High sensitivity in quantitative analysis of mixed-size polystyrene micro/nanoplastics in one step.

As emerging environmental pollutants, microplastics (MPs) and nanoplastics (NPs) pose a serious threat to human health. Owing to the lack of feasible and reliable analytical methods, the separation and identification of MPs and NPs of different sizes remains a challenge. In this study, a hyphenated method involving filtration and surface-enhanced Raman spectroscopy (SERS) for the separation and identification of MPs and NPs is reported. This method not only avoids the loss of MPs and NPs during the transfer process but also provides an excellent SERS substrate. The SERS substrate was fabricated by electrochemically depositing silver particles onto the reduced graphene oxide layer coated on stainless steel mesh. Results show that polystyrene (PS) MPs and NPs are efficiently separated on the SERS substrate via vacuum filtration, resulting in high retention rates (74.26 % ± 1.58 % for 100 nm, 81.06 % ± 1.49 % for 500 nm, and 97.73 % ±0.11 % for 5 µm) and low limit of detection (LOD). The LOD values of 100 nm, 500 nm, and 5 µm PS are 8.89 × 10-5, 3.39 × 10-5, and 1.57 × 10-4 µg/mL, respectively. More importantly, a linear relationship for uniform quantification of 100 nm, 500 nm, 3 µm and 5 µm PS was established, and the relationship is Y = 225.61 lgX + 1076.36 with R2 = 0.980. The method was validated for the quantitative analysis of a mixture of 100 nm, 500 nm PS NPs, 3 µm and 5 µm PS MPs in a ratio of 1:1:1:1, which successfully approaches the evaluation of evaluated PS NPs in the range of 10-4-10 µg/mL with an LOD value of approximately 7.82 × 10-5 µg/mL. Moreover, this method successfully detected (3.87 ± 0.06) × 10-5 µg MPs and NPs per gram of oyster tissue.

A spatiotemporal atlas of cholestatic injury and repair in mice.

Cholestatic liver injuries, characterized by regional damage around the bile ductular region, lack curative therapies and cause considerable mortality. Here we generated a high-definition spatiotemporal atlas of gene expression during cholestatic injury and repair in mice by integrating spatial enhanced resolution omics sequencing and single-cell transcriptomics. Spatiotemporal analyses revealed a key role of cholangiocyte-driven signaling correlating with the periportal damage-repair response. Cholangiocytes express genes related to recruitment and differentiation of lipid-associated macrophages, which generate feedback signals enhancing ductular reaction. Moreover, cholangiocytes express high TGFß in association with the conversion of liver progenitor-like cells into cholangiocytes during injury and the dampened proliferation of periportal hepatocytes during recovery. Notably, Atoh8 restricts hepatocyte proliferation during 3,5-diethoxycarbonyl-1,4-dihydro-collidin damage and is quickly downregulated after injury withdrawal, allowing hepatocytes to respond to growth signals. Our findings lay a keystone for in-depth studies of cellular dynamics and molecular mechanisms of cholestatic injuries, which may further develop into therapies for cholangiopathies.

Single cell multi-omics reveal intra-cell-line heterogeneity across human cancer cell lines.

Human cancer cell lines have long served as tools for cancer research and drug discovery, but the presence and the source of intra-cell-line heterogeneity remain elusive. Here, we perform single-cell RNA-sequencing and ATAC-sequencing on 42 and 39 human cell lines, respectively, to illustrate both transcriptomic and epigenetic heterogeneity within individual cell lines. Our data reveal that transcriptomic heterogeneity is frequently observed in cancer cell lines of different tissue origins, often driven by multiple common transcriptional programs. Copy number variation, as well as epigenetic variation and extrachromosomal DNA distribution all contribute to the detected intra-cell-line heterogeneity. Using hypoxia treatment as an example, we demonstrate that transcriptomic heterogeneity could be reshaped by environmental stress. Overall, our study performs single-cell multi-omics of commonly used human cancer cell lines and offers mechanistic insights into the intra-cell-line heterogeneity and its dynamics, which would serve as an important resource for future cancer cell line-based studies.

