Polystyrene nanoplastics cause reproductive toxicity in zebrafish: PPAR mediated lipid metabolism disorder.

The ubiquitous presence of micro-and nanoplastics (MNPs) in the environment and everyday products has attracted attention due to their hazardous risks. However, the effects of MNPs on reproduction and the underlying mechanisms remain unclear. The present study investigated the impact of polystyrene (PS) nanoplastics of 80, 200 and 500 nm diameters on zebrafish reproduction at an environmentally relevant concentration of 0.5 mg/L. Exposure to PS delayed spermatogenesis and caused aberrant follicular growth, resulting in dysgenesis in F0 adults and impacting F1 embryo development. Notably, the reproductive toxicity exhibited size-dependency, with the 500 nm PS being the most detrimental. Combined analyses of transcriptomics and metabolomics in ovary tissue revealed that treatment with 500 nm PS affected the peroxisome proliferator-activated receptor (PPAR) signaling pathway, dysregulated lipid transport, binding and activity processes, and led to dysgenesis in zebrafish. Specifically, the ovulatory dysfunction induced by PS exposure resembled clinical manifestations of polycystic ovary syndrome (PCOS) and can be attributed to lipid metabolism disorder involving glycerophospholipid, sphingolipid, arachidonic acid, and alpha-linolenic acid. Collectively, our results provide new evidence revealing the molecular mechanisms of PS-induced reproductive toxicity, highlighting that MNPs may pose a risk to female reproductive health.

Environmentally relevant exposure to cotinine induces neurobehavioral toxicity in zebrafish (Danio rerio): A study using neurobehavioral and metabolomic approaches.

As an important psychoactive substance, cotinine is ubiquitous in aquatic environment and poses a threat to aquatic organisms. However, the mechanism of its adverse health impacts remains unclear. We evaluated the effects of cotinine exposure at environmentally relevant concentrations on the development and locomotor behavior of zebrafish (Danio rerio) larvae using neurotransmitters and whole endogenous metabolism. Mild developmental toxicity and significant neurobehavior disorder, such as spontaneous movement (1-1000 µg/L), 48 hpf tactile response (50, 100, and 1000 µg/L), and 144 hpf swimming speed (1, 10, 100, 500, and 1000 µg/L), were observed in zebrafish. Exposure to cotinine led to significant alterations in 11 neurotransmitters, including homogentisic acid, serotonin, glutamic acid and aspartic acid, etc. 298 metabolites were identified and two pathways - linoleic acid metabolism and taurine and hypotaurine metabolism - were delineated. In addition, amino acid neurotransmitters were significantly correlated with metabolites such as arachidonic acid as well as its derivatives, steroidal compounds, and amino acids. Serotonin demonstrates a noteworthy correlation with 31 out of 40 differentially expressed neurotransmitters, encompassing lipids, amino acids, and other compounds. These novel findings contribute to a comprehensive understanding of the ecological risks associated with cotinine contamination in surface waters.

DDT and titanium dioxide nanoparticle coexposure induced neurobehavioral deficits in zebrafish.

Both dichlorodiphenyltrichloroethane (DDT) and titanium dioxide nanoparticle (TiO2 NP) have worldwide-scale commercial applications, resulting in their co-pollution in the ecosystems and posing combined health risks. However, there is a lack of toxicity studies for the interactions of DDT and TiO2 NP in the environmental relevant concentrations. In this study, we characterized the coexposures using a zebrafish waterborne exposure approach and evaluated the neurotoxicity response of the treated embryos or adults. Our results showed that DDT/TiO2 NP coexposure enhanced the DDT accumulation in vivo and increased the larval locomotor. The chronic DDT/TiO2 NP coexposure did not affect the overall survival rate, sex ratio and growth. However, DDT/TiO2 NP coexposure severely affected the adult locomotor activity, social contact, shoaling and aggressive behaviors compared to single treatment groups or controls. These adult behavioral deficits were accompanied by changes in neurotransmitter acetylcholine (ACH) level in the brain and muscle tissues, as well as neural development genes expression activation of growth-associated protein 43 (gap43) and synaptic vesicle glycoprotein 2 (sv2) in the brain. The significantly increased ACH level and the activated neural genes expression in the DDT/TiO2 NP co-exposed fish may account for the observed hyperactivity and social deficits.

