fold after 30 min, suggesting a proinflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception., FCT-MCTES is acknowledged for PhD fellowship SFRH/BD/52225/2013 to J.P. Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) is acknowledged for funding: call H2020-MSCA-RISE-2014, Grant agreement 644167, 2015-2019.

Cathorops spixii is the most common venomous fish on the Brazilian coast. Apart from the involvement with defense against pathogens, the possible contribution of skin mucus components to the development of injuries caused by venomous fish species has not been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. Our results show that sting venom and skin mucus have distinct constituents that distinguished them like structural proteins, chaperones, ion transport, carbohydrate metabolism, oxidoreductase, cell cycle and protein binding present in sting venom and like tropomyosin 3 isoform 2 and energy metabolim proteins in skin mucus. But in a group of common 13 proteins we identified and isolated a WAP65 protein. The peptide fractions caused more harmful effects, such as venular stasis, hemorrhage and changes in the arteriolar wall diameter, and the protein fractions produced a typical inflammatory process in post-capillary venules. And finally we showed for the first time the presence WAP65 in sting venom and skin mucus of C. spixii using LC/MS/MS and also we purified this protein in the sting venom. Wap65 shows inflammatory action, working at different doses inducing an increase in the number of leukocytes rolling and adhering to the endothelium.

Considerable efforts are currently focused on the biology of DC in view of their possible clinical use as adjuvant for the generation of antigen-specific immunity and lifelong immunologic memory or for the treatment of tumors. We assessed the role of Nattectin a C-type lectin identified in the Thalassophryne nattereri fish venom in DC maturation. Nattectin induced a significant neutrophilic recruitment into peritoneal cavity of mice, followed by macrophages, with lipidic mediators and IL-12 p70 synthesis. Macrophages derived from 7day-Nattectin mice were CD11c+CD11b(low)Ly6(high)F4/80R(high) and express high levels of MHC class II and CD80 molecules. Culture of peritoneal exudates derived macrophages from 7day Nattectin-mice and immature BMDCs with Nattectin markedly increased the surface expression of CD40, CD80, CD86, and MHC class II in a dose-dependent manner, and the production of MMP-2 and MMP-9 distributed in nucleus and cytoplasm of cells, that was associated with strong activity in the culture supernatant. Nattectin treated DCs secreted IL-12 p70 and IL-10. The Nattectin-treated BMDC or macrophage-derived DCs were highly efficient at Ag capture. The specific immune response elicited by Nattectin was characterized by the production of specific antibodies IgG1 and mainly IgG2a with IL-10 and IFN-γ synthesis by splenic cells. These results enable us to address that Nattectin induces the recruitment of Ly6C(high) monocytes into the peritoneum, which exhibit a pro-inflammatory profile, where they differentiate into proliferating F4/80R(high) macrophages. Macrophage-derived DCs mature in the presence of the cytokine milieu generated against Nattectin, exhibiting T cell co-stimulatory molecule expression and induced a Th1 polarized response.

Amphibian skin secretions are a source of potential new drugs with medical and biotechnological applications. Rich in peptides produced by holocrine-type serous glands in the integument, these secretions play different roles, either in the regulation of physiological skin functions or in the defense against predators or microorganisms. The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of Phyllomedusa nordestina. In order to achieve this goal, the crude skin secretion of this frog was pre-fractionated by solid phase extraction and separated by reversed-phase chromatography. The fractions were screened for low-molecular-mass peptides and sequenced by mass spectrometry. It was possible to identify three novel bradykinin-related peptides, namely: KPLWRL-NH2 (Pnor 3), RPLSWLPK (Pnor 5) and VPPKGVSM (Pnor 7) presenting vascular activities as assessed by intravital microscopy. Pnor 3 and Pnor 7 were able to induce vasodilation. On the other hand, Pnor 5 was a potent vasoconstrictor. These effects were reproduced by their synthetic analogues.

Amphibian skin secretions contain several bioactive compounds such as biogenic amines, alkaloids, steroids, proteins and peptides; being peptides a continuously growing field of interest. This work aims to describe the main physiopathological properties of the tree frog Phyllomedusa hypochondrialis skin secretion, obtained by manual stimulation of the dorsal skin surface. Intravenous skin secretion administration provoked lethal effect in mice after 5 min. Low doses induced significant systemic and local effects like edema and nociception in mice and topic administration induced myonecrosis in the endothelium of cremaster mice. The presence of phospholipase A2 activity, proteolytic activity and creatine kinase activity (in the plasma of treated mice) are reported and are very likely to be related to the physiopathological (edematic and myotoxic) activities observed. These data provide in vivo evidence of the complex toxic effects of the P. hypochondrialis skin secretion as well as possible mechanisms of action for these effects.

© Springer-Verlag Wien 2015, Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same., Fundação para a Ciência e Tecnologia (Portugal) is acknowledged for funding (Grant Nos. SFRH/BD/65709/2009, PTDC/BIA-PRO/104378/2008 and PEst-OE/EQB/LA0004/2011). Katia Conceição and Vasanthakumar Ramu were recipients of Marie Curie IAPP fellowships. Marie Curie Industry-Academia Partnerships and Pathways (European Commission) is acknowledged for funding (FP7-PEOPLE-2007-3-1-IAPP. Project 230654). The financial contributions of the Deutsche Forschungsgemeinschaft (to MM), the Agence Nationale de la Recherche (projects TRANSPEP, ProLipIn, membrane DNP and the LabEx Chemistry of Complex Systems), the University of Strasbourg, the CNRS, the Région Alsace and the RTRA International Center of Frontier Research in Chemistry are gratefully acknowledged.

