Zebrafish Larvae Research Articles

Design, Synthesis, and Evaluation of the Antitubercular Activity of 5-Phenyl Substituted-5, 6-dihydropyrido[2, 3-d]pyrimidine-4, 7(3H, 8H)-dione Compounds.

Tuberculosis (TB) remains a major public health challenge, with research on new anti-TB drugs crucial for global TB elimination efforts. Here, we report a novel class of anti-TB agents. Especially, compounds 5b and 5j exhibited the highest activity [minimum inhibitory concentration (MIC) H37Rv: 0.16 and 0.12 μg/mL]. Chiral resolution was performed on compounds 5b and 5j; the isomers were evaluated for their activity and safety, confirming that the R-isomer 5bb and 5jb displayed significant anti-TB activity (MIC H37Rv: 0.03-0.06 μg/mL; MDR-Mtb: 0.125-0.06 μg/mL) and low hERG toxicity. Further evaluations on 5bb and 5jb demonstrated good metabolic stability, favorable kinetic parameters and oral bioavailability (F: 56.7 and 63.8%, respectively). The results of in vivo activity assessment indicate that 5bb and 5jb exhibit protective and therapeutic effects on zebrafish larvae and adult zebrafish infected with Mycobacterium marinum. Based on these results, compounds 5bb and 5jb are considered promising candidates for further in-depth studies.

Sodium thiosulfate treatment rescues hyperglycaemia-induced pronephros damage in zebrafish by upregulating nitric oxide signalling.

Sodium thiosulfate (STS) is gaining increasing attention in research for its potential therapeutic applications across a spectrum of disease processes beyond its current uses. However, the precise mechanisms of action remain incompletely understood. We investigated the efficacy of STS in treating hyperglycaemia-induced pronephros damage in zebrafish to gain further insight into the underlying mechanisms. Hyperglycaemia was induced in zebrafish by suppressing the pdx1 transcription factor, which plays a crucial role in maintaining physiological pancreatic function. STS was administered by introducing it into the medium of zebrafish larvae. The pronephros structure was analysed at 48h post-fertilization. Metabolomic profiling and RNA sequencing were conducted on groups exposed to various experimental conditions. Our findings reveal a downregulation of nitric oxide (NO) signalling in zebrafish with a knocked-down pdx1 gene, both metabolomically and transcriptionally. Notably, treatment with STS led to a compensatory upregulation of the NO signalling, ultimately resulting in the rescue of the pronephros structure. Our study provides compelling evidence that targeting NO metabolism by the administration of STS offers a promising strategy for addressing hyperglycaemia-induced organ damage. These findings underscore the potential of STS as a promising therapeutic agent for diabetic complications and warrant further investigation of its clinical applications. KEY POINTS: Sodium thiosulfate (STS) is increasingly drawing attention in research for its potential therapeutic applications across a spectrum of disease processes. Here, we demonstrate that STS treatment rescues hyperglycaemia-induced pronephros damage in zebrafish. We identified upregulation of nitric oxide signalling as the major driver behind STS-mediated rescue. Our data suggest that STS offers a promising strategy for addressing hyperglycaemia-induced organ damage, including diabetic nephropathy.

Ecotoxicological risk assessment of the novel psychoactive substance Esketamine: Emphasis on fish skeletal, behavioral, and vascular development

Novel psychoactive substances (NPS), such as Esketamine (Esket), often contaminate the aquatic ecosystems following human consumption, raising concerns about the residues and potential ecological hazards to non-target organisms. The study used zebrafish as a model organism to investigate the developmental toxicity and ecotoxicological effects of acute Esket exposure. Our findings demonstrate that exposure to Esket significantly affected the early development and angiogenesis of zebrafish embryos/larvae. The mandible length was significantly decreased, and the angles between the pharyngeal arch cartilages were narrowed compared to the control group (all P < 0.05). Additionally, Esket resulted in a decrease of 47.6–89.8 % in the number of neural crest cells (NCC). Transcriptome analysis indicated altered expression of genes associated with cartilage and osteoblast growth. Moreover, Esket significantly inhibited swimming ability in zebrafish larvae and was accompanied by behavioral abnormalities and molecular alterations in the brain. Potential mechanisms underlying Esket-induced behavioral disorders involve neurotransmitter system impairment, abnormal cartilage development and function, aberrant vascular development, as well as perturbations in oxidative stress and apoptosis signaling pathways. Notably, the dysregulation of skeleton development through the bone morphogenetic protein (BMP) signaling pathway is identified as the primary mechanistic behind Esket-induced behavioral disorder. This study enhances our understanding of Esket's ecotoxicology profile and provides a comprehensive assessment of the environmental risks associated with NPS.