Polystyrene nanoplastics induced size-dependent developmental and neurobehavioral toxicities in embryonic and juvenile zebrafish.

Because of widespread environmental contamination, there is growing concern that nanoplastics may pose a risk to humans and the environment. Due to their small particle size, nanoplastics may cross the blood-nerve barrier and distribute within the nervous system. The present study systematically investigated the uptake/distribution and developmental/neurobehavioral toxicities of different sizes (80, 200, and 500 nm) of polystyrene nanoplastics (PS) in embryonic and juvenile zebrafish. The results indicate that all three sizes of PS could cross the chorion, adsorb by the yolk, and distribute into the intestinal tract, eye, brain, and dorsal trunk of zebrafish, but with different patterns. The organ distribution and observed developmental and neurobehavioral effects varied as a function of PS size. Although all PS exposures induced cell death and inflammation at the cellular level, only exposures to the larger PS resulted in oxidative stress. Meanwhile, exposure to the 80 nm PS increased the expression of neural and optical-specific mRNAs. Collectively, these studies indicate that early life-stage exposures to PS adversely affect zebrafish neurodevelopment and that the observed toxicities are influenced by particle size.

DDT exposure induces tremor-like behavior and neurotoxicity in developmental stages of embryonic zebrafish.

Dichlorodiphenyltrichloroethane (DDT) is a broad-spectrum insecticide, widely detected in environments due to its high stability characteristic and long natural half-life period. The adverse impact of DDT exposure on organisms and humans has attracted great concern worldwide. The current study explored the developmental and neurobehavioral toxicity response of DDT in embryonic zebrafish. The embryos were treated with DDT (0, 0.1, 1, 2.5 and 5 µM) during 6 h post fertilization (hpf) to 144 hpf. Our result indicated that DDT exposures increased the embryo hatching rate at 48 and 60 hpf, the larval malformation rate at 120 hpf and mortality rate at 144 hpf. The manifested malformations included uninflated swim bladder, bent spine and tail, deformed liver, and pericardial edema. The 120 hpf larval organs size of the gut and swim bladder was decreased in higher exposed concentration groups. Besides, DDT exposure resulted in hyperactivity for the embryo spontaneous movement at 24 hpf and tremor like movement measured by the free larval activity at 72 hpf, as well as the larval activity at 96 hpf under light-dark transition stimulus. Mechanistic examinations at 120 hpf revealed DDT exposure elevated oxidative stress through MDA formation increase, ATP level decrease as well as antioxidant enzyme genes (sod1 and gpx1a) expression decrease. DDT exposure induced abnormal neurotransmitters expression with DA level increase, 5-HT and NOS level decrease. DDT exposure suppressed the gene expressions involved in axon development (rab33a and nrxn2a) and potassium channel (kcnq2 and kcnq3). Our results suggest that the hyperactivity and tremor like movement in DDT-exposed embryos/larvae may result from oxidative stress involved with neuronal damage.

The neurodevelopmental toxicity induced by combined exposure of nanoplastics and penicillin in embryonic zebrafish: The role of aging processes.

