Characterization of an autonomous pathway complex that promotes flowering in Arabidopsis.

Although previous studies have identified several autonomous pathway components that are required for the promotion of flowering, little is known about how these components cooperate. Here, we identified an autonomous pathway complex (AuPC) containing both known components (FLD, LD and SDG26) and previously unknown components (EFL2, EFL4 and APRF1). Loss-of-function mutations of all of these components result in increased FLC expression and delayed flowering. The delayed-flowering phenotype is independent of photoperiod and can be overcome by vernalization, confirming that the complex specifically functions in the autonomous pathway. Chromatin immunoprecipitation combined with sequencing indicated that, in the AuPC mutants, the histone modifications (H3Ac, H3K4me3 and H3K36me3) associated with transcriptional activation are increased, and the histone modification (H3K27me3) associated with transcriptional repression is reduced, suggesting that the AuPC suppresses FLC expression at least partially by regulating these histone modifications. Moreover, we found that the AuPC component SDG26 associates with FLC chromatin via a previously uncharacterized DNA-binding domain and regulates FLC expression and flowering time independently of its histone methyltransferase activity. Together, these results provide a framework for understanding the molecular mechanism by which the autonomous pathway regulates flowering time.

[Effects of post-traumatic stress disorder on the excitability of glutamatergic and GABAergic neurons in dorsal and ventral hippocampus in mice].

The purpose of this study was to investigate the effects of post-traumatic stress disorder (PTSD) on electrophysiological characteristics of glutamatergic and GABAergic neurons in dorsal hippocampus (dHPC) and ventral hippocampus (vHPC) in mice, and to elucidate the mechanisms underlying the plasticity of hippocampal neurons and memory regulation after PTSD. Male C57Thy1-YFP/GAD67-GFP mice were randomly divided into PTSD group and control group. Unavoidable foot shock (FS) was applied to establish PTSD model. The spatial learning ability was explored by water maze test, and the changes in electrophysiological characteristics of glutamatergic and GABAergic neurons in dHPC and vHPC were examined using whole-cell recording method. The results showed that FS significantly reduced the movement speed, and enhanced the number and percentage of freezing. PTSD significantly prolonged the escape latency in localization avoidance training, shortened the swimming time in the original quadrant, extended the swimming time in the contralateral quadrant, and increased absolute refractory period, energy barrier and inter-spike interval of glutamatergic neurons in dHPC and GABAergic neurons in vHPC, while decreased absolute refractory period, energy barrier and inter-spike interval of GABAergic neurons in dHPC and glutamatergic neurons in vHPC. These results suggest that PTSD can damage spatial perception of mice, down-regulate the excitability of dHPC and up-regulate the excitability of vHPC, and the underlying mechanism may involve the regulation of spatial memory by the plasticity of neurons in dHPC and vHPC.

The CCR4-NOT complex component NOT1 regulates RNA-directed DNA methylation and transcriptional silencing by facilitating Pol IV-dependent siRNA production.

Small interfering RNAs (siRNAs) are responsible for establishing and maintaining DNA methylation through the RNA-directed DNA methylation (RdDM) pathway in plants. Although siRNA biogenesis is well known, it is relatively unclear about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana, we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. We demonstrated that NOT1 is required for Pol IV-dependent siRNA accumulation and DNA methylation at a subset of RdDM target genomic loci. Furthermore, we revealed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex by immunoprecipitation combined with mass spectrometry and demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Therefore, our work establishes that the CCR4-NOT complex regulates the biogenesis of Pol IV-dependent siRNAs, and hence facilitates DNA methylation and transcriptional silencing in Arabidopsis.

Folate polyglutamylation is involved in chromatin silencing by maintaining global DNA methylation and histone H3K9 dimethylation in Arabidopsis.

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.

A Pre-mRNA-splicing factor is required for RNA-directed DNA methylation in Arabidopsis.

Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation.

Four undescribed pyrethrins from seeds of Pyrethrum cinerariifolium and their aphidicidal activity.

