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International Journal of Molecular... Feb 2024Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting...
Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.
Topics: Animals; Drug-Related Side Effects and Adverse Reactions; Chemical and Drug Induced Liver Injury; Bioprinting; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds
PubMed: 38339088
DOI: 10.3390/ijms25031811 -
Scientific Reports Apr 2024Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation...
Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.
Topics: Animals; Mice; alpha-Synuclein; Disease Models, Animal; DNA-Binding Proteins; Dopamine; Neurodegenerative Diseases; Parkinson Disease; Plasminogen; Serine Proteases; tau Proteins; Dopaminergic Neurons
PubMed: 38615036
DOI: 10.1038/s41598-024-59090-8 -
Toxins Oct 2023For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species....
For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels () as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5-75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves (21 µg kg) and (63 µg kg), respectively. However, mean concentrations for the bivalve group were shallow (2-6 µg kg). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6-44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions.
Topics: Animals; Humans; Okadaic Acid; Marine Toxins; Shellfish; Shellfish Poisoning; Chromatography, Liquid; Tandem Mass Spectrometry; Bivalvia; Mytilus
PubMed: 37999494
DOI: 10.3390/toxins15110631 -
Life (Basel, Switzerland) Dec 2022Toxins of the OA-group (okadaic acid, OA; dinophysistoxin-1, DTX-1) are the most prevalent in the fjords of southern Chile, and are characterized by their potential...
Toxins of the OA-group (okadaic acid, OA; dinophysistoxin-1, DTX-1) are the most prevalent in the fjords of southern Chile, and are characterized by their potential harmful effects on aquatic organisms. The present study was carried out to determine the acute toxicity of OA/DTX-1 on oxidative stress parameters in medaka () larvae. Medaka larvae were exposed to different concentrations (1.0-30 μg/mL) of OA/DTX-1 for 96 h to determine the median lethal concentration. The LC value after 96 h was 23.5 μg/mL for OA and 16.3 μg/mL for DTX-1 (95% confidence interval, CI was 22.56, 24.43 for OA and 15.42, 17.17 for DTX-1). Subsequently, larvae at 121 hpf were exposed to acute doses (10, 15 and 20 μg/mL OA and 5.0, 7.5 and 11.0 μg/mL DTX-1) for 96 h and every 6 h the corresponding group of larvae was euthanized in order to measure the activity levels of biochemical biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx; and glutathione reductase, GR) as well as the levels of oxidative damage (malondialdehyde, MDA; and carbonyl content). Our results showed that acute doses caused a decrease in SOD (≈25%), CAT (≈55%), and GPx and GR (≈35%) activities, while MDA levels and carbonyl content increased significantly at the same OA/DTX-1 concentrations. This study shows that acute exposure to OA-group toxins tends to simultaneously alter the oxidative parameters that induce sustained morphological damage in medaka larvae. DTX-1 stands out as producing greater inhibition of the antioxidant system, leading to increased oxidative damage in medaka larvae. Considering that DTX-1 is the most prevalent HAB toxin in southern Chile, these findings raise the possibility of an important environmental impact on the larval stages of different fish species present in the southern fjords of the South Pacific.
PubMed: 36675964
DOI: 10.3390/life13010015 -
Comparative Biochemistry and... Sep 2023Globally around 24 million elderly population are dealing with dementia, and this pathological characteristic is commonly seen in people suffering from Alzheimer's...
Globally around 24 million elderly population are dealing with dementia, and this pathological characteristic is commonly seen in people suffering from Alzheimer's disease (AD). Despite having multiple treatment options that can mitigate AD symptoms, there is an imperative call to advance our understanding of the disease pathogenesis to unfold disease-modifying treatments/therapies. To explore the driving mechanisms of AD development, we stretch out further to study time-dependant changes after Okadaic acid (OKA)-induced AD-like conditions in zebrafish. We evaluated the pharmacodynamics of OKA at two-time points, i.e., after 4-days and 10-days exposure to zebrafish. T-Maze was utilized to observe the learning and cognitive behaviour, and inflammatory gene expressions such as 5-Lox, Gfap, Actin, APP, and Mapt were performed in zebrafish brains. To scoop everything out from the brain tissue, protein profiling was performed using LCMS/MS. Both time course OKA-induced AD models have shown significant memory impairment, as evident from T-Maze. Gene expression studies of both groups have reported an overexpression of 5-Lox, GFAP, Actin, APP, and OKA 10D group has shown remarkable upregulation of Mapt in zebrafish brains. In the case of protein expression, the heatmap suggested an important role of some common proteins identified in both groups, which can be explored further to investigate their mechanism in OKA-induced AD pathology. Presently, the preclinical models available to understand AD-like conditions are not completely understood. Hence, utilizing OKA in the zebrafish model can be of great importance in understanding the pathology of AD progression and as a screening tool for drug discovery.