As ubiquitous contaminants, nanoplastics and antibiotics are frequently co-presence and widely detected in the freshwater environment and biota, posing a high co-exposure risk to aquatic organisms and even humans. More importantly, how the aging process of nanoplastics affects the joint toxic potential of nanoplastics and antibiotics has not been explored. Here, we generated two aged polystyrene nanoplastics (PS) by UV radiation (UV-PS) and ozonation (O3-PS). Non-teratogenic concentrations of pristine PS (80 nm) and antibiotics penicillin (PNC) co-exposure synergistically suppressed the embryo heart beating and behaviors of spontaneous movement, touch response, and larval swimming behavioral response. Pristine PS and aged UV-PS, but not aged O3-PS, showed similar effects on zebrafish embryo/larval neurodevelopment. However, when co-exposure with PNC, both aged PS, but not pristine PS, showed antagonistic effects. In late-stage juvenile social behavior testing, we found that PS decreased the exploration in light/dark preference assay. The synergistic effect of aged PS with PNC was further explored, including cellular apoptosis, ROS formation, and neurotransmitter metabolite regulation. Mechanistically, aged UV-PS but not O3-PS significantly increased the adsorption rate of PNC compared to pristine PS, which may account for the toxicity difference between the two aged PS. In conclusion, our results confirmed that PS served as a carrier for PNC, and the environmental aging process changed their neurobehavioral toxicity pattern in vivo.

Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma.

In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss- or gain-of-function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding p53 variant effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet mutations when present are associated with worse prognosis. Employing a kRASG12D-driven ERMS tumor model and tp53 null (tp53-/-) zebrafish, we define wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumorigenesis, where tp53 loss expands tumor initiation from <35% to >97% of animals. Characterizing three patient-specific alleles reveals that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, whereas TP53P153Δ and TP53Y220C encode two structurally related proteins with gain-of-function effects that predispose to head musculature ERMS. TP53P153Δ unexpectedly also predisposes to hedgehog-expressing medulloblastomas in the kRASG12D-driven ERMS-model.

6PPD and its metabolite 6PPDQ induce different developmental toxicities and phenotypes in embryonic zebrafish.

The automobile tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite 6PPDQ have recently received much attention for their acute aquatic toxicity. The present study investigated the mechanistic developmental toxicity of 6PPD and 6PPDQ in embryonic zebrafish. Neither compound induced significant mortality but significantly decreased spontaneous embryo movement and heart rate. Both compounds induced malformations with different phenotypes; the 6PPD-exposed larvae manifested a myopia-like phenotype with a convex eyeball and fusion vessels, while the 6PPDQ-exposed embryonic zebrafish manifested enlarged intestine and blood-coagulated gut, activated neutrophils, and overexpressed enteric neurons. mRNA-Seq and quantitative real-time PCR assays showed that 6PPD- and 6PPDQ-induced distinct differential gene expression aligned with their toxic phenotype. 6PPD activated the retinoic acid metabolic gene cyp26a, but 6PPDQ activated adaptive cellular response to xenobiotics gene cyp1a. 6PPD suppressed the gene expression of the eye involved in retinoic acid metabolism, phototransduction, photoreceptor function and visual perception. In contrast, 6PPDQ perturbed genes involved in inward rectifier K+ and voltage-gated ion channels activities, K+ import across the plasma membrane, iron ion binding, and intestinal immune network for IgA production. The current study advances the present understanding the reason of why many fish species are so adversely impacted by 6PPD and 6PPDQ.

Structure-based developmental toxicity and ASD-phenotypes of bisphenol A analogues in embryonic zebrafish.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that has become more prevalent in recent years. Environmental endocrine disruptor bisphenol A (BPA) has been linked to ASD. BPA analogues (BPs) are structure-modified substitutes widely used as safer alternatives in consumer products, yet few studies have explored the developmental neurotoxicity (DNT) of BPA analogues. In the present study, we used the larval zebrafish model to assess the DNT effects of BPA and its analogues. Our results showed that many BPA analogues are more toxic than BPA in the embryonic zebrafish assay regarding teratogenic effect and mortality, which may partially due to differences in lipophilicity and/or different substitutes of structural function groups such as CF3, benzene, or cyclohexane. At sublethal concentrations, zebrafish embryos exposed to BPA or BPs also displayed reduced prosocial behavior in later larval development, evidenced by increased nearest neighbor distance (NND) and the interindividual distance (IID) in shoaling, which appears to be structurally independent. An in-depth analysis of BPA, bisphenol F (BPF), and bisphenol S (BPS) revealed macrocephaly and ASD-like behavioral deficits resulting from exposures to sublethal concentrations of these chemicals. The ASD-like behavioral deficits were characterized by hyperactivity, increased anxiety-like behavior, and decreased social contact. Mechanistically, accelerated neurogenesis that manifested by increased cell proliferation, the proportion of newborn mature neurons, and the number of neural stem cells in proliferation, as well as upregulated genes related to the K+ channels, may have contributed to the observed ASD-like morphological and behavioral alterations. Our findings indicate that BPF and BPS may also pose significant risks to ASD development in humans and highlight the importance of a comprehensive assessment of DNT effects for all BPA analogues in the future.

