Evaluation of the Biological Activities of Peptides from Epidermal Mucus of Marine Fish Species from Chilean Aquaculture.

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.

Alpha-helical domain from IL-8 of salmonids: Mechanism of action and identification of a novel antimicrobial function.

In this work, the potential antimicrobial role and mechanism of action of α-helix domain of trout and salmon IL-8 against Eschericia coli, Pseudomonas aeruginosa and Staphylococcus aureus was investigated. By an in silico analysis of the primary structure of IL-8 from Oncorhynchus mykiss and salmo salar, it was evidenced that γ-core motif was present, as in the vast majority of kinocidins. The α-helix domain of IL-8 (αIL-8) was synthesized by solid phase peptide synthesis and showed a tendency to form an α-helix conformation, as revealed by circular dichroism. Additionally, it was demonstrated that αIL-8 from both species showed antimicrobial activity against E. coli, P. aeruginosa and S. aureus. Membrane permeabilization and co-localization assay, as well as scanning electron microscopy, showed that these peptides were accumulated on the cell surface and in the cytoplasm, suggesting that they were capable of permeabilizing and disrupt the bacterial membranes and interact with cytoplasmic components. Our results represent the first analysis on the antimicrobial function of IL-8-derived peptide from salmonids.

Chemical Synthesis and Functional Analysis of VarvA Cyclotide.

Cyclotides are circular peptides found in various plant families. A cyclized backbone, together with multiple disulfide bonds, confers the peptides' exceptional stability against protease digestion and thermal denaturation. In addition, the features of these antimicrobial molecules make them suitable for use in animal farming, such as aquaculture. Fmoc solid phase peptide synthesis on 2-chlorotrityl chlorine (CTC) resin using the "tea-bag" approach was conducted to generate the VarvA cyclotide identified previously from Viola arvensis. MALDI-TOF mass spectrometry determined the correct peptide amino acid sequence and the cyclization sites-critical in this multicyclic compound. The cyclotide showed antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens present in Chilean aquaculture. The highest antimicrobial activity was found to be against Flavobacterium psychrophilum. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and the Sytox Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that this compound can be proposed for the control of fish farming infections.

Ultrasonic Sensor: A Fast and Non-Destructive System to Measure the Viscosity and Density of Molecular Fluids.

This study presents the design and development of an ultrasonic sensor as a fundamental tool for characterizing the properties of fluids and biofluids. The analysis primarily focuses on measuring the electrical parameters of the system, which correlate with the density and viscosity of the solutions, in sample volumes of microliters and with high temporal resolution (up to 1 data point per second). The use of this sensor allows the fast and non-destructive evaluation of the viscosity and density of fluids deposited on its free surface. The measurements are based on obtaining the impedance versus frequency curve and the phase difference curve (between current and voltage) versus frequency. In this way, characteristic parameters of the transducer, such as the resonance frequency, phase, minimum impedance, and the quality factor of the resonant system, can characterize variations in density and viscosity in the fluid under study. The results obtained revealed the sensor's ability to identify two parameters sensitive to viscosity and two parameters sensitive to density. As a proof of concept, the unfolding of the bovine albumin protein was studied, resulting in a curve that reflects its unfolding kinetics in the presence of urea.

First Insights about Antiparasitic and Action Mechanisms of the Antimicrobial Peptide Hepcidin from Salmonids against Caligus rogercresseyi.

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.

Development of a sandwich ELISA for quantifying hepcidin in Rainbow trout.

One of the most widespread antimicrobial peptides (AMPs) in fish is the hepcidins, which have potent, broad-spectrum activity against viruses, bacteria, fungi, and parasites. Moreover, they play the role of central regulation of iron metabolism and their expression is over-regulated by bacterial and viral infections, inflammation and vaccination. Quantification of their expression is an important factor in understanding their function. We therefore generated two polyclonal antibodies using synthetic peptides in order to measure hepcidin expression via sandwich ELISA. The specificity of both antibodies was confirmed by identifying an absence of cross-reactivity with other peptides that have similar pI and with the detection by Western blot of only one 9.6 kDa immunoreactive band corresponding to the hepcidin prepropeptide. The sensitivity of the sandwich ELISA was in the order of 0.005 ng/µL of hepcidin, which allowed analysis of the presence of the peptide and its variation in different tissues of Oncorhynchus mykiss. With the sandwich ELISA it could be seen that hepcidin expression in rainbow trout challenged with Aeromonas salmonicida was increased twofold over the untreated fish in head kidney samples, in correlation with the increase in the observed transcriptional level in the head kidney cells. These results provide the first evidence for quantifying the presence of active hepcidin and may be a useful indicator of disease susceptibility, providing a new, sensitive tool for rapid screening of population health.