GA3 Treatment Delays the Deterioration of 'Shixia' Longan during the On-Tree Preservation and Room-Temperature Storage and Up-Regulates Antioxidants.

Gibberellic acids had been proven to improve the fruit quality and storability by delaying deterioration and maintaining the antioxidant system. In this study, the effect of GA3 spraying at different concentrations (10, 20, and 50 mg L-1) on the quality of on-tree preserved 'Shixia' longan was examined. Only 50 mg L-1 GA3 significantly delayed the decline of soluble solids (22.0% higher than the control) and resulted in higher total phenolics content (TPC), total flavonoid content (TFC), and phenylalanine ammonia-lyase activity in pulp at the later stages. The widely targeted metabolome analysis showed that the treatment reprogrammed secondary metabolites and up-regulated many tannins, phenolic acids, and lignans during the on-tree preservation. More importantly, the preharvest 50 mg L-1 GA3 spraying (at 85 and 95 days after flowering) led to significantly delayed pericarp browning and aril breakdown, as well as lower pericarp relative conductivity and mass loss at the later stages of room-temperature storage. The treatment also resulted in higher antioxidants in pulp (vitamin C, phenolics, and reduced glutathione) and pericarp (vitamin C, flavonoids, and phenolics). Therefore, preharvest 50 mg L-1 GA3 spraying is an effective method for maintaining the quality and up-regulating antioxidants of longan fruit during both on-tree preservation and room-temperature storage.

A Comprehensive Analysis of Physiologic and Hormone Basis for the Difference in Room-Temperature Storability between 'Shixia' and 'Luosanmu' Longan Fruits.

Although the effects of phytohormones (mainly salicylic acid) on the storability of longan fruit have been reported, the relationship between postharvest hormone variation and signal transduction and storability remains unexplored. The basis of physiology, biochemistry, hormone content and signalling for the storability difference at room-temperature between 'Shixia' and 'Luosanmu' longan fruit were examined. 'Luosanmu' longan exhibited faster pericarp browning, aril breakdown and rotting during storage. 'Luosanmu' pericarp exhibited higher malondialdehyde but faster decreased total phenolics, flavonoid, glutathione, vitamin C, catalase activity and gene expression. Higher H2O2 and malondialdehyde but lower glutathione, glutathione-reductase and peroxidase activities, while higher activities and gene expressions of polygalacturonase, ß-galactosidase and cellulose, lower covalent-soluble pectin, cellulose and hemicellulose but higher water-soluble pectin were observed in 'Luosanmu' aril. Lower abscisic acid and methyl jasmonate but higher expressions of LOX2, JAZ and NPR1 in pericarp, while higher abscisic acid, methyl jasmonate and salicylic acid together with higher expressions of ABF, JAZ, NPR1 and PR-1 in 'Luosanmu' aril were observed. In conclusion, the imbalance between the accumulation and scavenging of active oxygen in 'Luosanmu' longan might induce faster lipid peroxidation and senescence-related hormone signalling and further the polymerization of phenolics in pericarp and polysaccharide degradation in aril.

Spatiotemporal mapping of gene expression landscapes and developmental trajectories during zebrafish embryogenesis.

A major challenge in understanding vertebrate embryogenesis is the lack of topographical transcriptomic information that can help correlate microenvironmental cues within the hierarchy of cell-fate decisions. Here, we employed Stereo-seq to profile 91 zebrafish embryo sections covering six critical time points during the first 24 h of development, obtaining a total of 152,977 spots at a resolution of 10 × 10 × 15 µm3 (close to cellular size) with spatial coordinates. Meanwhile, we identified spatial modules and co-varying genes for specific tissue organizations. By performing the integrated analysis of the Stereo-seq and scRNA-seq data from each time point, we reconstructed the spatially resolved developmental trajectories of cell-fate transitions and molecular changes during zebrafish embryogenesis. We further investigated the spatial distribution of ligand-receptor pairs and identified potentially important interactions during zebrafish embryo development. Our study constitutes a fundamental reference for further studies aiming to understand vertebrate development.