Cold stress during the perinatal period leads to developmental and neurobehavioral toxicity in zebrafish larvae.

In nature, cold stress is a core threat to aquatic organisms. But the neurodevelopmental effects of cold stress during the perinatal period on the offspring development were unknown. In the present study, adult zebrafish were cold-stressed at 18 °C for five days before spawning, and then the fertilized eggs were raised at 18, 24, or 28 °C from 0 to 120 h post fertilization (hpf). The resulting embryos and larvae were assessed for developmental and neurobehavioral responses. Our findings showed that embryos raised at 18 °C (Cold+++) suffered hatching failure and death, at 24 °C (Cold++) had decreased hatching, while those raised at 28 °C (Cold+) exhibited no developmental adversity. The neurobehavioral assessment showed that embryos from Cold+ and Cold++ groups displayed decreased motor behaviors, including spontaneous movement at 20-24 hpf, touch response at 48 hpf, and swimming speed at 120 hpf. In addition, cold stress during perinatal stage irreversibly affected larval social behaviors examined during 10-13 days post fertilization (dpf), such as unconsolidated shoaling, increased mirror attacks, and decreased social contacts. Notably, behavioral adversity was more pronounced in larvae from the Cold ++ group than those from the Cold+ group. Mechanistically, cold stress increased cell apoptosis, evidenced by increased acridine orange positive cells at 24 hpf and upregulation of casp8 at 120 hpf, increased oxidative stress (upregulation of cat and nos1) at 120 hpf, delayed motor neuron extension at 72 hpf, and upregulated nrxn2 and rab33a at 120 hpf. Our data indicate that cold stress during the perinatal period impaired neural development in zebrafish larvae, showing high mental health risk. These findings highlight cold stress should be avoided during the perinatal period for both aquatic fish or even humans.

Antibiotic artificial bone implantation for the treatment of infection after internal fixation of tibial plateau fractures.

OBJECTIVE: To explore the clinical effect of antibiotic artificial bone (Calcium phosphate) in the treatment of infection after internal fixation of tibial plateau fractures. METHODS: We retrospectively reviewed the clinical data of 32 patients with infection after internal fixation of tibial plateau fractures treating from March 2010 to October 2021. There were 18 males and 14 females, aged from 23 to 70 (average 49.66 ± 10.49), 19 cases of the left side and 13 cases of the right side. Among them, 7 cases were open fractures with initial injury and 25 cases were closed fractures. On the basis of thorough debridement and implanting antibiotic artificial bone, the internal fixation of 18 patients were tried to be preserved and the internal fixation of 14 patients were removed completely. In order to provide effective fixation, 14 patients also received external fixation. Postoperative wound healing, infection control, Hospital for Special Surgery knee scores (HSS), related inflammatory indicators and bone healing time were recorded and followed up. RESULTS: Thirty-two patients were followed up for 12 ~ 82 months (average 36.09 ± 19.47 months). The redness, swelling and pain of pin site occurred in 2 patients, which returned to normal after applying antibiotics and continuous dressing change. One patient retained the internal fixation during the first-stage operation. Redness and swelling of incision, subcutaneous undulation occurred after two months. In order to avoid the recurrence of infection, the internal fixation was removed completely and antibiotic artificial bone was filled again. The infection was controlled and fracture healed. Four patients' wounds could not be closed directly due to soft tissue defect and was covered with skin flap. After the first-stage operation, 12 patients received second-stage autologous iliac bone grafting due to residual bone defects and poor healing of the fracture end. The bone healing time was 4 ~ 16 months (average 7.31 ± 2.79 months). Inflammatory indicators including CRP, ESR, and WBC returned to normal levels within 2 ~ 10 weeks (average 4.97 ± 2.58 weeks). The HSS of all patients were 54 ~ 86 points (average 73.06 ± 8.44 points) at the last follow-up. CONCLUSION: Implantation of antibiotic artificial bone, retention or removal of internal fixation according to infection and fracture healing, application of external fixation timely is an effective method to treat infection after internal fixation of tibial plateau fractures, which can control infection effectively and promote functional recovery.