The Effects of Kynurenic Acid in Zebrafish Embryos and Adult Rainbow Trout.

Kynurenic acid (KYNA) is a metabolite of tryptophan formed on the kynurenine pathway. Its pharmacological effects are relatively well characterized in mammals, whereas its role in fish is poorly understood. Therefore, the aim of the study was to expand the knowledge of KYNA's presence inside a fish's body and its impact on fish development and function. The study was performed on zebrafish larvae and adult rainbow trout. We provide evidence that KYNA is present in the embryo, larva and mature fish and that its distribution in organs varies considerably. A study of KYNA's effect on early larval development suggests that it can accelerate larval maturation, especially under conditions that are suboptimal for fish growth. Moreover, KYNA in concentrations over 1 mM caused morphological impairment and death of larvae. However, long-lasting exposure of larvae to subtoxic concentrations of KYNA does not affect the behavior of 5-day-old larvae kept under standard optimal conditions. We also show that ingestion of KYNA-supplemented feed can lead to KYNA accumulation, particularly in the pyloric caeca of mature trout. These results shed new light on the relevance of KYNA and provide new impulse for further research on the importance of the kynurenine pathway in fish.

Ellagic acid-enhanced biocompatibility and bioactivity in multilayer core-shell gold nanoparticles for ameliorating myocardial infarction injury

BackgroundMyocardial infarction (MI) is the main contributor to most cardiovascular diseases (CVDs), and the available post-treatment clinical therapeutic options are limited. The development of nanoscale drug delivery systems carrying natural small molecules provides biotherapies that could potentially offer new treatments for reactive oxygen species (ROS)-induced damage in MI. Considering the stability and reduced toxicity of gold-phenolic core-shell nanoparticles, this study aims to develop ellagic acid-functionalized gold nanoparticles (EA-AuNPs) to overcome these limitations.ResultsWe have successfully synthesized EA-AuNPs with enhanced biocompatibility and bioactivity. These core-shell gold nanoparticles exhibit excellent ROS-scavenging activity and high dispersion. The results from a label-free imaging method on optically transparent zebrafish larvae models and micro-CT imaging in mice indicated that EA-AuNPs enable a favorable excretion-based metabolism without overburdening other organs. EA-AuNPs were subsequently applied in cellular oxidative stress models and MI mouse models. We found that they effectively inhibit the expression of apoptosis-related proteins and the elevation of cardiac enzyme activities, thereby ameliorating oxidative stress injuries in MI mice. Further investigations of oxylipin profiles indicated that EA-AuNPs might alleviate myocardial injury by inhibiting ROS-induced oxylipin level alterations, restoring the perturbed anti-inflammatory oxylipins.ConclusionsThese findings collectively emphasized the protective role of EA-AuNPs in myocardial injury, which contributes to the development of innovative gold-phenolic nanoparticles and further advances their potential medical applications.Graphical

Metabolite profiling and bioactivity assessment of cyanobacteria from the Azores reveals unique producers of cytotoxic and lipid reducing compounds