Stability of ACE2 Peptide Mimetics and Their Implications on the Application for SARS-CoV2 Detection.

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.

Poly(ethylene imine)-chitosan carbon dots: study of its physical-chemical properties and biological in vitro performance.

Carbon dots (CDs) have been quickly extended for nanomedicine uses because of their multiple applications, such as bioimaging, sensors, and drug delivery. However, the interest in increasing their photoluminescence properties is not always accompanied by cytocompatibility. Thus, a knowledge gap exists regarding their interactions with biological systems linked to the selected formulations and synthesis methods. In this work, we have developed carbon dots (CDs) based on poly (ethylene imine) (PEI) and chitosan (CS) by using microwave irradiation, hydrothermal synthesis, and a combination of both, and further characterized them by physicochemical and biological means. Our results indicate that synthesized CDs have sizes between 1 and 5 nm, a high presence of amine groups on the surface, and increased positive ζ potential values. Further, it is established that the choice and use of different synthesis procedures can contribute to a different answer to the CDs regarding their optical and biological properties. In this regard, PEI-only CDs showed the longest photoluminescent emission lifetime, non-hemolytic activity, and high toxicity against fibroblast. On the other hand, CS-only CDs have higher PL emission, non-cytotoxicity associated with fibroblast, and high hemolytic activity. Interestingly, their combination using the proposed methodologies allow a synergic effect in their CDs properties. Therefore, this work contributes to developing and characterizing CD formulations based on PEI and CS and better understanding the CD's properties and biological interaction.

Immune Modulation Ability of Hepcidin from Teleost Fish.

Antimicrobial peptides (AMP) play an essential role in the innate immune system, modulating the defense response. In a previous study, we demonstrated the antimicrobial activity of synthetic hepcidin (hep20) from rainbow trout (Oncorhynchus mykiss), and its protective effect in European sea bass (Dicentrarchus labrax) challenged with Vibrio anguillarum. Additionally, we described the uptake and distribution of hep20 in different tissues and leukocyte cells. Interestingly, various AMPs characterized in high vertebrates, called host defense peptides (HDPs), also possess immunomodulation activity. For that reason, the present study explores the immunomodulatory abilities of hep20 through in vitro and in vivo studies. First, a monocyte/macrophage RTS-11 cell line from rainbow trout was used to evaluate hep20 effects on pro- and anti-inflammatory cytokines in fish leukocyte cells. Next, the European sea bass juveniles were used to determine if hep20 can regulate the expression of cytokines in fish immune tissues. The results show that hep20 was uptake inner to RTS-11 cells and was able to induce the expression of IL-10, IL-1ß, and TNFα at transcriptional and protein levels. Then, the European sea bass juveniles were given intraperitoneal injections of the peptide. At 1, 3, 7, 14, and 21 days post-injection (dpi), IL-10, IL -1ß, and TNFα mRNA were quantified in the anterior gut, spleen, and head kidney. The hep20 was able to up-regulate cytokine gene expression in these tissues, mainly in the head kidney. Furthermore, the evaluated cytokines showed a cyclical tendency of higher to lesser expression. Finally, a bioinformatics analysis showed that the structure adopted by hep20 is similar to the γ-core domain described for cysteine-stabilized AMP, defined as immunomodulatory and antimicrobial, which could explain the ability of hep20 to regulate the cytokine expression. This study provides new insights into immunomodulatory function complementary to the previously established antimicrobial activity of hep20, suggesting a role as an HDP in teleost fish. These facts are likely to be associated with molecular functions underpinning the protective effect of fish hepcidin against pathogens.

Effect of Fish Stock Density on Hormone Genes Expression from Brain and Gastrointestinal Tract of Salmo salar.

