Myclobutanil induces neurotoxicity by activating autophagy and apoptosis in zebrafish larvae (Danio rerio).

Myclobutanil (MYC), a typical broad-spectrum triazole fungicide, is often detected in surface water. This study aimed to explore the neurotoxicity of MYC and the underlying mechanisms in zebrafish and in PC12 cells. In this study, zebrafish embryos were exposed to 0, 0.5 and 1 mg/L of MYC from 4 to 96 h post fertilization (hpf) and neurobehavior was evaluated. Our data showed that MYC decreased the survival rate, hatching rate and heart rate, but increased the malformation rate and spontaneous movement. MYC caused abnormal neurobehaviors characterized by decreased swimming distance and movement time. MYC impaired cerebral histopathological morphology and inhibited neurogenesis in HuC:egfp transgenic zebrafish. MYC also reduced the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and downregulated neurodevelopment related genes (gfap, syn2a, gap43 and mbp) in zebrafish and PC12 cells. Besides, MYC activated autophagy through enhanced expression of the LC3-II protein and suppressed expression of the p62 protein and autophagosome formation, subsequently triggering apoptosis by upregulating apoptotic genes (p53, bax, bcl-2 and caspase 3) and the cleaved caspase-3 protein in zebrafish and PC12 cells. These processes were restored by the autophagy inhibitor 3-methyladenine (3-MA) both in vivo and in vitro, indicating that MYC induces neurotoxicity by activating autophagy and apoptosis. Overall, this study revealed the potential autophagy and apoptosis mechanisms of MYC-induced neurotoxicity and provided novel strategies to counteract its toxicity.

Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown. RESULTS: We carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis. CONCLUSIONS: These results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

Diversity of fungal community and quality evaluation of Spatholobus Suberectus Dunn during the process of mildew.

Spatholobus suberectus Dunn as a traditional Chinese herbal medicine, which is susceptible to being infected by molds during storage. In order to explore the diversity characteristics of fungal community and the quality evaluation of Spatholobus suberectus Dunn during the process of mildew. The study used high-throughput sequencing technology to detect the diversity characteristics of fungal community, high-performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV-spectrophotometry) methods to detect the content of flavonoids, and enzyme-linked immunosorbent assay (ELISA) method to detect the content of Aflatoxins B1 (AFB1). The result showed that the fungi of all samples belonged to 14 phyla, 336 genera, and the dominant fungi at the early stage of mildew was not obvious, while that at middle and late stages of mildew was Aspergillus. The species diversity of fungal community was the highest at the early stage of mildew, while the species richness of fungal community was the highest at the late stage of mildew. The content of AFB1 showed an upward trend, while the content of flavonoids showed a downward trend during the process of mildew. In brief, the diversity of fungal community decreased gradually, and the number of dominant fungi increased gradually, and the quality of Spatholobus suberectus Dunn decreased gradually during the process of mildew.

Design of a dual Ir-Eu tag for fluorescent visualization and ICP-MS quantification of SIRPα and its host cells.

With the expansion of ICP-MS application into the field of bioanalysis, there is an urgent need for novel element tags today. Here, we report the design of a dual-element Ir-Eu tag, opening the door to simultaneous fluorescent imaging and ICP-MS quantification. The ratio of 153Eu/193Ir may serve as a precision control of the labeling process, allowing internal validation of the quantitative results obtained. As for SIRPα and its host cell analysis exemplified here, the Ir-Eu tag demonstrated superior figures of ICP-MS quantification with the LOD (3σ) down to 0.5 (153Eu) and 1.1 (193Ir) pM SIRPα and 220 (153Eu) and 830 (193Ir) RAW264.7 cells more than 130 times more sensitive compared with the LOD (3σ) of 65.2 pM SIRPα at 612 nm using fluorometry. Not limited to these demonstrations, we believe that the design ideas of the dual Ir-Eu tags should be applicable to various cases of bioanalysis when dual optical profiling and ICP-MS quantification are indispensable.

Diversity and correlation analysis of different root exudates on the regulation of microbial structure and function in soil planted with Panax notoginseng.