Topics: Aged; Animals; Humans; Alzheimer Disease; Zebrafish; Proteomics; Actins; Brain; Okadaic Acid; Genomics; Disease Models, Animal
PubMed: 37100105
DOI: 10.1016/j.cbpc.2023.109636 -
Toxins May 2023The successful cultivation of Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its...
The successful cultivation of Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its toxin content and production for the first time. Maintaining the strains at a high abundance (>2000 cells per mL) for more than 20 months was achieved by feeding them with the ciliate Lohmann, 1908, along with the addition of the cryptophyte (W.Conrad) D.R.A.Hill, 1992. Toxin production was examined using seven established strains. At the end of the one-month incubation period, the total amounts of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged between 132.0 and 375.0 ng per mL (n = 7), and 0.7 and 3.6 ng per mL (n = 3), respectively. Furthermore, only one strain was found to contain a trace level of okadaic acid (OA). Similarly, the cell quota of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged from 60.6 to 152.4 pg per cell (n = 7) and 0.5 to 1.2 pg per cell (n = 3), respectively. The results of this study indicate that toxin production in this species is subject to variation depending on the strain. According to the growth experiment, exhibited a long lag phase, as suggested by the slow growth observed during the first 12 days. In the growth experiment, grew very slowly for the first 12 days, suggesting they had a long lag phase. However, after that, they grew exponentially, with a maximum growth rate of 0.56 divisions per day (during Days 24-27), reaching a maximum concentration of 3000 cells per mL at the end of the incubation (Day 36). In the toxin production study, the concentration of DTX1 and PTX2 increased following their vegetative growth, but the toxin production still increased exponentially on Day 36 (1.3 ng per mL and 154.7 ng per mL of DTX1 and PTX2, respectively). The concentration of OA remained below detectable levels (≤0.010 ng per mL) during the 36-day incubation period, with the exception of Day 6. This study presents new information on the toxin production and content of , as well as insights into the maintenance and culturing of this species.
Topics: Marine Toxins; Japan; Bays; Dinoflagellida; Okadaic Acid; Ciliophora
PubMed: 37235353
DOI: 10.3390/toxins15050318 -
Journal of the American Chemical Society Feb 2023The dinoflagellate-derived polyether prorocentin is a co-metabolite of the archetypical serine/threonine phosphatase inhibitor okadaic acid. Whereas a structural...
The dinoflagellate-derived polyether prorocentin is a co-metabolite of the archetypical serine/threonine phosphatase inhibitor okadaic acid. Whereas a structural relationship cannot be missed and a biosynthetic link was proposed, it is currently unknown whether there is any parallel in the bioactivity profile of these natural products. However, it was insinuated in the past that the structure assigned to prorocentin might need to be revised. Indeed, re-examination of the published spectra cast doubts as to the constitution of the fused/spirotricyclic BCD-ring system in the core. To clarify this issue, a flexible synthesis blueprint was devised that allowed us to obtain the originally proposed structure as well as the most plausible amended structure. The key to success was late-stage gold-catalyzed spirocyclization reactions that furnished the isomeric central segments with excellent selectivity. The lexicon of catalytic transformations used to make the required cyclization precursors comprised a titanium-mediated ester methylenation/metathesis cascade, a rare example of a gold-catalyzed allylic substitution, and chain extensions via organocatalytic asymmetric aldehyde propargylation. A wing sector to be attached to the isomeric cores was obtained by Krische allylation, followed by a superbly selective cobalt-catalyzed oxidative cyclization of the resulting di-unsaturated alcohol with the formation of a 2,5--disubstituted tetrahydrofuran; the remaining terminal alkene was elaborated into an appropriate handle for fragment coupling by platinum-catalyzed asymmetric diboration/oxidation. The assembly of the different building blocks to the envisaged isomeric target compounds proved that the structure of prorocentin needs to be revised as disclosed herein.
PubMed: 36652728
DOI: 10.1021/jacs.2c12529 -
EXCLI Journal 2023The marine biotoxin okadaic acid (OA) is produced by dinoflagellates and enters the human food chain by accumulating in the fatty tissue of filter-feeding shellfish....
