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Polysaccharides from wood-rooting fungi have attracted attention due to their broad pharmacological properties. Herein, we report the antitumor and immunomodulatory activities of acid polysaccharides isolated from fungi Gloeosoma mirabile. The polysaccharide extracts displayed significant antiproliferative activity against cancer cell lines (MCF-7, HCT-116, U-937) in a dose-dependent manner and induction of IL-6 in macrophage RAW 264.7. Furthermore, flow cytometry analysis showed that high polysaccharide concentrations induced apoptosis by 83% in HL-60 cells. Based on gas chromatography-mass spectrometry (GC-MS) and Fourier transform infra-red (FT-IR) spectroscopy studies, acidic polysaccharides from G. mirabile were mainly composed of arabinose, α-D-galactopyranose and methyl ß-D-galactopyranoside.

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NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

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Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

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Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1ß) and anti-inflammatory (IL-10 and TGF-ß) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.

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The intensive salmon farming is associated with massive outbreaks of infections. The use of antibiotics for their prevention and control is related to damage to the environment and human health. Antimicrobial peptides (AMPs) have been proposed as an alternative to the use of antibiotics for their antimicrobial and immunomodulatory activities. However, one of the main challenges for its massive clinical application is the high production cost and the complexity of chemical synthesis. Thus, recombinant DNA technology offers a more sustainable, scalable, and profitable option. In the present study, using an AMPs function prediction methodology, we designed a chimeric peptide consisting of sequences derived from cathelicidin fused with the immunomodulatory peptide derived from flagellin. The designed peptide, CATH-FLA was produced by recombinant expression using an easy pre-purification system. The chimeric peptide was able to induce IL-1ß and IL-8 expression in Salmo salar head kidney leukocytes, and prevented Piscirickettsia salmonis-induced cytotoxicity in SHK-1 cells. These results suggest that pre-purification of a recombinant AMP-based chimeric peptide designed in silico allow obtaining a peptide with immunomodulatory activity in vitro. This could solve the main obstacle of AMPs for massive clinical applications.

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Various microorganisms are able to synthesize pigments, which usually present antioxidant properties. The aim of this work was to evaluate the antiproliferative activity of bacterial pigments against cancer cells Neuro-2a, Saos-2 and MCF-7. Pigments were obtained from Deinococcus sp. UDEC-P1 and Arthrobacter sp. UDEC-A13. Both bacterial strains were isolated from cold environments (Patagonia and Antarctica, respectively). Pigments were purified and analyzed by HPLC. Antiproliferative activity was evaluated by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT) assay. Deinoxanthin carotenoid obtained from Deinococcus sp. UDEC-P1 was able to reduce significatively the viability of Saos-2 (37.1%), while no effect was observed against MCF-7 and Neuro-2a. Pigments obtained from Arthrobacter sp. UDEC-A13 showed a significant viability reduction of three tumour cells (20.6% Neuro-2a, 26.3% Saos-2 and 13.2% MCF-7). Therefore, carotenoid pigments produced by extremophilic bacteria Deinococcus sp. UDEC-P1 and Arthrobacter sp. UDEC-A13 could be proposed as novel complementary compounds in anticancer chemotherapy.

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There are two official PSP detection methods (mouse bioassay and HLPC-FLD) and a number of alternative methods. Ethical considerations have led to regulations being adopted in some countries that limit or prohibit the application of mouse bioassay. Analytical methodologies (e.g. HPLC-FLD or LC-MSMS) have the disadvantages of not being able to detect new toxins or analogues or reflecting the overall toxicity of the sample. In addition, they require highly trained personnel and expensive equipment, which are not always available. In this work, we have evaluated a method based on the Neuro-2a cell-based assay to detect substances that inhibit voltage-dependent sodium channels (Manger's method). We tested PSP standards and natural samples contaminated with PSP. Here we demonstrate that the adapted Manger's method is suitable for calculating Toxicity Equivalency Factors (TEF) for STX-analogues. The method was shown to be useful for screening contaminated natural samples in concentrations above the regulatory limit for these toxins (80 µg STX equivalents/100 g shellfish). We were able to detect PSP in 19 natural mollusc samples from South Chile despite the presence of other marine toxins. These preliminary results suggest that the method could be used as a first step in screening programmes.

