Thermo Fisher Scientific (China) Co., Ltd.

Flocoumafen (FCF), a second-generation anticoagulant rodenticide (SGAR), has limited toxicological data regarding its effects on aquatic organisms. In this study, we exposed zebrafish embryos to FCF solutions at concentrations of 0.2, 0.4, and 0.8 mg/L until 120 h post-fertilization (hpf). The results revealed a decrease in survival rates. Notably, FCF exposure significantly reduced the frequency of spontaneous tail coils at 24 hpf, while shortened body length and induced spinal curvature at 120 hpf. Furthermore, zebrafish larvae exhibited craniofacial abnormalities and incomplete bone mineralization at 120 hpf following FCF exposure. In Tg(HuC:EGFP) transgenic strains, neuronal loss was observed. Additionally, FCF-exposed zebrafish larvae showed a marked reduction in locomotor ability, activity levels, and turning capacity. The qPCR and enzyme activity assays revealed significant changes in gene expression associated with the Notch signaling pathway, accompanied by increased levels of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA). Astaxanthin (ASTA) partially alleviated the toxicities induced by FCF. These findings suggest that FCF may induce skeletal and neurological toxicities by affecting oxidative stress, disrupting the normal expression of skeletal and nervous system-related genes in the Notch signaling pathway, and ultimately leading to behavioral abnormalities. Our findings may provide new insights into a comprehensive evaluation of FCF toxicology in aquatic organisms, and may assist the government in formulating and implementing regulatory policies regarding the application of FCF.

Single-cell proteomics (SCP) promises to revolutionize biomedicine by providing an unparalleled view of the proteome in individual cells. Here, we present a high-sensitivity SCP workflow named Chip-Tip, identifying >5,000 proteins in individual HeLa cells. It also facilitated direct detection of post-translational modifications in single cells, making the need for specific post-translational modification-enrichment unnecessary. Our study demonstrates the feasibility of processing up to 120 label-free SCP samples per day. An optimized tissue dissociation buffer enabled effective single-cell disaggregation of drug-treated cancer cell spheroids, refining overall SCP analysis. Analyzing nondirected human-induced pluripotent stem cell differentiation, we consistently quantified stem cell markers OCT4 and SOX2 in human-induced pluripotent stem cells and lineage markers such as GATA4 (endoderm), HAND1 (mesoderm) and MAP2 (ectoderm) in different embryoid body cells. Our workflow sets a benchmark in SCP for sensitivity and throughput, with broad applications in basic biology and biomedicine for identification of cell type-specific markers and therapeutic targets.

Dai-Bai-Jie, the root of the plant Marsdenia tenacissima from the Asclepiadaceae family, is well-known for its therapeutic effects in clearing heat, detoxifying, reducing swelling, and relieving pain as one of the most commonly used Dai medicine. Due to numerous structurally similar C21 steroidal compounds in Dai-Bai-Jie, chemical composition profiling has been substantially challenged. In this study, an offline two-dimensional chromatographic method (LC × SFC separation system) was developed to address these issues. Using the Hypersil Gold (^1stD LC column) and 2-PIC (^2ndD SFC column) based on 40 reference standards, the orthogonality was as high as 83.83 %. Most profiled ion peaks were tentatively identified through quadrupole time-of-flight mass spectrometry and a self-built compound virtual library. Consequently, the integrated method effectively addressed and resolved the issues associated with co-elution, thus significantly expanding the peak capacity. This advancement identified 362 C21 steroidal components, 319 of which were speculated to be potentially novel compounds. Furthermore, 86 groups of isomeric compounds were distinguished. This method provides a comprehensive understanding of chemical composition of Dai-Bai-Jie and an integrated qualitative analysis method for the C21 steroids.