Cyanobacteria are a group of photosynthetic bacteria and a rich secondary metabolites source. The Bank of Algae and Cyanobacteria of the Azores (BACA) culture collection holds a significant number of strains, including many novel genera and species. 56 strains from freshwater, brackish, and thermal habitats were selected, and grown under standard conditions. Biomass was extracted with methanol, and cytotoxicity was assessed on two carcinoma cell lines, HepG2 and HCT116. The reduction of lipids was tested in zebrafish larvae, and in a steatosis model with fatty acid overloaded human liver cells. The cyanobacterial metabolome was analyzed by HR-ESI-LC-MS/MS and compared using CompareMS2. High similarities were observed in strains of the same genus when isolated from similar habitats, clustering in concordance to the taxonomical order, while no relation could be observed between strains from different genera originated from the same habitat. The extracts of Cyanobium sp. BACA0019, Pseudocalidococcus azoricus BACA0433 and Pegethrix atlantica BACA0077 reduced neutral lipids >40 % in zebrafish at 25 μg/mL, while from Symphyonema sp. BACA0090 and Aliinostoc sp. BACA0355 induced mortality. Lipid reduction in the steatosis model was observed in many strains, with significant results varying from 50 % to 100 %. Several strains reduced cell viability with the strongest effects from Scytonematopsis sp. BACA0005 (HepG2, 59.8 % and HCT116, 68.1 %), Aliinostoc sp. BACA0035 (HepG2, 43.3 %, and HCT116, 59.4 %) and Aliinostoc sp. BACA0355 (HepG2, 46.2 %, and HCT116, 75.5 %). The feature-based molecular networking identified several cluster of mass peaks related to the observed bioactivities. Chlorophyll derivatives and glycerolipids from Cyanobium sp. BACA0019, Pseudocalidococcus azoricus BACA0433 and Pegethrix atlantica BACA0077 were correlated with the reduction of lipids in zebrafish larvae, while several oligopeptides and fatty amides of Symphyonema sp. BACA0090 and Aliinostoc sp. BACA0355 with toxicity. Many clusters associated to the bioactivities remained unidentified, which may represent novel compounds, highlighting the chemodiversity of the BACA culture collection.

Ammonia exposure impairs bone mineralization in zebrafish (Danio rerio) larvae

Ammonia is a major pollutant of freshwater environments. Previous studies have indicated that ammonia exposure adversely affects the physiology of freshwater fish. However, its effect on bone mineralization in freshwater fish larvae remains unclear. In this study, zebrafish larvae were used as a model to investigate the effects of different ammonia levels (0, 2.5, 5, and 10 mM NH4Cl) on the survival rate, body length, and bone mineralization of fish. The survival rate of zebrafish embryos exposed to different NH4Cl concentrations for 8 days was not affected. In contrast, the body length and bone mineralization of zebrafish larvae at 8 days post fertilization (dpf) were significantly reduced at 5 and 10 mM NH4Cl exposure. Further investigations revealed that ammonia exposure decreased the mRNA expression of osteoblast-related genes and increased that of osteoclast-related genes. Additionally, exposure to 5 mM and 10 mM NH4Cl induced the production of reactive oxygen species (ROS). 10 mM—but not 5 mM—NH4Cl exposure reduced the calcium and phosphorus content in 8 dpf zebrafish larvae. In conclusion, ammonia exposure induces bone resorption, while decreasing the calcium and phosphorus content of the whole body and bone formation, resulting in impaired bone mineralization in fish larvae.

Screening of Lipid-Reducing Activity and Cytotoxicity of the Exometabolome from Cyanobacteria.

Cyanobacteria are rich producers of secondary metabolites, excreting some of these to the culture media. However, the exometabolome of cyanobacteria has been poorly studied, and few studies have dwelled on its characterization and bioactivity assessment. In this work, exometabolomes of 56 cyanobacterial strains were characterized by HR-ESI-LC-MS/MS. Cytotoxicity was assessed on two carcinoma cell lines, HepG2 and HCT116, while the reduction in lipids was tested in zebrafish larvae and in a steatosis model with fatty acid-overloaded human liver cells. The exometabolome analysis using GNPS revealed many complex clusters of unique compounds in several strains, with no identifications in public databases. Three strains reduced viability in HCT116 cells, namely Tolypotrichaceae BACA0428 (30.45%), Aphanizomenonaceae BACA0025 (40.84%), and Microchaetaceae BACA0110 (46.61%). Lipid reduction in zebrafish larvae was only observed by exposure to Dulcicalothrix sp. BACA0344 (60%). The feature-based molecular network shows that this bioactivity was highly correlated with two flavanones, a compound class described in the literature to have lipid reduction activity. The exometabolome characterization of cyanobacteria strains revealed a high chemodiversity, which supports it as a source for novel bioactive compounds, despite most of the time being overlooked.

Characterizing the adult zebrafish model of Parkinson's disease: a systematic review of dynamic changes in behavior and physiology post-MPTP administration.