Four undescribed pyrethrins C-F (1-4) as well as four known pyrethrins (5-8) were isolated from seeds of Pyrethrum cinerariifolium Trev. The structures of compounds 1-4 were elucidated by UV, HRESIMS, and NMR (1H and 13C NMR, 1H-1H COSY, HSQC, HMBC and ROESY), among which the stereostructure of compound 4 was determined by calculated ECD. Furthermore, compounds 1-4 were evaluated for their aphidicidal activities. The insecticidal assay results showed that 1-4 exhibited moderate aphidicidal activities at the concentration of 0.1 mg/mL with the 24 h mortality rates ranging from 10.58 to 52.98%. Among them, pyrethrin D (2) showed the highest aphidicidal activity, with the 24 h mortality rate of 52.98%, which was slightly lower than the positive control (pyrethrin II, 83.52%).

Accumulation of nanoplastics in human cells as visualized and quantified by hyperspectral imaging with enhanced dark-field microscopy.

Nanoplastic (NP) pollution is receiving increasing attention regarding its potential effects on human health. The identification and quantification of intracellular NPs are prerequisites for an accurate risk assessment, but appropriate methods are lacking. Here we present a label-free technique to simultaneously visualize and quantify the bioaccumulation of NPs based on hyperspectral imaging with enhanced dark-field microscopy (HSI-DFM). Using polystyrene NPs (PS NPs) as representative particles, the construction of a hyperspectral library was optimized first with more accurate NP identification achieved when the library was based on intracellular instead of extracellular PS NPs. The PS NPs used herein were labeled with a green fluorescent dye so that the accuracy of HSI-DFM in identifying and quantifying intracellular NPs can be evaluated, by comparing the results with those obtained by fluorescence microscopy and flow cytometry. The validation of HSI-DFM for use in determinations of the NP concentration at the single-cell level allows analyses of the accumulation kinetics of NPs in single living cells. The utility of HSI-DFM in different cell lines and with NPs differing in their chemical composition was also demonstrated. HSI-DFM therefore provides a new approach to studies of the accumulation and distribution of NPs in human cells.

Potential effects of Ag ion on the host by changing the structure of its gut microbiota.

Silver (Ag) can change the structure of the gut microbiota (GM), but how such change may affect host health is unknown. In this study, mice were exposed to silver acetate daily for 120 days. During this period, Ag accumulation in the liver was measured, its effects on GM structure were analyzed, and potential metabolic changes in liver and serum were examined. Although Ag accumulation remained unchanged in most treatments, the ratio of Firmicutes to Bacteroidetes at the phylum level increased and changes in the relative abundance of 33 genera were detected, suggesting that Ag altered the energy metabolism of mice via changes in the gut GM. In serum and liver, 34 and 72 differentially expressed metabolites were identified, respectively. The KEGG pathways thus enriched mainly included those involving the metabolism of amino acids, organic acids, lipids, and purine. Strong correlations were found between 33 % of the microorganisms with altered relative abundances and 46 % of the differentially expressed metabolites. The resulting clusters yielded two communities responsible for host inflammation and energy metabolism. Overall, these results demonstrate potential effects of Ag on the host, by changing its GM structure, and the need to consider them when evaluating the health risk of Ag.

Changes in Gut Microbiota Structure: A Potential Pathway for Silver Nanoparticles to Affect the Host Metabolism.

Silver nanoparticles (AgNPs) are one of the most widely used NPs. Their adverse effects on either the host or its gut microbiota (GM) have been examined. Nevertheless, whether the GM plays any role in AgNP toxicity to the host remains unclear. In the present study, AgNPs were administered to mice by oral gavage once a day for 120 days. A significant dose-dependent accumulation of Ag in the liver was observed, with a steady state reached within 21 days. The AgNPs changed the structure of the GM, mainly with respect to microorganisms involved in the metabolism of energy, amino acids, organic acids, and lipids, as predicted in a PICRUST analysis. Effects of the AgNPs on liver metabolism were also demonstrated, as a KEGG pathway analysis showed the enrichment of pathways responsible for the metabolism of amino acids, purines and pyrimidine, lipids, and energy. More interestingly, the changes in GM structure and liver metabolism were highly correlated, evidenced by the correlation between ∼23% of the differential microorganisms at the genus level and ∼60% of the differential metabolites. This implies that the metabolic variations in liver as affected by AgNPs were partly attributable to NP-induced changes of GM structure. Therefore, our results demonstrate the importance of considering the roles of GM in the toxicity of NPs to the host in evaluations of the health risks of NPs.