Introduction: Specific interactions between root exudates and soil microorganisms has been proposed as one of the reasons accounting for the continuous cropping obstacle (CCO) of Panax notoginseng. However, rotation of other crops on soils planted with P. notoginseng (SPP) did not show CCO, suggesting that root exudates of different crops differentially regulate soil microorganisms in SPP. Methods: Here, we investigated the microbial community structure and specific interaction mechanisms of the root exudates of the four plant species, P. notoginseng (Pn), Zea mays (Zm), Nicotiana tabacum (Nt) and Perilla frutescens (Pf), in SPP by static soil culture experiment. Results: The results showed that the chemical diversity of root exudates varied significantly among the four plant species. Pn had the highest number of unique root exudates, followed by Nt, Zm and Pf. Terpenoids, flavonoids, alkaloids and phenolic acids were the most abundant differentially accumulated metabolites (DAMs) in Pn, Nt, Zm and Pf, respectively. However, lipids were the most abundant common DAMs among Zm Nt and Pf. Pn root exudates decreased the relative abundance of bacteria, but increased that of fungi. While specific DAMs in Pn enriched Phenylobacterium_zucineum, Sphingobium_yanoikuyae, Ophiostoma_ulmi and functional pathways of Nucleotide excision repair, Streptomycin biosynthesis, Cell cycle-Caulobacter and Glycolysis/Gluconeogenesis, it inhibited Paraburkholderia _caledonica and Ralstonia_pickettii. However, common DAMs in Zm, Nt and Pf had opposite effects. Moreover, common DAMs in Zm, Nt and Pf enriched Ralstonia_pseudosolanacearum and functional pathway of Xylene degradation; unique DAMs in Zm enriched Talaromyces_purcureogeneus, while inhibiting Fusarium_tricinctum and functional pathways of Nucleotide excision repair and Alanine, aspartate and glutamate metabolism; unique DAMs in Pf enriched Synchytrium_taraxaci. Discussion: The core strains identified that interact with different root exudates will provide key clues for regulation of soil microorganisms in P. notoginseng cultivation to alleviate CCO.

Protein Recognition Based on Temperature-Stimulated Multiparameter Response Virtual Array Sensing Strategy.

In the field of array sensing, researchers are committed to miniaturizing array sensing systems while ensuring the acquisition of multiple sensing information. Here, a new strategy called "stimulus responsive array sensing" was presented to obtain virtual multiple sensing without constructing multiple physical sensing units. Based on bioluminescence resonance energy transfer, where luciferase acts as the donor and temperature stimulus response polymers act as the receptors, by using only one sensing unit to output multiple stimulus responsive sensing signals in temperature dimension, an equivalent array sensing could be achieved. This strategy can distinguish and quantify a variety of proteins. More importantly, glucose responsive monomers were doped in polymers; thus, more virtual sensing units can be further increased to obtain more sensing signals, greatly increasing the accuracy of protein recognition, and it can also be used to differentiate several compositions of protein under different glucose concentrations in urine caused by different renal diseases. The results show the potential of the "stimulus responsive array sensing" for analyzing molecular compositions in complex biological systems and show a new tack in array sensing.

Curcumin protects against fenvalerate-induced neurotoxicity in zebrafish (Danio rerio) larvae through inhibition of oxidative stress.

Fenvalerate (FEN), a typical type II pyrethroid pesticide, is widely used in agriculture. FEN has been detected in the environment and human body. However, the neurotoxicity of FEN has not been well elucidated. This study aimed to explore the mechanisms underlying FEN-induced neurotoxicity using the zebrafish (Danio rerio) model. We also investigated whether curcumin (CUR), a polyphenol antioxidant that exhibits neuroprotective properties, can prevent FEN-induced neurotoxicity. Here, zebrafish embryos were exposed to 0, 3.5, 7 and 14 µg/L of FEN from 4 to 96 h post fertilization (hpf) and neurotoxicity was assessed. Our results showed that FEN decreased the survival rate, heart rate, body length and spontaneous movement, and increased malformation rate. FEN caused neurobehavioral alterations, including decreased swimming distance and velocity, movement time and clockwise rotation times. FEN also suppressed neurogenesis in transgenic HuC:egfp zebrafish, reduced cholinesterase activity and downregulated the expression of neurodevelopment related genes (elavl3, gfap, gap43 and mbp). In addition, FEN enhanced oxidative stress via excessive reactive oxygen species and antioxidant enzyme inhibition, then triggered apoptosis by upregulation of apoptotic genes (p53, bcl-2, bax and caspase 3). These adverse outcomes were alleviated by CUR, indicating that CUR mitigated FEN-induced neurotoxicity by inhibiting oxidative stress. Overall, this study revealed that CUR ameliorated FEN-induced neurotoxicity via its antioxidant, indicating a promising protection of CUR against environmental pollutant-induced developmental anomalies.