Adult zebrafish are increasingly used in Parkinson's disease (PD) research due to their well-characterized dopaminergic system. Among the toxin-based models, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely utilized to induce parkinsonism in adult zebrafish. Therefore, this review presents an overview of the procedures and the dynamic changes in behavior and physiology observed in the adult zebrafish PD model following a single intraperitoneal injection of MPTP. A systematic literature search in the PubMed and Google Scholar databases was conducted to identify relevant articles. Of the 165 articles identified, 9 were included in this review. These chosen articles are original works published before March 2024, all of which utilized adult zebrafish induced with MPTP as the model for PD. Other articles were excluded based on factors such as limited relevance, utilization of zebrafish embryos or larvae instead of adults, and variations in MPTP deliveries. Studies indicated that the ideal model entails the utilization of mixed gender zebrafish aged between 4 and 6 months from the wild-type strain. The acceptable MPTP doses ranges between 20 μg/g (lowest) and 225 μg/g (highest) and doses above 292 μg/g are lethal. Furthermore, noticeable parkinsonian symptoms appear 1 day after administration and persist for more than 1 week. Mitochondrial dysfunction precedes dopaminergic neurodegeneration within this experimental regime. A single administration of MPTP effectively induces PD in adult zebrafish. This study aids in crafting the adult zebrafish PD model, outlining the progressive behavioral and physiological changes ensuing from MPTP administration.

Cloning of down-regulated genes under cold stress and identification of important genes related to cold tolerance in zebrafish (Danio rerio)

Low-temperature stress poses a significant risk to the survival of both cultivated and wild fish populations. Existing studies have found that the pre-acclimation of fishes to moderate cold stress can stimulate the activation of acclimation pathways, thereby enhancing their tolerance to cold stress. The fitness of fish relies heavily on appropriately controlled transcriptional reactions to environmental changes. Despite previous characterization of gene expression profiles in various fish species during cold acclimation, the specific genes responsible for essential functions in this process remain largely unknown, particularly the down-regulated genes induced by cold acclimation. To investigate the genes involved in cold acclimation, this study employed real-time quantitative PCR (RT-qPCR), molecular cloning, microinjection techniques, and cold stress experiments to determine the genes that play an essential part in cold acclimation. Consequently, 18 genes were discovered to be down-regulated in larval zebrafish experiencing cold stress. All 18 genes successfully detected overexpression in zebrafish at 96 and 126 hpf (fold change ≥3), which declined with the growth of zebrafish. Following microinjection, it was observed that her8a, cyp51, lss, txnipb, and bhlha9 had an adverse impact on the survival rate of zebrafish larvae under cold stress. These genes have been identified to play significant roles in various biological processes. For instance, bhlha9 has been found to be involved in both limb development and temperature sensing and her8a has been implicated in neural development. Additionally, cyp51 and lss have been identified as participants in the cholesterol synthesis pathway. Txnipb has been reported to induce cell apoptosis, thereby potentially influencing the survival rate of zebrafish larvae under cold stress. These findings offered crucial data for the analysis of molecular processes related to cold tolerance and the development of cold-resistant fish breeding.

Multi-targeted action of rooibos may protect against ischaemic stroke-induced neurological deficit and endothelial dysfunction

Ethnopharmacological relevanceIndigenous use communities in the Western Cape (South Africa) where Aspalathus linearis (Brum.f) R.Dahlgren - or rooibos - grows naturally, has a long history of using rooibos for medicinal purposes. Apart from its well-known antioxidant effect, the Cederberg community in particular has been using rooibos as a treatment for high blood pressure. Given the detrimental effects of high blood pressure on endothelial cells, rooibos may either directly or indirectly affect vascular health. This, together with more recent reports of neuroprotective effects, may position rooibos as complementary medicine in related vascular conditions such as ischaemic stroke. Aims of the studyThe study aimed to evaluate the potential benefit of acute administration of unfermented rooibos, on vascular health in a larval zebrafish model of stroke. Materials and methodsStroke was induced via 24-h ponatinib exposure, in the presence or absence of an aqueous solution of an ethanolic extract of unfermented Rooibos (GreenOxithin™). The magnitude of stroke was assessed by monitoring larval locomotion and thrombus formation. In terms of specific mechanisms probed, changes in redox status (MDA and TEAC), neurological markers (TH and NeuroD1) and endothelial health (tight/adhesion junction protein expression) were assessed. ResultsRooibos treatment limited thrombus formation and prevented stroke-induced deficits on larval motility. In terms of redox status, rooibos treatment prevented lipid peroxidation 3 days after initial stroke induction, reducing the need for significant upregulation of endogenous antioxidant mechanisms. Stroke-induced changes in neuronal (NeuroD1 and TH) protein expression were normalized in the presence of rooibos, suggesting a neuroprotective role. In terms of tight junction proteins, stroke-related decreases in ZO-1 expression were again prevented by rooibos treatment. In addition, rooibos treatment may beneficially modulate levels of claudin-5 and VE-cadherin, to indirectly limit stroke-associated vascular dysfunction. ConclusionsTaken together, activity data and physiological assessments suggest that unfermented rooibos may indeed have benefit in the context of stroke, via action at multiple targets. Thus, current data further our understanding of the mechanisms of actions of rooibos and warrant future research to confirm sufficient bioavailability of rooibos in target tissues, in mammalian systems.