A label-free technique to quantify and visualize gold nanoparticle accumulation at the single-cell level.

Despite their wide bioapplications, potential health risks of gold nanoparticles (AuNPs) remain unclear. As a determinant of their risks, AuNP accumulation within a cell population is subject to cell-to-cell heterogeneity. Methods to simultaneously quantify and visualize intracellular AuNPs at the single-cell level are, however, lacking. Here we developed a novel label-free technique, based on hyperspectral imaging with enhanced darkfield microscopy (HSI-DFM), to visualize and quantify AuNP accumulation at the single-cell level. The identification ability of the hyperspectral libraries derived from extra- and intracellular AuNPs was compared. The spectral number in the libraries was optimized to maximize their identification ability while minimizing the identification time. In addition, a filtration method was established to merge spectral libraries from different cell lines based on their similarity. The intracellularly accumulated AuNPs as determined by HSI-DFM well correlated with those detected by inductively coupled plasma mass spectrometry. This validation allowed us to calculate the intracellular concentration of AuNPs at the single-cell level and to monitor the accumulation kinetics of AuNPs in living cells. The label-free method developed herein can be applied to other types of AuNPs differing in their physicochemical properties as well as other NPs, as long as they are detectable by HSI-DFM.

[Study on testing methods of Al content of polyimide hybrid films with Al2O3].

The inorganic particles hybrid polyimide films are newly emerging advanced materials with excellent corona-resistant and have been widely used in frequency control motor. The Al2O3/PI hybrid films with different Al2O3 contents were prepared by superfine aluminum power treated by coupling agent and polyamide acid (PAA). Qualitative analysis and quantitative analysis on the inorganic content to it were conducted using Fourier Transform Infrared Spectroscope (FTIR), X-ray photoelectron spectroscopy (XPS), Inductively coupled plasma spectrometry (ICP), Gravimetric method, Thermogravimetric Analysis (TG/air atmosphere). It was found that FTIR and XPS are good qualitative analysis methods. FTIR is used to inference possible components by analyzing the structure of material. The method has a lot of advantages such as easily operational, good repeatability, high accuracy and so on. XPS is mainly used to get information of elements contained in the material, it provides information about the core level binding energies and oxidation states of complexes. It can be used to identify the species and valence states of elements, measure the relative content of elements. The relative standard deviations (RSDs) of XPS and TG are too great to perform quantitative analysis, the RSDs of XPS are all above 5.0%, and TG's RSDs are also above 2.0%. So they can be only used as semiquantitative analysis methods. On the contrary, ICP and Gravimetric method are two excellent quantitative analysis methods, their RSDs are all below 1.0%. Moreover Gravimetric method only can be used to analyze single inorganic constituent complex material, although its measured value is closest approach to theoretical value. ICP is the most accurate method and it can be used to analyze multi inorganic components in complex material, this method proved to be easily operational, rapid, highly sensitive, and accurate, and can be adopted as the method of determining many elements simultaneously. So a method was got to analysis of inorganic constituent in complex material from the conclusion upward. Firstly, components in complex material are defined by using FTIR and XPS as qualitative analysis methods and then using the result of XPS as a reference, exactly quantitative analysis of inorganic constituent in complex material was performed by using Gravimetric method and ICP.

[Characterization of inorganic nano-alundum composite film of polyimide].

The key to the study on the regularity about the mechanical, thermology and electricities property of the inorganic nano-mingled organic composition thin film is to understand the incorporated quantity, the particle size and distribution of nano-inorganic matter in the membrane quickly and accurately. In the present paper, the chemical structure, surface morphology and the actual content of nano-Al2O3 of the nano Al2O3-composite film of polyimide were characterized by X-ray atomic fluorescent spectroscopy (XRF), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic forced microscope (AFM). The results are that the organic phase of PI and the inorganic phase of Al2O3 formed a complex composite hybrid system of bond-to-bond pattern, the nano-Al2O3 particles in the film of PI are dispersed homogeneously, and the diameter of the particle is smaller than 50 nm; the weight content of Al2O3 is 7.9% by XRF. The approach we used is an effective way of analyzing the inorganic component of the organic composite film materials doped with the inorganic nano-phase materials with the merits of no pretreatment, no fed charge (for analysis of insulation materials), no-contagion, no destruction, high speed and high accuracy, etc.