A variety of long-term stress conditions may exist in fish cultivation, some of which are so severe that fish can no longer reestablish homeostasis. In teleost fish, the brain and gastrointestinal tract integrate signals that include the perception of stress factors regulating physiological responses, such as social stress by fish population density, where peripheral and central signals, such as peptide hormones, are the main regulators. Therefore, we proposed in this study to analyze the effect of different stock densities (SD) in the gene expression of brain neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP), together with the gastrointestinal peptide hormones leptin (Lep), vasointestinal peptide (VIP), and protachykinin-1 (Prk-1) in Salmo salar post-smolt. The coding sequence of S. salar VIP and Prk-1 precursors were firstly cloned and characterized. Then, the mRNA expression of these genes, together with the NPY, Lep, and CGRP genes, were evaluated in post-smolts kept at 11 Kg/m3, 20 Kg/m3, and 40 Kg/m3. At 14 days of culture, the brain CGRP and liver leptin mRNA levels increased three and tenfold in the post-smolt salmons kept at the highest SD, respectively. The high levels of leptin were kept during all the fish culture experiments. In addition, the highest expression of intestine VIP mRNA was obtained on Day 21 in the group of 40 Kg/m3 returning to baseline on Day 40. In terms of stress biochemical parameters, cortisol levels were increased in the 20 Kg/m3 and 40 Kg/m3 groups on Day 40 and were the highest in the 20 Kg/m3 group on Day 14. This study provides new insight into the gastrointestinal signals that could be affected by chronic stress induced by high stock density in fish farming. Thus, the expression of these peptide hormones could be used as molecular markers to improve production practices in fish aquaculture.

New Drug Design Avenues Targeting Alzheimer's Disease by Pharmacoinformatics-Aided Tools.

Neurodegenerative diseases (NDD) have been of great interest to scientists for a long time due to their multifactorial character. Among these pathologies, Alzheimer's disease (AD) is of special relevance, and despite the existence of approved drugs for its treatment, there is still no efficient pharmacological therapy to stop, slow, or repair neurodegeneration. Existing drugs have certain disadvantages, such as lack of efficacy and side effects. Therefore, there is a real need to discover new drugs that can deal with this problem. However, as AD is multifactorial in nature with so many physiological pathways involved, the most effective approach to modulate more than one of them in a relevant manner and without undesirable consequences is through polypharmacology. In this field, there has been significant progress in recent years in terms of pharmacoinformatics tools that allow the discovery of bioactive molecules with polypharmacological profiles without the need to spend a long time and excessive resources on complex experimental designs, making the drug design and development pipeline more efficient. In this review, we present from different perspectives how pharmacoinformatics tools can be useful when drug design programs are designed to tackle complex diseases such as AD, highlighting essential concepts, showing the relevance of artificial intelligence and new trends, as well as different databases and software with their main results, emphasizing the importance of coupling wet and dry approaches in drug design and development processes.

Immunodetection of rainbow trout IL-8 cleaved-peptide: Tissue bioavailability and potential antibacterial activity in a bacterial infection context.

Chemokines such as IL-8 are part of an important group of proinflammatory response molecules, as well as cell recruitment. However, it has been described in both higher vertebrates and fish that IL-8 has an additional functional role by acting as an antimicrobial effector, either directly or by cleavage of a peptide derived from its C-terminal end. Nevertheless, it is still unknown whether this fragment is released in the context of infection by bacterial pathogens and if it could be immunodetected in tissues of infected salmonids. Therefore, the objective of this research was to demonstrate that the C-terminal end of IL-8 from Oncorhynchus mykiss is cleaved, retaining its antibacterial properties, and that is detectable in tissues of infected rainbow trout. SDS-PAGE and mass spectrometry demonstrated the cleavage of a fragment of about 2 kDa when the recombinant IL-8 was subjected to acidic conditions. By chemical synthesis, it was possible to synthesize this fragment called omIL-8α80-97 peptide, which has antibacterial activity against Gram-negative and Gram-positive bacteria at concentrations over 10 µM. Besides, by fluorescence microscopy, it was possible to locate the omIL-8α80-97 peptide both on the cell surface and in the cytoplasm of the bacteria, as well as inside the monocyte/macrophage-like cell. Finally, by indirect ELISA, Western blot, and mass spectrometry, the presence of the fragment derived from the C-terminal end of IL-8 was detected in the spleen of trout infected with Piscirickettsia salmonis. The results reported in this work present the first evidence about the immunodetection of an antibacterial, and probably cell-penetrating peptide cleaved from the C-terminal end of IL-8 in monocyte/macrophage-like cell and tissue of infected rainbow trout.

New insight to the rol of α-enolase (Eno-1) as immunological marker in rainbow trout fry.