Deep autoencoder-based behavioral pattern recognition outperforms standard statistical methods in high-dimensional zebrafish studies.

Zebrafish have become an essential model organism in screening for developmental neurotoxic chemicals and their molecular targets. The success of zebrafish as a screening model is partially due to their physical characteristics including their relatively simple nervous system, rapid development, experimental tractability, and genetic diversity combined with technical advantages that allow for the generation of large amounts of high-dimensional behavioral data. These data are complex and require advanced machine learning and statistical techniques to comprehensively analyze and capture spatiotemporal responses. To accomplish this goal, we have trained semi-supervised deep autoencoders using behavior data from unexposed larval zebrafish to extract quintessential "normal" behavior. Following training, our network was evaluated using data from larvae shown to have significant changes in behavior (using a traditional statistical framework) following exposure to toxicants that include nanomaterials, aromatics, per- and polyfluoroalkyl substances (PFAS), and other environmental contaminants. Further, our model identified new chemicals (Perfluoro-n-octadecanoic acid, 8-Chloroperfluorooctylphosphonic acid, and Nonafluoropentanamide) as capable of inducing abnormal behavior at multiple chemical-concentrations pairs not captured using distance moved alone. Leveraging this deep learning model will allow for better characterization of the different exposure-induced behavioral phenotypes, facilitate improved genetic and neurobehavioral analysis in mechanistic determination studies and provide a robust framework for analyzing complex behaviors found in higher-order model systems.

Toxicity and Metabolomic Dysfunction Invoked by Febrifugin, a Harmful Component of Edible Nut of Swietenia macrophylla.

Swietenia macrophylla fruit is a valuable and historically significant medicinal plant with anti-hypertension and anti-diabetes. We identified a toxic component, Febrifugin, from the edible part of the nut following zebrafish toxicity-guided isolation. Febrifugin is a mexicanolide-type limonoid compound. The toxic factor induced acute toxicity in zebrafish, including yolk sac edema and pericardial edema, reduced body length, decreased melanin deposition, and presented acute skeletal developmental issues. Further exploration of the acute toxicity mechanism through metabolomics revealed that Febrifugin caused significant changes in 13 metabolites in zebrafish larvae, which are involved in the pentose phosphate, tricarboxylic acid (TCA) cycle, and amino acid biosynthesis. The bioassay of oxidative stress capacity and qRT-PCR measurement showed that the compound significantly affected the h6pd gene in the pentose phosphate pathway and the mRNA expression of cs, idh3a, fh, and shda genes in the TCA cycle, leading to reactive oxygen species (ROS) accumulation and a notable decrease in glutathione (GSH) activity in zebrafish. These findings provide a basis for the rational use of S. macrophylla as a medicinal plant and raise awareness of the safety of medicinal plants.