Antibiotic artificial bone implantation and external fixation for the treatment of infection after intramedullary nail fixation: a retrospective study of 33 cases.

OBJECTIVE: To explore the clinical effect of antibiotic artificial bone implantation and external fixation in the treatment of infection after intramedullary nail fixation. METHODS: We retrospectively reviewed the clinical data of patients with infection after intramedullary nail fixation treated from March 2010 to August 2020. There were 27 males and 6 female, aged from 12 to 67 years (average 42.27 years), 18 cases on the left side and 15 cases on the right side. Among them, 20 cases were open fractures with initial injury and 13 cases were closed fractures. All patients were treated with intramedullary nail removal, local debridement, antibiotic artificial bone implantation and external fixation. Because of bone defects, 19 patients underwent secondary autologous cancellous bone grafting after infection control. Postoperative wound healing, related inflammatory indicators, fixation time, and bone healing time were recorded and followed up. RESULTS: The 33 patients were followed up with period of 10 ~ 98 months (average 62.7 months). One patients failed to control the infection effectively after treatment, so received antibiotics artificial bone implantation again. Two patients also received antibiotic artificial bone implants again due to the recurrence of the infection. After treatment, infection was controlled and the fracture healed well. One patient received vacuum sealing drainage (VSD) due to persistent postoperative exudation, and five patients were also cured successfully after continuous dressing. Two patients had sinus tract after surgery, and the wound was cured by continuous dressing change. Nineteen patients received autogenous iliac bone grafts for healing due to bone defects ranging from 3 to 6.5 cm (average 4.15 cm) after infection control. The external fixation time of 33 patients ranged from 4 to 16 months (average 7.79 months), the bone healing time ranged from 4 to 13 months (average 6.67 months), and the related inflammatory indexes returned to normal within 2-8 weeks (average 4.48 weeks). CONCLUSION: Antibiotic artificial bone implantation and external fixation is an effective method for the treatment of infection after intramedullary nail fixation.

Leachate from plastic food packaging induced reproductive and neurobehavioral toxicity in zebrafish.

The present study mimicked daily life exposure to plastic food package bags and evaluated its effects on the reproductive and neurobehavioral responses using zebrafish model. Gas chromatography-mass spectrometer (GC/MS) full scan analysis revealed that phthalic acid, isobutyl octyl ester (DEHP) and its metabolites were the main leachate from plastic bags. Our results demonstrated that during the eight weeks exposure, leaching from plastic bags treated with boiling water (P-high group) significantly affected the spawn egg production, embryo hatching and larval malformation rate. Cross-spawning trails between zebrafish collected from the controls and P-high group at the end of eight weeks showed that these adverse effects were more severe in the offspring derived from paternal exposure than those derived from the maternal exposure, suggesting leached chemicals may have a more pronounced effect in sperm than in eggs. In addition, P-high group male testis weight, sperm motility and sperm swimming velocities were decreased significantly. After eight weeks treatment, neurobehavioral tests demonstrated significant changes in the swimming speed during free swimming and light-dark stimulation in the adult zebrafish from P-high group, with the effects being more severe in the males than females. P-high group males also showed altered response in the light/dark explore and mirror attacks assays.

A comparison of the vancomycin calcium sulfate implantation versus fenestration decompression for the treatment of sclerosing osteomyelitis.