High-sensitivity fiber SPR strain sensor based on n-type structure.

At present, fiber strain sensors are mainly of the grating type and interference type, while there is relatively little research on fiber surface plasmon resonance (SPR) strain sensors. In this Letter, we propose a highly sensitive fiber SPR strain sensor based on an n-type structure. The strain changes the shape of the fiber n-type structure, causing the transmission mode of light in the fiber to change, thereby changing the SPR incidence angle and causing the SPR resonance valley wavelength to shift, achieving highly sensitive SPR strain sensing. The test results indicate that the strain sensing sensitivity of the proposed sensor reaches 21.33 pm/µÎµ, and two n-type structures are connected in series to obtain a double n-type structure, further enhancing the strain sensing sensitivity to 33.44 pm/µÎµ. This fiber strain sensor has advantages of high sensitivity, low temperature cross talk, strong structural stability, and low production cost, and is expected to become a new solution for wearable intelligent monitoring equipment and strain sensors in the aerospace field.

Hypomethylation of DYRK4 in peripheral blood is associated with increased lung cancer risk.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. It is urgent to identify new biomarkers for the early detection of LC. DNA methylation in peripheral blood has been reported to be associated with cancers. We conducted two independent case-control studies and a nested case-control study (168 LC cases and 167 controls in study Ⅰ, 677 LC cases and 833 controls in study Ⅱ, 147 precancers and 21 controls in the nested case-control study). The methylation levels of DYRK4 CpG sites were measured using mass spectrometry and their correlations with LC were analyzed by logistic regression and nonparametric tests. Bonferroni correction was used for the multiple comparisons. LC-related decreased DYRK4 methylation was discovered in Study I and validated in Study II (the odds ratios [ORs] for the lowest vs. highest quartile of all three DYRK4 CpG sites ranged from 1.64 to 2.09, all p < 0.001). Combining the two studies, hypomethylation of DYRK4 was observed in stage I cases (ORs per -10% methylation ranged from 1.16 to 1.38, all p < 5.9E-04), and could be enhanced by male gender (ORs ranged from 1.77 to 4.17 via interquartile analyses, all p < 0.017). Hypomethylation of DYRK4_A_CpG_2 was significantly correlated with tumor size, length, and stage (p = 0.034, 0.002, and 0.002, respectively) in LC cases. Our study disclosed the association between DYRK4 hypomethylation in peripheral blood and LC, suggesting the feasibility of blood-based DNA methylation as new biomarker for LC detection.

Clinical impact of hepatic steatosis on chronic hepatitis B patients in Asia: A systematic review and meta-analysis.

Chronic hepatitis B (CHB) and hepatic steatosis (HS) are two prevalent chronic liver diseases in Asia. The incidence of CHB combined with HS is increasing due to the rising obesity rates. However, the impact of HS on CHB remains a topic of debate. Hereby, this meta-analysis aims to examine the effect of HS on Asian patients with CHB. Searches were conducted on four databases to identify articles published from 2005 to 2023. The random-effects or fixed-effects model was used to calculate pooled odds ratios (ORs), weighted mean difference (WMD), and confidence intervals (CIs) for the included articles. Of the 15,959 records screened, 88 studies were included in the analysis of HS prevalence in Asian CHB patients with a prevalence of 36.5% (95% CI: 33.7%-39.3%). In addition, age, sex, body mass index (BMI), waist circumference (WC), alanine aminotransferase (ALT) and combined metabolic diseases have varying degrees of impact on HS in CHB patients. Furthermore, the coexistence of HS was negatively associated with the response to antiviral therapy, including hepatitis B surface antigen (HBeAg) seroconversion (OR = 0.69, 95% CI: 0.53-0.89) and ALT normalization (OR = 0.75, 95% CI: 0.61-0.92) in CHB patients after 48 weeks of treatment. Regarding disease prognosis, HS was not significantly associated with fibrosis or cirrhosis in CHB patients, while an inverse association was observed between HS and hepatocellular carcinoma (HCC) (OR = 2.93, 95% CI: 1.23-6.99). This implies that the coexistence of HS in CHB patients may exacerbate the progression of HCC, which needs to be verified by further studies.