α-Enolase is an enzyme of the glycolytic pathway that has also been involved in vertebrate inflammatory processes through its interaction with plasminogen. However, its participation in the immune response of lower vertebrates during early life development is unknown. Opportunistic pathogens in salmon farming are the principal cause of mortality in the fry stage. For that reason, molecular indicators of their immunological status are required to ensure the success of the large-scale cultivation. Thus, the objective of this work was to analyze if ENO-1 is involved in the immune response of rainbow trout fry. For this purpose, the coding sequence of trout ENO-1 was characterized, identifying the plasminogen-binding domain that has been described for homologs of this enzyme in higher vertebrates. A peptide-epitope of α-enolase was used for producing mice antiserum. The specificity of polyclonal antibodies was confirmed by dot blot, ELISA and Western blot. Then, the antiserum was used to evaluate α-enolase expression in fry between 152 and 264 degree-days post-hatching after 2, 8, and 12 h of challenge with lipopolysaccharide from Pseudomona auroginosa. The expression of α-enolase at both transcriptional (RT-qPCR) and protein (ELISA) levels was significantly increased after 8 h post-challenge with lipopolysaccharide. These results were confirmed by proteomic analysis by 2D-difference gel electrophoresis (DIGE). This work provides the first evidence of the involvement of α-enolase in the early immune response of salmonids. Future research will be required to understand the possible interaction of α-enolase with plasminogen in cells and tissues of the salmonid immune system.

Co0 superparamagnetic nanoparticles stabilized by an organic layer coating with antimicrobial activity.

Cobalt (Co) is one of the most promising materials in nanotechnology due to its superior magnetic properties. However, due to the high cytotoxicity of cobalt, the activity in biological systems has been little studied. In this work, we report the structural, morphological, and magnetic properties of cobalt nanoparticles stabilized with an organic layer (Co0@C-NPs) and its potential antimicrobial activity. The Co0@C-NPs were obtained from solvothermal conditions and characterized by X-ray powder diffraction, electronic microscopy, and magnetic measurements. The organic layer was analysed by thermogravimetric analysis, Scanning Electron Microscopy, Energy Dispersive Spectrometer, and Fourier Transform Infrared Spectroscopy. From the TEM image, an organic coating layer is observed around Co0 where this coating prevents NPs from oxidation allowing it to remain stable until 400 °C. Surface composition studies by SEM/EDS allowed the identification of carbon, oxygen, and cobalt elements present in the organic layer. This result was corroborated later by FITR analysis. Preliminary antibacterial properties were also investigated, which showed that the cobalt nanoparticles are active against Staphylococcus aureus after 1 h of exposure. The superparamagnetic properties and organic coating Co0@C-NPs could be biocompatible with biological systems, but more research is needed to apply these nanoparticles in biomedical products.

Hepcidin, Cathelicidin-1 and IL-8 as immunological markers of responsiveness in early developmental stages of rainbow trout.

During the early developmental stage of salmonids, high mortality occurs largely as a result of pathogens. These cause low immune competence in fry, producing disease, decreasing production and finally leading to economic losses. Therefore, the aim of this study was to characterise the developmental stages in which rainbow trout acquires immune response capability when challenged with LPS from Pseudomona aeruginosa for 8 h, studying the hepcidin, cathelicidin-1 and IL-8. Total RNA was extracted from fry at 34, 42, 56 and 66 days post hatching (dph). Hepcidin and cathelicidin-1 transcripts were detected only at days 34 and 42, whereas the IL-8 transcript was detected from day 34 to day 66. To analyse the protein expression in the fry, polyclonal anti-peptide antibodies were generated in rabbit. These three immune sera demonstrated the ability to recognise the whole molecule in biological samples. Immunofluorescence showed that skin, gills and intestine mainly responded to the LPS challenge, indicating that these portals of pathogen entry are capturing LPS. This study constitutes a valuable approach, since it has the potential to identify molecules with biological activity that can be used to evaluate the status of fry in culture.

Detection of the hepcidin prepropeptide and mature peptide in liver of rainbow trout.

Hepcidin is a small, cationic peptide which displays antimicrobial activities and iron regulatory function. Originally identified in mammals, this peptide is also present in fish. Hepcidin mRNA is predominantly expressed in liver and is regulated by iron and pathogen infection. In this work, we characterized the expression of trout hepcidin at protein level using rabbit antisera. Results showed that the prepropeptide of hepcidin can be detected by Western Blot in liver tissue from trout injected with iron or lipopolysaccharide. The mature hepcidin peptide was detected at the ionized state 5+(m/z 577.2) by HPLC-ESI-MS in acid extracts from liver tissue. Moreover, hepcidin peptide was located in trout liver imprints by immunofluorescence. These results showed that hepcidin peptide is up-regulated by iron and bacterial components in the trout liver. This up-regulation could be a potential indicator of disease susceptibility, suggesting that hepcidin regulates iron homeostasis in salmonids.