OBJECTIVE: To compare the clinical efficacy of vancomycin calcium sulfate implantation and fenestration decompression in the treatment of sclerosing osteomyelitis. METHOD: A retrospective analysis for 46 cases of sclerosing osteomyelitis were admitted to our department between June 2010 to June 2020. Twenty-one patients were treated with fenestration decompression, twenty-five patients were treated with vancomycin calcium sulfate implantation. The postoperative hospital stay, days of drainage tube placement, visual analogue scale scores, C-reactive protein and erythrocyte sedimentation rate were compared between the two groups. RESULTS: The visual analogue scale scores of both groups were significantly lower than before treatment (p < 0.05), but the difference between them was not statistically significant. Patients treated by vancomycin calcium sulfate implantation had shorter postoperative hospital stay and days of drainage tube placement compared to those treated by fenestration decompression (p < 0.05). C-reactive protein and erythrocyte sedimentation rate in both groups were significantly lower than before treatment, but the improvement effect of vancomycin calcium sulfate implantation was better (p < 0.05). CONCLUSION: Both treatment methods can relieve pain effectively. Compared with fenestration decompression, vancomycin calcium sulfate implantation can shorten the treatment time effectively, control the infection better.

Developmental titanium dioxide nanoparticle exposure induces oxidative stress and neurobehavioral changes in zebrafish.

The widespread commercial application of titanium dioxide nanoparticles (TiO2 NPs) leads to ubiquitous presence of TiO2 NPs in the aquatic environment, which highlights the necessity to determine their potential adverse effects on aquatic organisms. The developing nerve system is particularly susceptible to environment perturbation. However, few studies have explored the developmental neurobehavioral toxicity of TiO2 NPs, especially at smaller particle size ranges (≤20 nm) that have relatively longer retention time in the water column. In this study, zebrafish embryos were exposed to non-teratogenic concentrations of 0.1 and 1 mg/L TiO2 NPs (average size of 14-20 nm) from 8 to 108 h post-fertilization (hpf) followed by various assessments at different time points up to 12 days post-fertilization (dpf). Our findings revealed that 1 mg/L TiO2 NPs perturbed the motor and social behaviors in larval zebrafish. These behavioral changes were characterized by decreased swimming speed in a locomotor response test at 5 dpf, increased travel distance in a flash stimulus test at 5 dpf, increased preference to the light zone in a light/dark preference test at 10 dpf, and increased mirror attack and percent time spent in the mirror zone in a mirror stimulus response assay at 12 dpf. Mechanistic examinations at 5 dpf revealed elevated cell apoptosis and oxidative stress. Cell apoptosis was characterized by increased acridine orange (AO) positive cells in the olfactory region and neuromasts of the lateral line system. Oxidative stress was characterized by increased lipid peroxidation, increased ROS production, and upregulated catalase (cat) gene expression. In addition, TiO2 NP exposure also upregulated genes associated with the developmental nervous system such as the growth associated protein 43 (gap43) and proliferating cell nuclear antigen (pcna). Our results suggest that the neurobehavioral changes in larvae exposed to 1 mg/L TiO2 NPs during early development may result from cell apoptosis and oxidative stress induced neuronal damages.

SNAI2-Mediated Repression of BIM Protects Rhabdomyosarcoma from Ionizing Radiation.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

Early life stage transient aristolochic acid exposure induces behavioral hyperactivity but not nephrotoxicity in larval zebrafish.