Psychological capital and perceived supervisor social support as mediating roles between role stress and work engagement among Chinese clinical nursing teachers: a cross-sectional study.

OBJECTIVE: This study aimed to investigate the work engagement of clinical nursing teachers and examine whether psychological capital and perceived supervisor social support mediates the association between role stress and work engagement among clinical nursing teachers. DESIGN: A cross-sectional study design was used. SETTING: This study was conducted in five tertiary hospitals affiliated with medical universities in Shandong Province, China. PARTICIPANTS: A total of 412 clinical nursing teachers were recruited out of which 406 completed the questionnaires effectively. METHODS: Questionnaires were administered offline and the items consisted of social demographic characteristics, scale related to role stress, psychological capital, perceived supervisor social support and work engagement. T-tests, one-way analysis of variance and Pearson's correlation analysis were conducted using SPSS, and mediation analysis was performed using structural equation modelling. RESULTS: The work engagement scores of clinical nursing teachers were high, with an average score of 4.76±0.69. Structural equation modelling showed a good fit of the data to the hypothesised model. Role stress did not directly affect work engagement (ß=0.057, p>0.001). However, a strong indirect effect of role stress on work engagement (ß=-0.373, p<0.001) mediated by psychological capital and perceived supervisor social support was observed, with path coefficient of -0.319 (p<0.01) and -0.054 (p<0.01), respectively. The model explains 57% of the variance in work engagement. CONCLUSION: Psychological capital and perceived supervisor social support fully mediated the relationship between role stress and work engagement among clinical nursing teachers. Thus, to promote the work engagement of clinical nursing teachers, their psychological capital needs to be improved. Furthermore, superiors should provide more support, particularly to clinical nursing teachers experiencing high role stress.

Hypomethylation of ABCG1 in peripheral blood as a potential marker for the detection of coronary heart disease.

BACKGROUND: Novel molecular biomarkers for the risk assessment and early detection of coronary heart disease (CHD) are urgently needed for disease prevention. Altered methylation of ATP-binding cassette subfamily G member 1 (ABCG1) has been implicated in CHD but was mostly studied in Caucasians. Exploring the potential relationship between ABCG1 methylation in blood and CHD among the Chinese population would yield valuable insights. METHODS: Peripheral blood samples were obtained from a case-control study (287 CHD patients vs. 277 controls) and a prospective nested case-control study (171 CHD patients and 197 matched controls). DNA extraction and bisulfite-specific PCR amplification techniques were employed for sample processing. Quantitative assessment of methylation levels was conducted using mass spectrometry. Statistical analyses involved the utilization of logistic regression and nonparametric tests. RESULTS: We found hypomethylation of ABCG1 in whole blood was associated with the risk of CHD in both studies, which was enhanced in heart failure (HF) patients, female and younger subjects. When combined with baseline characteristics, altered ABCG1 methylation showed improved predictive effect for differentiating CHD cases, ischemic cardiomyopathy (ICM) cases, younger than 60 years CHD cases, and female CHD cases from healthy controls (area under the curve (AUC) = 0.68, 0.71, 0.74, and 0.73, respectively). CONCLUSIONS: We demonstrated a robust link between ABCG1 hypomethylation in whole blood and CHD risk in the Chinese population and provided novel evidence indicating that aberrant ABCG1 methylation in peripheral blood can serve as an early detection biomarker for CHD patients.

Multi working mode SPR chip laboratory for high refractive index detection.

The Fiber SPR chip laboratory has become a popular choice in biochemical detection. To meet the needs of different kinds of analytes for the detection range and number of channels of the chip, we proposed a multi-mode SPR chip laboratory based on microstructure fiber in this paper. The chip laboratory was integrated with microfluidic devices made from PDMS and detection units made of bias three-core fiber and dumbbell fiber. By injecting light into different cores of a bias three-core fiber, different detection areas of dumbbell fiber can be selected, enabling the chip laboratory to enter high refractive index detection, multi-channel detection and other working modes. In the high refractive index detection mode, the chip can detect liquid samples with a refractive index range of 1.571-1.595. In multi-channel detection mode, the chip can achieve dual parameter detection of glucose and GHK-Cu, with sensitivities of 4.16 nm/(mg/mL) and 9.729 nm/(mg/mL), respectively. Additionally, the chip can switch to temperature compensation mode. The proposed multi working mode SPR chip laboratory, based on micro structured fiber, offers a new approach for the development of portable testing equipment that can detect multiple analytes and meet multiple requirements.