Analyzing the neurotoxic effects of anatoxin-a and saxitoxin in zebrafish larvae

Global warming due to climate change, as well as freshwater eutrophication caused by anthropogenic activities are responsible, among other factors, for an increasing occurrence of harmful algal blooms (HABs) in aquatic systems. These can lead to the generation of cyanotoxins, secondary metabolites coming from cyanobacteria, producing adverse effects in living organisms including death. This research aims to study the effects that two neurotoxins, anatoxin-a (ATX-a) and saxitoxin (STX), have on living organisms. Once the stability of both compounds in water was determined for a 24 h period using ultra-high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UPLC-MS/MS), zebrafish larvae were exposed to different levels of toxins (1 ng L−1, 10 ng L−1, 100 ng L−1 and 1 μg L−1) during 24 h. Behavioral studies including vibrational startle response (VSR), habituation to vibrational stimuli, basal locomotor activity (BLM) and visual motor response (VMR) were performed using Danio Vision system, and neurotransmitters (NTs) from 15-head pools of control and exposed zebrafish larvae were extracted and analyzed by UPLC-MS/MS. Both compounds induced hypolocomotion in the individuals, while 10 and 100 ng L−1 of ATX-a significantly increased methionine (120 % and 126 %, respectively) and glutamate levels (118 % and 129 %, respectively). Saxitoxin enhanced 3-metoxytyramine (3-MT) levels at 1 ng L−1 by 185 %. The findings of this study show that both studied cyanotoxins influence the behavior of zebrafish larvae as well as their metabolism.

Brain-wide circuitry underlying altered auditory habituation in zebrafish models of autism.

Auditory processing is widely understood to occur differently in autism, though the patterns of brain activity underlying these differences are not well understood. The diversity of autism also means brain-wide networks may change in various ways to produce similar behavioral outputs. We used larval zebrafish to investigate auditory habituation in four genetic lines relevant to autism: fmr1, mecp2, scn1lab and cntnap2. In free-swimming behavioral tests, we found each line had a unique profile of auditory hypersensitivity and/or delayed habituation. Combining the optical transparency of larval zebrafish with genetically encoded calcium indicators and light-sheet microscopy, we then observed brain-wide activity at cellular resolution during auditory habituation. As with behavior, each line showed unique alterations in brain-wide spontaneous activity, auditory processing, and adaptation in response to repetitive acoustic stimuli. We also observed commonalities in activity across our genetic lines that indicate shared circuit changes underlying certain aspects of their behavioral phenotypes. These were predominantly in regions involved in sensory integration and sensorimotor gating rather than primary auditory areas. Overlapping phenotypes include differences in the activity and functional connectivity of the telencephalon, thalamus, dopaminergic regions, and the locus coeruleus, and excitatory/inhibitory imbalance in the cerebellum. Unique phenotypes include loss of activity in the habenula in scn1lab, increased activity in auditory regions in fmr1, and differences in network activity over time in mecp2 and cntnap2. Comparing these distinct but overlapping brain-wide auditory networks furthers our understanding of how diverse genetic factors can produce similar behavioral effects through a range of circuit- and network-scale mechanisms.

Neural Basis of Number Sense in Larval Zebrafish.

Number sense, the ability to discriminate the quantity of objects, is crucial for survival. To understand how neurons work together and develop to mediate number sense, we used two-photon fluorescence light sheet microscopy to capture the activity of individual neurons throughout the brain of larval Danio rerio, while displaying a visual number stimulus to the animal. We identified number-selective neurons as early as 3 days post-fertilization and found a proportional increase of neurons tuned to larger quantities after 3 days. We used machine learning to predict the stimulus from the neuronal activity and observed that the prediction accuracy improves with age. We further tested ethanol's effect on number sense and found a decrease in number-selective neurons in the forebrain, suggesting cognitive impairment. These findings are a significant step towards understanding neural circuits devoted to discrete magnitudes and our methodology to track single-neuron activity across the whole brain is broadly applicable to other fields in neuroscience.

The Human Pathogen Mycobacterium tuberculosis and the Fish Pathogen Mycobacterium marinum Trigger a Core Set of Late Innate Immune Response Genes in Zebrafish Larvae.

Zebrafish is a natural host of various Mycobacterium species and a surrogate model organism for tuberculosis research. Mycobacterium marinum is evolutionarily one of the closest non-tuberculous species related to M. tuberculosis and shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos with M. tuberculosis and performed drug treatment of the infected larvae. In the present study, we examined for how long M. tuberculosis can be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Bacterial aggregates carrying fluorescently labeled M. tuberculosis could be detected up to 9 days post-infection. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the inflammatory response of zebrafish larvae infected with the surrogate species M. marinum. We conclude that M. tuberculosis can be propagated in zebrafish larvae for at least one week after infection and provide further evidence that M. marinum is a good surrogate model for M. tuberculosis. The generated extensive transcriptome data sets will be of great use to add translational value to zebrafish as a model for infection of tuberculosis using the M. marinum infection system. In addition, we identify new marker genes such as dusp8 and CD180 that are induced by M. tuberculosis infection in zebrafish and in human macrophages at later stages of infection that can be further investigated.

