A rapid protocol for inducing acute pancreatitis in zebrafish models.

Acute pancreatitis (AP) is an inflammatory disorder that occurs in the exocrine pancreas associated with tissue injury and necrosis. Experimental models of AP typically involve rodents, such as rats or mice. However, rodents exhibit divergent pathophysiological responses after the establishment of AP between themselves and in comparison, with human. The experiments conducted for this manuscript aimed to standardize a new AP model in zebrafish and validate it. Here, we provide a protocol for inducing AP in zebrafish through intraperitoneal injections of synthetic caerulein. Details are provided for solution preparation, pre-injection procedures, injection technique, and monitoring animal survival. Subsequently, validation was performed through biochemical and histological analyses of pancreatic tissue. The administered dose of caerulein for AP induction was 10 µg/kg applied four times in the intraperitoneal region. The histological validation study demonstrated the presence of necrosis within the first 12 h post-injection, accompanied by an excess of zymogen granules in the extracellular milieu. These observations align with those reported in conventional rodent models. We have standardized and validated the AP model in zebrafish. This model can contribute to preclinical and clinical studies of new drugs for AP treatment. Therefore, this novel model expands the toolkit for exploring faster and more effective preventive and therapeutic strategies for AP.

The tissue-specific nature of physiological zebrafish mitochondrial bioenergetics.

Zebrafish are a powerful tool to study a myriad of experimental conditions, including mitochondrial bioenergetics. Considering that mitochondria are different in many aspects depending on the tissue evaluated, in the zebrafish model there is still a lack of this investigation. Especially for juvenile zebrafish. In the present study, we examined whether different tissues from zebrafish juveniles show mitochondrial density- and tissue-specificity comparing brain, liver, heart, and skeletal muscle (SM). The liver and brain complex IV showed the highest O2 consumption of all ETC in all tissues (10x when compared to other respiratory complexes). The liver showed a higher potential for ROS generation. In this way, the brain and liver showed more susceptibility to O2- generation when compared to other tissues. Regarding Ca2+ transport, the brain showed greater capacity for Ca2+ uptake and the liver presented low Ca2+ uptake capacity. The liver and brain were more susceptible to producing NO. The enzymes SOD and Catalase showed high activity in the brain, whereas GPx showed higher activity in the liver and CS in the SM. TEM reveals, as expected, a physiological diverse mitochondrial morphology. The essential differences between zebrafish tissues investigated probably reflect how the mitochondria play a diverse role in systemic homeostasis. This feature may not be limited to normal metabolic functions but also to stress conditions. In summary, mitochondrial bioenergetics in zebrafish juvenile permeabilized tissues showed a tissue-specificity and a useful tool to investigate conditions of redox system imbalance, mainly in the liver and brain.

Performance of collagen-based matrices from Nile tilapia skin: A pilot proteomic study in a murine model of wound healing.

Full-thickness cutaneous trauma, due to the lack of dermis, leads to difficulty in epithelialization by keratinocytes, developing a fibrotic scar, with less elasticity than the original skin, which may have disorders in predisposed individuals, resulting in hypertrophic scar and keloids. Biomedical materials have excellent characteristics, such as good biocompatibility and low immunogenicity, which can temporarily replace traditional materials used as primary dressings. In this work, we developed two dermal matrices based on Nile tilapia collagen, with (M_GAG) and without (M) glycosaminoglycans, using a sugarcane polymer membrane as a matrix support. To assess the molecular mechanisms driving wound healing, we performed qualitative proteomic analysis on the wound bed in an in vivo study involving immunocompetent murine models at 14 and 21 days post-full-thickness skin injury. Gene Ontology and Pathway analysis revealed that both skins were markedly represented by modulation of the immune system, emphasizing controlling the acute inflammation response at 14 and 21 days post-injury. Furthermore, both groups showed significant enrichment of pathways related to RNA and protein metabolism, suggesting an increase in protein synthesis required for tissue repair and proper wound closure. Other pathways, such as keratinization and vitamin D3 metabolism, were also enriched in the groups treated with M matrix. Finally, both matrices improved wound healing in a full post-thick skin lesion. However, our preliminary molecular data reveals that the collagen-mediated healing matrix lacking glycosaminoglycan (M) exhibited a phenotype more favorable to tissue repair, making it more suitable for use before skin grafts.