Comparative metabolomics reveals differences in primary and secondary metabolites between "Shixia" and "Chuliang" longan (Dimocarpus longan Lour.) pulp.

Longan was a characteristic fruit for both medicine and food in China, which was rich in primary and secondary metabolites. Comprehensive high-throughput identification and comparison of metabolites in longan pulp among different varieties were still lacked. "Shixia" (SX) and "Chuliang" (CL) were the biggest major cultivars of longan in China. In this study, the content of total soluble solid, total flavonoid, and total phenolics indicated the difference of sweetness and bioactive compound content between the SX and CL pulp. Through a widely targeted metabolome, a total of 514 metabolites were identified and categorized into 23 groups mainly including flavonoids, amino acids & derivatives, lipids, phenolic acids, nucleotides & derivatives, alkaloids, organic acids and sugars & derivatives. A total of 89 metabolites with significantly differential accumulation (variable importance in projection (VIP) value ≧1, p-value <.05) over 1.2 fold were found between SX and CL, which were mainly enriched into pathways including flavone and flavonol biosynthesis, glycolysis/gluconeogenesis, and arginine and proline metabolism. Higher leveled hexose and hexose-phosphate (i.e., ß-D-glucose, D(+)-glucose, glucose-1-phosphate and glucose-6-phosphate), dominant organic acids (i.e., citric acid, succinic acid, D-malic acid, and citramalate), and essential amino acids (L-threonine, L-valine, L-isoleucine, L-leucine, L-phenylalanine and L-lysine) in SX pulp might be contributed to the taste and flavor difference between SX and CL. Moreover, the greatly differential accumulated secondary metabolites especially flavonoids and phenolic acids might result in different medicinal and nutritional characteristic between SX and CL. In conclusion, this study provided a systemic metabolic basis for understanding the nutritional differences between SX and CL and would help deepen the molecular biology and pharmacology research on characteristic metabolites in longan pulp.

Postharvest melatonin treatment inhibited longan (Dimocarpus longan Lour.) pericarp browning by increasing ROS scavenging ability and protecting cytomembrane integrity.

Postharvest melatonin treatments have been reported to improve the quality and storability, especially to inhibit browning in many fruits, but the effect had not been systematically investigated on longan fruit. In this study, the effect of 0.4 mM melatonin (MLT) dipping on the quality and pericarp browning of longan fruits stored at low temperature was investigated. The MLT treatment did not influence the TSS content of longan fruits but lead to increased lightness and h° value while decreased a* value of pericarp. More importantly, the treatment significantly delayed the increase in electrolyte leakage and malonaldehyde accumulation, inhibited the activities of polyphenol oxidase and peroxidase, and thus retarded pericarp browning. In addition, the treatment significantly inhibited the production of O2 •- and H2O2 while promoted the accumulation of glutathione, flavonoids, and phenolics at earlier storage stages in longan pericarp. Interestingly, the activities of ascorbate peroxidase (APX) and superoxide dismutase (SOD) were significantly upregulated but activities of catalase were downregulated in the MLT-treated longan pericarp. MLT treatment effectively enhanced APX and SOD activities, increased flavonoid, phenolics, and glutathione content, protected cytomembrane integrity, inhibited the production of O2 •- and H2O2 and browning-related enzymes, and thus delayed the longan pericarp browning.

Self-assembly and mineralization of full-length human amelogenin and its functional fragments in vitro.