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In this work Talaromyces australis and Penicillium murcianum pigment production in liquid cultures and the cytotoxic effect of such pigments on skin model cells were studied. Response surface methodology (RSM) was used to optimize culture conditions aiming to increase pigment production in malt extract and peptone-glucose-yeast extract medium. Cytotoxicity of fungal pigments and also from lixiviates of wool fabrics dyed with T. australis and P. murcianum pigment was evaluated on mammalian cell lines HEK293 and NIH/3T3. Results showed that variations on initial pH, NaCl and peptone, resulted in increments up to 188.2% for red pigment of T. australis and 107.4% for yellow pigment of P. murcianum, regarding non-optimized conditions. Tested fungi also showed great differences in culture conditions for the maximum pigment production, with P. murcianum requiring an alkaline medium (initial pH 9) supplemented with NaCl and T. australis an acidic medium (initial pH 5) without addition of salt. The cytotoxicity assays provided evidences on the safe nature of these natural pigments when used for textile applications. The cytotoxicity assay showed that the threshold of toxicity, given by the lowest IC50 value (0.21 g L-1) was more than double of the concentration of pigment required to dye the wool samples. In addition, cytotoxicity of lixiviates depicted no toxic effect over tested cells.

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Didymosphenia geminata (Lyngbye) Schmidt, also referred to as Didymo, is an invasive diatom that forms nuisance mats. Since it was first reported in our country in approximately 2010, Didymo has expanded and colonized different rivers in the Zona Austral region of Chile. Its biology and effects on ecosystems are still being studied because Didymo is an invasive algal mat that forms in a range of systems from oligotrophic austral rivers to more subtropical systems. We aimed to evaluate the viability of two salmonid cell lines, CHSE-214 and SHK-1 (somatic and embryonic cell lines, respectively), in dilutions of river water alone and in river water contaminated with Didymo or polyphenols extracted from Didymo under controlled conditions. We developed an artificial river system (2 aquariums/replicate) from five different rivers from the central area (Bio-Bio) and Patagonia area (Futaleufú) of Chile to maintain Didymo in the benthic phase. The Didymo populations were maintained for six months in the water from the rivers, after which samples were obtained. Following the extraction of polyphenols from the Didymo samples maintained in the artificial rivers, toxicity assays (10 assays) were performed to determine cell viability. Our results indicated that the CHSE-214 cells were highly sensitive to increasing concentrations of Didymo extracts. We observed a 50% reduction in cell viability after 24 h of exposure to a 0.01 V/V dilution, and this treatment further reduced the proliferative capacity by 70% after 120 h. The SHK-1 cells were less responsive, showing only a 20% decrease in viability at 24 h and a lower cell proliferation rate (45%) after 120 h, which remained higher than that of the CHSE-214 cells. We conclude that certain cell types are sensitive to Didymo in rivers, suggesting that there are chronic effects on several aquatic species following exposure to these diatom substances. These effects should be further studied using this laboratory model to understand the full impact of Didymo on river ecosystems.

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Despite the advance of knowledge about the factors and potential mechanisms triggering the ichthyotoxicity in microalgae, these remain unclear or are controversial for several species (e.g. Heterosigma). Neither typical toxicity tests carried out with cell extracts nor direct exposure to harmful species were proved suitable to unravel the mechanism of harm. Ichthyotoxic species show a complex harmful effect on fish, which is mediated through various mechanisms depending on the species. In this work, we present a method to study sub-lethal effects triggered by reactive oxygen species of a population of harmful algae in vivo over a fish cell line. To that end, Transwell co-cultures in which causative and target species are separated by a 0.4 µm pore membrane were carried out. This allowed the evaluation of the effect of the released molecules by cells in a rapid and compact test. In our method, the harmful effect was sensed through the transcriptional activation of sub-lethal marker Hsp70b in the CHSE214 salmon cell line. The method was tested with the raphidophyte Heterosigma akashiwo and Dunaliella tertiolecta (as negative control). It was shown that superoxide intracellular content and its release are not linked in these species. The methodology allowed proving that reactive oxygen species produced by H. akashiwo are able to induce the transcriptional activation of sub-lethal marker Hsp70b. However, neither loss of viability nor apoptosis was observed in CHSE214 salmon cell line except when exposed to direct contact with the raphidophyte cells (or their extract). Consequently, ROS was not concluded to be the main cause of ichthyotoxicity in H. akashiwo.

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