Shrimp laccase degrades polycyclic aromatic hydrocarbons from an oil spill disaster in Brazil: A tool for marine environmental bioremediation.

Our work aims to purify, characterize and evaluate a laccase from by-products of the shrimp farming industry (Litopenaeus vannamei) for the degradation of Polycyclic Aromatic Hydrocarbons (PAHs) from 2019 oil spill in Brazilian coast. The enzyme was purified by affinity chromatography and characterized as thermostable, with activity above 90 °C and at alkaline pH. In addition, the laccase was also tolerant to copper, lead, cadmium, zinc, arsenic, hexane and methanol, with significant enzymatic activation in acetone and 10 mM mercury. Concerning PAHs' degradation, the enzyme degraded 42.40 % of the total compounds, degrading >50 % of fluorene, C4-naphthalenes, C3-naphthalenes, C2-naphthalenes, anthracene, acenaphthene, 1-methylnaphthalene and 2-methylnaphthalene. Thus, this laccase demonstrated important characteristics for bioremediation of marine environments contaminated by crude oil spills, representing a viable and ecological alternative for these purposes.

Binding Mechanism between Acetylcholinesterase and Drugs Pazopanib and Lapatinib: Biochemical and Biophysical Studies.

Tyrosine kinase inhibitors (TKIs) are antitumor compounds that prevent the phosphorylation of proteins in a biological environment. However, the multitarget performance of TKIs promotes them as possible candidates for drug repositioning. In this work, interaction and inhibition studies through spectroscopic and computational techniques to evaluate the binding effectiveness of lapatinib and pazopanib TKIs to acetylcholinesterase (AChE) are reported. The results indicated potent inhibition at the µM level. The types of inhibition were identified, with pazopanib acting through non-competitive inhibition and lapatinib through acompetitive inhibition. The fluorescence suppression studies indicate a static mechanism for lapatinib-AChE and pazopanib-AChE systems, with a binding constant in the order of 105 M-1. The obtained thermodynamic parameters reveal interactions driven by van der Waals forces and hydrogen bonds in the lapatinib-AChE system (ΔH° and ΔS° < 0). In contrast, the pazopanib-AChE system shows positive ΔH° and ΔS°, characteristic of hydrophobic interactions. The Foster resonance energy transfer study supports the fluorescence studies performed. The 3D fluorescence studies suggest changes in the microenvironment of the tryptophan and tyrosine residues of the protein in contact with lapatinib and pazopanib. The results suggest effective inhibition and moderate interaction of the drugs with AChE, making them interesting for conducting more in-depth repositioning studies as AChE inhibitors.

Non-clinical repeated dose 28-day oral toxicity, reproductive toxicity and cytotoxicity studies of the polar fraction of Parkinsonia aculeata aerial parts extract.

This study was aimed to evaluate toxicity in repeated doses for 28 days, reproductive toxicity and cytotoxicity of a polar fraction obtained from the hydroethanolic extract of Parkinsonia aculeata (PfrHEPA) in experimental models. To perform the toxicity test in repeated doses for 28 days, male and female Wistar rats were treated via orogastric for 28 days with PfrHEPA (35, 70 or 140 mg/kg) according to the guidelines established by the Organisation for Economic Co-operation and Development (OECD) number 407 (1995). For assessment, the impact of PfrHEPA on the reproductive output various parameters were measured, including maternal weight, no. of pregnant females, female fertility index (%), gestation lengthtime, implantation sites, litter size and placental index of test animals. The cytotoxicity of PfrHEPA was performed on the tumor lines NCI-H292 (human lung carcinoma), HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer). In the repeated dose toxicity test for 28 days, no mortality was observed in the male and female rats treated with PfrHEPA as well as morphological changes and biochemical and hematological parameters. In the reproductive toxicity test, no abnormalities were observed related to the toxicological parameters in both mothers and offspring. Regarding the cytotoxicity assay, the PfrHEPA fraction did not demonstrate significant cytotoxic effect on the cell lines analyzed. The present results suggest the use of PfrHEPA is safe and well tolerated in rats. Further studies are planned to identify and purify the active compounds for subsequent in vivo evaluation.

Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: A highly thermostable enzyme with promising features for estuarine biomonitoring.

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ±â€¯0.20 mM for gills and 0.43 ±â€¯0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ±â€¯1.72 and 27.71 ±â€¯1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol-1 (gills) and 11.87 kcal mol-1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.