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by 'de novo' mass spectrometry and grouped into Dermaseptins (DPh-1).

Antimicrobial molecules are important components of the innate immune system in vertebrates. They have been studied widely in several fishes, but little is known about these defence factors in stingrays, which are thought to have less sophisticated adaptive immune systems when compared to other teleosts. Stingrays from the specie Potamotrygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the cause of numerous envenomations occurring in the dry seasons.In a previous study, we reported that the mucus of the stingray P. cf. henlei shows antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf. henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-MS, and Edman degradation. A protein similar to the β-chain of hemoglobin was identified, isolated and partially sequenced by Edman degradation. This protein has a molecular weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new protein in the microcirculation environment were also evaluated. The results obtained provide fundamental information for future basic research, clinical diagnosis and development of new therapies to accident treatment. To the best of our knowledge, this is the first description of a bioactive polypeptide from the mucus of a stingray.

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout the rivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posing a serious public health problem even if not properly reported. The accidents usually involve fishermen and bathers, and to date there is no effective treatment for the injured. Considering these facts and limitations of studies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describe the principal pharmacological and certain biochemical properties of the mucus and sting venom. We found that mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Our results also indicate that the inflammatory cellular influx observed could be triggered by the venom and mucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested for antimicrobial activity in which only the mucus presented activity. It could be inferred from the present study that P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically active components.

In order to investigate the relationship between the primary structure of Orpotrin, a vasoactive peptide previously isolated from the freshwater stingray Potamotrygon gr. orbignyi, and its microcirculatory effects, three Orpotrin analogs were synthesized. The analogs have a truncated N-terminal with a His residue deletion and two substituted amino acid residues, where one Nle is substituted for one internal Lys residue and the third analog has a substitution of a Pro for an Ala (Orp-desH(1) , Orp-Nle and Orp-Pro/Ala, respectively). Only Orp-desH(1) could induce a lower vasoconstriction effect compared with the natural Orpotrin, indicating that besides the N-terminal, the positive charge of Lys and the Pro residues located at the center of the amino acid chain is crucial for this vasoconstriction effect. Importantly, the suggestions made with bioactive peptides were based on the molecular modeling and dynamics of peptides, the presence of key amino acids and shared activity in microcirculation, characterized by intravital microscopy. Moreover, this study has demonstrated that even subtle changes in the primary structure of Orpotrin alter the biological effects of this native peptide significantly, which could be of interest for biotechnological applications.

Brazilian freshwater stingrays, Potamotrygon gr. orbigyni, are relatively common in the middle-western regions of Brazil, where they are considered an important public health threat. In order to identify some of their naturally occurring toxin peptides available in very low amounts, we combine analytical protocols such as reversed-phase high-performance liquid chromatography (RP-HPLC), followed by a biological microcirculatory screening and mass spectrometry analysis. Using this approach, one bioactive peptide was identified and characterized, and two analogues were synthesized. The natural peptide named Porflan has the primary structure ESIVRPPPVEAKVEETPE (MW 2006.09 Da) and has no similarity with any bioactive peptide or protein found in public data banks. Bioassay protocols characterized peptides as presenting potent activity in a microcirculatory environment. The primary sequences and bioassay results, including interactions with the membrane phospholipids, suggest that these toxins are a new class of fish toxins, directly involved in the inflammatory processes of a stingray sting.

Bradykinin potentiating peptides (BPPs) from Bothrops jararaca venom were first described in the middle of 1960s and were the first natural inhibitors of the angiotensin-converting enzyme (ACE). BPPs present a classical motif and can be recognized by their typical pyroglutamyl (Pyr)/proline rich sequences presenting, invariably, a proline residue at the C-terminus. In the present study, we describe the isolation and biological characterization of a novel BPP isolated from the skin secretion of the Brazilian tree-frog Phyllomedusa hypochondrialis. This new BPP, named Phypo Xa presents the sequence Pyr-Phe-Arg-Pro-Ser-Tyr-Gln-Ile-Pro-Pro and is able to potentiate bradykinin activities in vivo and in vitro, as well as efficiently and competitively inhibit ACE. This is the first canonical BPP (i.e. Pyr-Aaan-Gln-Ile-Pro-Pro) to be found not only in the frog skin but also in any other natural source other than the snake venoms.

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97-105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms.

Phyllomedusa hypochondrialis skin secretion can cause both systemic and local effects. In this study, we describe the pattern of local acute inflammatory response after P. hypochondrialis skin secretion injection. The inflammatory reaction in the mice footpad was analysed, including the leukocyte recruitment into local tissue from the peripheral blood, in a mouse model of tissue injury. We also investigated the release of the cytokines IL-1, IL-6 and TNF- α , chemokines KC and MCP-1 and the eicosanoids LTB 4 and PGE 2 in mice. The present findings support the ability of P. hypochondrialis skin secretion to induce local inflammation. In addition, these skin secretion components play a role in the initial rolling and slowing of recruited leukocytes and the transition from rolling to adhesion. Levels of the proinflammatory cytokine IL-6, chemokines KC and MCP-1 as well as the eicosanoid PGE 2 were significantly increased after injection of a skin secretion of P. hypochondrialis (0.6 μg/30 μl intraplantar), whereas no changes in other parameters were observed. Finally, the mechanisms involved in the local inflammatory process induced by P. hypochondrialis skin secretion is one of the questions of relevance related to the complex pathophysiology induced by this particular secretion and other toxins.