Aristolochic acids (AA) are nitrophenanthrene carboxylic acids found in plants of the Aristolochiaceae family. Humans are exposed to AA by deliberately taking herbal medicines or unintentionally as a result of environmental contamination. AA is notorious for its nephrotoxicity, however, fewer studies explore potential neurotoxicity associated with AA exposure. The developing nervous system is vulnerable to xenobiotics, and pregnant women exposed to AA may put their fetuses at risk. In the present study, we used the embryonic zebrafish model to evaluate the developmental neurotoxicity associated with AA exposure. At non-teratogenic concentrations (≤ 4 µM), continuous AA exposure from 8 to 120 hours post fertilization (hpf) resulted in larval hyperactivity that was characterized by increased moving distance, elevated activity and faster swimming speeds in several behavioral assays. Further analysis revealed that 8-24 hpf is the most sensitive exposure window for AA-induced hyperactivity. AA exposures specifically increased motor neuron proliferation, increased apoptosis in the eye, and resulted in cellular oxidative stress. In addition, AA exposures increased larval eye size and perturbed the expression of vision genes. Our study, for the first time, demonstrates that AA is neurotoxic to the developmental zebrafish with a sensitive window distinct from its well-documented nephrotoxicity.

Production of bioactive recombinant human fibroblast growth factor 12 using a new transient expression vector in E. coli and its neuroprotective effects.

In recent years, an increasing number of studies have shown that fibroblast growth factor 12 (FGF12) plays important roles in regulating neural development and function. Importantly, changes of FGF12 expression are thought to be related to the pathophysiology of many neurological diseases. However, little research has been performed to explore the protective effect of FGF12 on nerve damage. This study aims to explore its neuroprotective effects using our recombinant humanized FGF12 (rhFGF12). The hFGF12 gene was cloned and ligated into an expression vector to construct a recombinant plasmid pET-3a-hFGF12. Single colonies were screened to obtain high expression engineering strains, and fermentation and purification protocols for rhFGF12 were designed and optimized. The biological activities and related mechanisms of rhFGF12 were investigated by MTT assay using NIH3T3 and PC12 cell lines. The in vitro neurotoxicity model of H2O2-induced oxidative injury in PC12 cells was established to explore the protective effects of rhFGF12. The results indicate that the beneficial effects of rhFGF12 were most likely achieved by promoting cell proliferation and reducing apoptosis. Moreover, a transgenic zebrafish (islet) with strong GFP fluorescence in the motor neurons of the hindbrain was used to establish a central injury model caused by mycophenolate mofetil (MMF). The results suggested that rhFGF12 could ameliorate central injury induced by MMF in zebrafish. In conclusion, we have established an efficient method to express and purify active rhFGF12 using an Escherichia coli expression system. Besides, rhFGF12 plays a protective effect of on nerve damage, and it provides a promising therapeutic approach for nerve injury. KEY POINTS: • Effective expression and purification of bioactive rhFGF12 protein in E. coli. • ERK/MAPK pathway is involved in rhFGF12-stimulated proliferation on PC12 cells. • The rhFGF12 has the neuroprotective effects by inhibiting apoptosis.

Zebrafish Tumor Graft Transplantation to Grow Tumors In Vivo That Engraft Poorly as Single Cell Suspensions.

Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. It can arise in vascular or lymphatic tissues, involve any part of the body, and aggressively spread locally or metastasize. Angiosarcomas spontaneously develop in the tp53 deleted (tp53del/del) zebrafish mutant. However, established protocols for tumor dissection and transplantation of single cell suspensions of angiosarcoma tumors result in inferior implantation rates. To resolve these complications, we developed a new tumor grafting technique for engraftment of angiosarcoma and similar tumors in zebrafish, which maintains the tumor microenvironment and has superior rates of engraftment.

External-oxidant-free amino-benzoyloxylation of unactivated alkenes of unsaturated ketoximes with O-benzoylhydroxylamines.

A new copper-catalyzed two-component amino-benzoyloxylation of unactivated alkenes of unsaturated ketoximes with O-benzoylhydroxylamines as the benzoyloxy sources is developed. Chemoselectivity of this method toward amino-benzoyloxylation or oxy-benzoyloxylation of alkenyl ketoximes relies on the position of the tethered olefins, and provides an external-oxidant-free alkene difunctionalization route that directly utilizes O-benzoylhydroxylamines as the benzoyloxy radical precursors and internal oxidants for the divergent synthesis of cyclic nitrones and isoxazolines.