Highly sensitive SPR curvature sensor based on graded-index fiber.

At present, fiber curvature sensors based on surface plasmon resonance (SPR) are mostly of the multimode fiber core type or cladding type. These types have many SPR modes, resulting that the sensitivity cannot be adjusted and is difficult to improve. In this Letter, a highly sensitive SPR curvature sensor based on graded-index fiber is proposed. The light-injecting fiber is eccentrically connected with the graded-index fiber to inject single-mode light. Due to the self-focusing effect, the light beam propagates in the graded-index multimode fiber with a cosine trajectory, and the cosine beam contacts the flat grooved sensing region fabricated on the graded-index fiber to generate SPR. Due to the single transmission mode of the proposed fiber SPR sensor, the curvature sensing sensitivity is greatly improved. By changing the light injection position of the graded-index multimode fiber, the sensitivity can be adjusted. The proposed curvature sensing probe has a high sensitivity and can identify the bending direction. When bending in the X direction, the sensitivity reaches 5.62 nm/m-1, and when bending in the - X direction, the sensitivity reaches 4.75 nm/m-1, which provides a new scheme for highly sensitive and directionally identifiable curvature measurement.

Chiral zinc oxide functionalized quartz crystal microbalance sensor for enantioselective recognition of amino acids.

Chiral transition metal oxides with tunable structures and multiple physicochemical features have been increasingly applied for chiral sensing and detection. In this work, chiral zinc oxide (ZnO) was first applied as selector to construct quartz crystal microbalance (QCM) sensor for enantioselective recognition of amino acids. The chiral ZnO was prepared by a methionine-induced self-assembly strategy and its high topological chirality was confirmed by several techniques such as circular dichroism spectrum. The chiral discrimination factors were calculated by frequency shifts in response to aspartic acid, phenylalanine, lysine and arginine on L-ZnO surface, achieving 1.89 ± 0.04, 1.76 ± 0.11, 1.66 ± 0.07 and 1.54 ± 0.09, respectively. Notably, L-enantiomers preferred stronger absorptions on L-ZnO surface as compared to D-forms. It was further found that this sensor was appropriate for quantitative analysis and enantiomer excess analysis and adsorption kinetics study. Furthermore, molecular docking revealed the recognition mechanism, where chiral distinction was caused by the different steric interactions between enantiomers and chiral ZnO. This method enjoyed merits of high enantioselectivity, simple preparation and low cost, offering newly chiral sensing method for other molecules.

Fiber SPR biosensor sensitized by MOFs for MUC1 protein detection.

The concentration of tumor markers is low, which needs a highly sensitive, stable and fast detection method. In this paper, we proposed and demonstrated a U-shape fiber SPR biosensor sensitized by MOFs materials. The surface of the U-shape SPR sensor was modified with MOFs materials to enhance the sensitivity, and the nucleic acid aptamer was immobilized on the sensor surface because of the biocompatibility of MOFs materials. By the high specificity of the nucleic acid aptamer, the MUC1 protein was recognized and detected. The testing results indicate that the sensor has a logarithmic linear response in the MUC1 protein concentration detection range of 1 pg/ml-100 µg/ml, its sensitivity and detection limit are 5.33 nm/log(µg/ml) and 0.16 pg/ml respectively. After being sensitized by MOFs, the detection sensitivity of the sensor can be increased by 1.62 times，the LOD can be decreased by 0.75 times. The sensor has high sensitivity and specificity, which has broad application prospects in clinical detection of tumor markers.

Ginsenoside Rg1 modulates vesicular dopamine storage and release during exocytosis revealed with single-vesicle electrochemistry.