Metabolic profile changes of zebrafish larvae in the single- and co-exposures of microplastics and phenanthrene

Microplastics (MPs) are ubiquitous in the environment, and can adsorb organic contaminants (OCs) and be taken by various microorganisms and organisms, which could eventually lead to risk to humans. In this study, the phenotypic changes and metabolic profile alternations of zebrafish in the single- and co-exposure of MPs and phenanthrene (Phe) were investigated. The results showed that significantly higher tail malformation rate and edema rate in zebrafish induced by MPs can be enhanced due to the co-existence of Phe. The metabolomic analysis revealed that both synergistic and antagonistic effects of MPs and Phe on the metabolic alternation of zebrafish larvae exist, since unique perturbations of metabolites or pathways were found in all of the three exposure scenarios. Based on Partial least squares-discriminant analysis, porphine, ribose, and L-glutamic acid were the most important metabolites resulting in the difference between the treated and control groups in the MP exposure, Phe exposure and co-exposure, respectively. Two dysregulated pathways namely d-glutamine and D-glutamate metabolism, and alanine, aspartate and glutamate metabolism were significantly affected in the co-exposure while not in either of the single exposure. These findings provide new insights into the toxic effects of MPs on aquatic organisms, and further studies on combined effects of MPs and OCs are suggested to be conducted.

Investigating recovery after a spontaneous intracerebral haemorrhage in zebrafish larvae.

Intracerebral haemorrhage is a debilitating stroke sub-type with high morbidity and mortality rates. For survivors, rehabilitation is a long process, and with no available therapeutics to limit the immediate pathophysiology of the haemorrhage, recovery is dependent on individual neuroplasticity. We have previously shown that zebrafish larvae can be used to model spontaneous brain haemorrhage. Zebrafish exhibit innate recovery mechanisms and are often used as a model system for investigation into regeneration after injury, including injury to the nervous system. Here, we investigate the spontaneous and immediate recovery in zebrafish larvae following an intracerebral haemorrhage at 2 days post-fertilisation, during pre-protected stages and over the first 3 weeks of life. We have shown that following the onset of bleed at ∼2 days post-fertilisation zebrafish are capable of clearing the haematoma through the ventricles. Brain cell damage associated with intracerebral haemorrhage is resolved within 48 h, and this recovery is associated with survival rates equal to wildtype and non-haemorrhaged sibling control animals. Larvae express more nestin-positive neural progenitor cells 24 h after injury when the most damage is observed, and through mass spectrometry analysis, we have determined that these cells are highly proliferative and may specially differentiate into oligodendrocytes. This study provides an insight into the haematoma resolution processes in a live, intact organism, and may suggest potential therapeutic approaches to support the recovery of intracerebral haemorrhage patients.

Effective paclitaxel: β-Cyclodextrin-based formulation boosts in vitro anti-tumor potential and lowers toxicity in zebrafish.

Paclitaxel (PCTX) is one of the most prevalently used chemotherapeutic agents. However, its use is currently beset with a host of problems: solubility issue, microplastic leaching, and drug resistance. Since drug discovery is challenging, we decided to focus on repurposing the drug itself by remedying its drawbacks and making it more effective. In this study, we have harnessed the aqueous solubility of sugars, and the high affinity of cancer cells for them, to entrap the hydrophobic PCTX within the hydrophilic shell of the carbohydrate β-cyclodextrin. We have characterized this novel drug formulation by testing its various physical and chemical parameters. Importantly, in all our in vitro assays, the conjugate performed better than the drug alone. We find that the conjugate is internalized by the cancer cells (A549) via caveolin 1-mediated endocytosis. Thereafter, it triggers apoptosis by inducing the formation of reactive oxygen species. Based on experiments on zebrafish larvae, the formulation displays lower toxicity compared to PCTX alone. Thus, our "Trojan Horse" approach, relying on minimal components and relatively faster formulation, enhances the anti-tumor potential of PCTX, while simultaneously making it more innocuous toward non-cancerous cells. The findings of this study have implications in the quest for the most cost-effective chemotherapeutic molecule.