The marine biotoxin okadaic acid (OA) is produced by dinoflagellates and enters the human food chain by accumulating in the fatty tissue of filter-feeding shellfish. Consumption of highly contaminated shellfish can lead to diarrheic shellfish poisoning. However, apart from the acute effects in the intestine, OA can also provoke toxic effects in the liver, as it is able to pass the intestinal barrier into the blood stream. However, molecular details of OA-induced hepatotoxicity are still insufficiently characterized, and especially at the proteomic level data are scarce. In this study, we used human HepaRG liver cells and exposed them to non-cytotoxic OA concentrations for 24 hours. Global changes in protein expression were analyzed using 2-dimensional gel electrophoresis in combination with mass-spectrometric protein identification. The results constitute the first proteomic analysis of OA effects in human liver cells and indicate, amongst others, that OA affects the energy homeostasis, induces oxidative stress, and induces cytoskeletal changes.
PubMed: 38054204
DOI: 10.17179/excli2023-6458 -
The Journal of Neuroscience : the... Jun 2021Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play...
Protein aggregation can induce explicit neurotoxic events that trigger a number of presently untreatable neurodegenerative disorders. Chaperones, on the other hand, play a neuroprotective role because of their ability to unfold and refold abnormal proteins. The progressive nature of neurotoxic events makes it important to discover endogenous factors that affect pathologic and molecular phenotypes of neurodegeneration in animal models. Here, we identified microtubule-associated protein tau, and chaperones Hsp70 (heat shock protein 70) and DNAJA1 (DJ2) as endogenous substrates of cereblon (CRBN), a substrate-recruiting subunit of cullin4-RING-E3-ligase. This recruitment results in ubiquitin-mediated degradation of tau, Hsp70, and DJ2. Knocking out CRBN enhances the chaperone activity of DJ2, resulting in decreased phosphorylation and aggregation of tau, improved association of tau with microtubules, and reduced accumulation of pathologic tau across brain. Functionally abundant DJ2 could prevent tau aggregation induced by various factors like okadaic acid and heparin. Depletion of CRBN also decreases the activity of tau-kinases including GSK3α/β, ERK, and p38. Intriguingly, we found a high expression of CRBN and low levels of DJ2 in neuronal tissues of 5XFAD and APP knock-in male mouse models of Alzheimer's disease. This implies that CRBN-mediated DJ2/Hsp70 pathway may be compromised in neurodegeneration. Being one of the primary pathogenic events, elevated CRBN can be a contributing factor for tauopathies. Our data provide a functional link between CRBN and DJ2/Hsp70 chaperone machinery in abolishing the cytotoxicity of aggregation-prone tau and suggest that mice serve as an animal model of resistance against tauopathies for further exploration of the molecular mechanisms of neurodegeneration.
Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line; Disease Models, Animal; HSP40 Heat-Shock Proteins; Humans; Male; Mice; Nerve Degeneration; Tauopathies; Ubiquitin-Protein Ligases; tau Proteins
PubMed: 33972400
DOI: 10.1523/JNEUROSCI.2494-20.2021 -
Aging Mar 2023Studies showed that thyroid function plays an important role in the pathology of Alzheimer's disease (AD). However, changes in brain thyroid hormone and related...
BACKGROUND
Studies showed that thyroid function plays an important role in the pathology of Alzheimer's disease (AD). However, changes in brain thyroid hormone and related receptors in the early stage of AD were rarely reported. The aim of this study was to explore the relationship between the early stage of AD and local thyroid hormone and its receptors in the brain.
METHODS
The animal model was established by stereotactic injection of okadaic acid (OA) into hippocampal region for the experiment, and 0.9% NS for the control. Blood sample from each mouse was collected and then the mice were sacrificed and the brain tissue was collected for detecting free triiodothyronine (FT3), free thyroid hormone (FT4), and thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH) and phosphorylated tau, amyloid-β (Aβ) and thyroid hormone receptors (THRs) in the hippocampus of the mice were detected as well.
RESULTS
Enzyme-linked immunosorbent assay showed that compared with the control, FT3, FT4, TSH and TRH in brain were significantly increased in the experimental group; in the serum, FT4, TSH and TRH were increased, while FT3 had no change; western blot analysis indicated that the expression of THR α and β in the hippocampus of the experimental group was significantly higher than that of the control.
CONCLUSION
Based on the results of this study, a mouse AD model can be established successfully by injecting a small dose of OA into the hippocampus. We speculate that early AD brain and circulating thyroid dysfunction may be an early local and systemic stress repair response.
Topics: Mice; Animals; Thyroid Gland; Alzheimer Disease; Thyroid Hormones; Thyrotropin; Thyrotropin-Releasing Hormone; Brain; Thyroxine
PubMed: 36897166
DOI: 10.18632/aging.204570