Use of chitosan coating in increasing the shelf life of liquid smoked Nile tilapia (Oreochromis niloticus) fillet.

The aim of this research was to evaluate the efficiency of liquid smoking and chitosan coating on the shelf life of Nile tilapia (Oreochromis niloticus) fillets. Fillets without liquid smoked and chitosan coating (control), liquid smoked fillets (LS), and liquid smoked and chitosan coated fillets (LSCh) were stored at 4 ± 1 °C for 30 days. The physicochemical (pH, moisture content, water activity-aw, color, texture, total volatile bases nitrogen-TVB-N and thiobarbituric acid reactive substances-TBARS) and microbiological analyses (mesophilic and psychrotrophic counts) and the electrophoresis profile of samples were carried out. Physicochemical parameters, such as TVB-N and TBARS, were reduced in the tilapia fillets with liquid smoking. The presence of the coating of chitosan was effective for the control of the microorganisms during storage. This work showed that the addition of a chitosan coating in liquid-smoked fillets further enhanced the effect of preservation.

Effect of Moringa oleifera flower extract on larval trypsin and acetylcholinesterase activities in Aedes aegypti.

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (ß-amyrin), sterol (ß-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality.

Purification and characterization of a mannose recognition lectin from Oreochromis niloticus (tilapia fish): cytokine production in mice splenocytes.

The aim of this work was to purify and partially characterize a mannose recognition lectin from Nile tilapia (Oreochromis niloticus) serum, named OniL. OniL was isolated through precipitation with ammonium sulfate and affinity chromatography (Concanavalin A-Sepharose 4B). In addition, we evaluated carbohydrate specificity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles, and in vitro immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production. The ammonium sulfate fraction F2 showed the highest specific hemagglutinating activity (331) and was applied to affinity matrix. Adsorbed proteins (OniL) were eluted with methyl-α-D: -mannopyranoside. OniL, a 17-kDa protein by SDS-PAGE constituted by subunits of 11 and 6.6 kDa, showed highest affinity for methyl-α-D: -mannopyranoside and D: -mannose. Immunological assays, in vitro, showed that OniL did not show cytotoxicity against splenocytes, induced higher IFN-γ production and lower IL-10 as well as nitrite release. In conclusion, OniL lectin was successfully purified and showed a preferential Th1 response in mice splenocytes.

Dose-dependent effects of astaxanthin on cortical spreading depression in chronically ethanol-treated adult rats.

BACKGROUND: The consumption of alcoholic drinks is a frequent drug-abuse situation, which is associated to a wide variety of pathological disturbances affecting several organs, including the brain. We have previously shown in the developing rat brain that ethanol intake facilitates the propagation of cortical spreading depression (CSD), an excitability-related neural phenomenon present in several animal species. This electrophysiological effect was attenuated by a shrimp (Litopenaeus vannamei) carotenoids extract. Here we investigated the effects of pure astaxanthin, the main carotenoid found in shrimp, on CSD. METHODS: Adult Wistar rats were treated per gavage, during 18 days, with 2.5, 10 or 90 microg/kg/d astaxanthin dissolved in ethanol (3 g/kg) and CSD was recorded on the cortical surface 1 to 3 days thereafter. Four groups, treated respectively with ethanol, distilled water and soybean oil with- and without astaxanthin were also studied for comparison with the ethanol + astaxanthin groups. RESULTS: Ethanol-treated rats displayed higher CSD-velocities (mean values, in mm/min, per hour of recording ranging from 4.08 +/- 0.09 to 4.12 +/- 0.16), compared to the distilled water-group (from 3.19 +/- 0.13 to 3.27 +/- 0.06). Addition of astaxanthin to ethanol lead to lower CSD-velocities in a dose-dependent manner, ranging from 3.68 +/- 0.09 to 3.97 +/- 0.22 for the 2.5 microg/kg/d-dose, from 3.29 +/- 0.09 to 3.32 +/- 0.07 for the 10 microg/kg/d-dose, and from 2.89 +/- 0.13 to 2.92 +/- 0.11 for the 90 microg/kg/d-dose. The velocities of the soybean oil groups (with and without astaxanthin) were not statistically different from the 10 microg/kg/d astaxanthin + ethanol and distilled water groups. CONCLUSION: The results demonstrate the antagonistic effect of astaxanthin against the ethanol-induced facilitation of CSD propagation. Probably carotenoid antioxidant properties are involved in such effects.