OBJECTIVES: To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) in vitro and its role in hydroxyapatite (HA) crystal formation. METHODS: The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified in vitro. The protein solution of 100 µg‧mL-1, pH=8, was prepared in Tris-HCl and incubated at room temperature for 1-15 min. Their self-assembly behaviors were observed and compared under a transmission electron microscope (TEM). The full-length AM was added to artificial saliva and incubated for 3 days. A scanning electron microscope (SEM) was used in observing the morphology of the induced new crystals. Then, TARP and LRAP were added. The resulting solution was incubated for 3 days and then observed again. RESULTS: When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor. CONCLUSIONS: The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals in vivo, providing a theoretical basis for the role of the fragments in the growth and maturation of HA crystals.

Widely targeted metabolic analysis revealed the changed pigmentation and bioactive compounds in the ripening Berchemia floribunda (Wall.) Brongn. fruit.

Berchemia plants were important materials for Chinese traditional medicines due to their special secondary metabolites. Unlike the root, stem and leaf tissues, Berchemia floribunda (Wall.) Brongn. fruit was lacked of systematic metabolic investigation. Biochemical analysis found that the total flavonoid and total phenolic content of Berchemia fruit pulp showed a peak value at red ripe stage, and then decreased, but the total anthocyanin content sharply increased along with the coloration. By widely targeted metabolomic analysis, 644 metabolites were identified and categorized into 23 groups mainly including flavonoid, organic acids, amino acids, lipids, phenylpropanoid, nucleotides, alkaloids, carbohydrates, alcohols, anthocyanins & proanthocyanidins, vitamins, terpenes, polyphenols, phenolamides, quinones, indole derivatives, and sterides. Among them, 111 metabolites and 123 metabolites respectively showed up- and down-regulation from break stage to full mature. KEGG enrichment analysis indicated that active secondary metabolism such as biosynthesis of phenylpropanoids, flavonoid, and alkaloids happened during Berchemia fruit ripening. More importantly, Cyanidin-3-O-galactoside and other 3 cyanidins were found to be the predominant pigments in mature Berchemia fruit and increased cyanidins and pelargonidins but decreased anthocyanins might be contributed to the purple pigmentation of Berchemia fruit. Interestingly, 29 pharmaceutical compounds previously reported in other Berchemia tissues were also detected in ripening Berchemia fruit pulp: 8 flavonoid, 2 quinones & sucrose showed up-regulated accumulation while 6 polyphenols, 5 flavonoid, 3 phenylpropanoid, 2 organic acids, 1 quinones and ß-sitosterol showed down-regulated accumulation In conclusion, our first comprehensive metabolic fingerprint will promote the further study of B. floribunda fruit and its medical and food application.

Copper-Catalyzed Inter/Intramolecular N-Alkenylation of Benzimidazoles via Tandem Processes Involving Selectively Mild Iodination of sp3 C-H Bond at α-Position of Ester.

Inter/intramolecular approaches to sp2 C-N bond formation of N-alkenyl benzimidazoles have been accomplished in the presence of an iodide anion associated with a copper catalyst. Both intermolecular and intramolecular reactions included tandem processes, in which selective iodination of sp3 C-H bond at the α-position of ester under mild conditions was demonstrated for the first time. Tandem reactions involving sp3 C-H activation via α-iodo ester intermediate under copper catalysis efficiently provided more than 20 novel azole compounds, and free radicals were not involved in this transformation.

Sequential Michael addition/retro-Claisen condensation of aromatic ß-diketones with α,ß-unsaturated esters: an approach to obtain 1,5-ketoesters.

A K2CO3-catalyzed one-pot protocol involving sequential C-C bond formation and cleavage of aromatic ß-diketones with α,ß-unsaturated esters is developed to obtain 1,5-ketoesters. The sequential reaction via Michael addition and retro-Claisen condensation proceeds smoothly under mild conditions in up to 98% isolated yield. The mechanism study disclosed that the cascade process involved C-C bond cleavage of aromatic ß-diketone as a phenacyl donor under alcoholic alkalescent conditions.