Ginsenoside Rg1, a tetracyclic triterpenoid derivative extracted from the roots of Panax ginseng C. A. Meyer, can enhance learning and memory and improve cognitive impairment. However, whether or how it affects vesicular dopamine storage and its release during exocytosis remains unknown. By using single-vesicle electrochemistry, we for the first time find out that Rg1 not only upregulates vesicular dopamine content but also increases exocytosis frequency and modulates dopamine release during exocytosis in PC12 cells, which may relate to the activation of protein kinases, causing a series of biological cascades. This finding offers the possible link between Rg1 and vesicular chemical storage and exocytotic release, which is of significance for understanding the nootropic role of Rg1 from the perspective of neurotransmission.

Three-dimensional micro displacement sensor based on fiber SPR mechanisms.

Three fiber micro displacement sensors can be combined to realize three-dimensional (3D) displacement sensing, but the system is complex. In this paper, a 3D displacement sensor based on fiber SPR was proposed, which was composed of displacement fiber and sensing fiber. By cascading the eccentric dual-core fiber and graded multimode fiber, the displacement fiber was realized. The V-groove was processed in the vertical and horizontal directions of the graded multimode fiber, and the inclined SPR sensing areas were fabricated to realize the sensing fiber. A straight beam from the middle core of the displacement fiber contacted the vertical V-groove inclined plane of the sensing fiber to realize the Y axis (up and down) direction micro displacement, contacted the horizontal V-groove inclined plane of the sensing fiber to realize the Z axis (front and back) direction micro displacement sensing. An oblique beam from the eccentric core of the displacement fiber cooperated with the sensing fiber to realize the micro displacement sensing in the X-axis (left and right) direction. The testing results indicate that the fiber SPR 3D micro displacement sensor can sense micro displacement in the X axis, Y axis and Z axis, and the wavelength sensitivity is 0.148 nm/µm, -3.724 nm/µm and 3.543 nm/µm, respectively. The light intensity sensitivity is -0.0014a.u./µm, -0.0458a.u./µm and -0.0494a.u./µm, respectively. When adjusting the parameters of eccentric dual-core fiber, the larger the core distance is, the greater the displacement sensitivity in the X-axis direction of the sensor is, and the smaller the detection range is. The proposed sensor can realize 3D micro displacement sensing by itself, which is expected to be used in the field of 3D micro displacement measurement and 3D space precision positioning.

Nano-TiO2 aggravates bioaccumulation and developmental neurotoxicity of difenoconazole in zebrafish larvae via oxidative stress and apoptosis: Protective role of vitamin C.

Titanium dioxide nanoparticles (n-TiO2) could enhance the bioavailability and toxicity of coexisting organic contaminants in the aquatic environment. This study attempted to investigate the combined effects of n-TiO2 and difenoconazole (DIF) on the neurodevelopment of zebrafish and the underlying mechanisms. In this study, zebrafish embryos were exposed to n-TiO2 (100 µg/L), DIF (0, 0.1 and 0.5 mg/L) and their mixtures from 4 to 96 h post fertilization (hpf) and neurotoxicity was evaluated. Our results indicated that n-TiO2 adsorbed DIF into the brain of zebrafish and significantly enhanced the bioaccumulation of DIF and n-TiO2 in the 0.5 mg/L co-exposure group. 100 µg/L n-TiO2 was not developmentally toxic to the zebrafish larvae, but it exacerbated DIF-induced neurobehavioral alterations in the zebrafish larvae. n-TiO2 also aggravated DIF-induced suppression of central nervous system (CNS) neurogenesis in Tg (HuC:egfp) zebrafish, motor neuron axon length in Tg (hb9:egfp) zebrafish, and downregulation of neurodevelopmental genes (elavl3, ngn1, gap43, gfap and mbp). In addition, DIF elevated oxidative stress by accumulation of reactive oxygen species (ROS) and inhibition of antioxidant enzymes, and triggered apoptosis by upregulation of p53, bax, bcl-2 and caspase-3, which were markedly intensified in the presence of n-TiO2. Moreover, vitamin C (VC) ameliorated n-TiO2/DIF-induced abnormal locomotor behaviors and neurotoxicity by inhibiting oxidative stress and apoptosis, indicating that oxidative stress and apoptosis are involved in n-TiO2/DIF-induced neurotoxicity. Taken together, our data indicated that n-TiO2 enhanced the accumulation of DIF and heightened oxidative stress and apoptosis, thereby inducing neurotoxicity. This study exemplifies the importance of the toxicity assessment of chemical mixtures and novel insights to mitigate their combined toxicity.