Quantifying the Growth Kinetics of Lithium Metal Reduced from Solid Ionic Conductors.

Investigating the growth kinetics of Li metal in solid-state batteries is crucial to both a fundamental understanding and practical application. Here, by directly observing the formation of Li metal from Ta-doped Li6.4La3Zr1.4Ta0.6O12 (LLZTO) in a transmission electron microscope, the growth kinetics is analyzed quantitatively. The growth kinetics of Li deposits shows a cubic-curve characteristic for LLZTO with Li-source-free. Instead, a linear growth process is observed with Li-source supplied. The impact of the illuminating electron dose rate on the growth kinetics is clarified, indicating that even low dose rates (1-3 e-/Å2/s) could affect Li growth, highlighting the significance of controlling dose rates. Furthermore, a new pathway for the formation of Li metal from Li-containing materials utilizing the field-emission effect is reported. This work has implications on the failure mechanism in solid batteries by using limited Li anodes and opens pathways for regulating Li growth in LLZTO at various scenarios, which can also extend to other ionic conductors.

Integrative Analysis of Metabolome and Transcriptome Reveals Molecular Insight into Metabolomic Variations during Hawthorn Fruit Development.

Hawthorn (Crataegus pinnatifida var. major), a cultivated fruit tree, is native and unique to China. Its fruits have high nutritional, health, and medicinal values. However, the development and ripening process of hawthorns is accompanied by dramatic changes in flavor, aroma, and bioactive phytochemicals, which are the fundamental factors that contribute to the potential health benefits and establishment of fruit quality. Therefore, an exploration of the dynamic changes in metabolites and their regulatory networks during the development of hawthorn fruits can elucidate the formation mechanisms of active substances in hawthorn fruits. In this study, we used a broad targeted metabolomics approach to identify and analyze the dynamics of metabolites in hawthorn fruits at five developmental stages. The results revealed 998 primary and secondary metabolites that were classified into 15 categories. The accumulation levels of most sugars increased during fruit development and then accelerated at the fruit ripening stage. The accumulation levels of a few organic acids (e.g., citric acid, isocitric acid, and quinic acid) continuously increased. Many organic acids exhibited significant decreasing trends. Among the 561 secondary metabolites detected, 189 were phenolic acids and 199 were flavonoids. The levels of many flavonoids were significantly reduced at later stages of fruit development; in contrast, the levels of two anthocyanins significantly increased during fruit ripening. Correlation analysis revealed that there is a certain correlation within and between primary as well as secondary metabolites during fruit development. Furthermore, the integration of metabolomic and transcriptomic data in this study revealed that changes in the expression of some differentially expressed genes (DEGs) were associated with the accumulation of metabolites such as sugars, organic acids, and flavonoids, e.g., the upregulated expression levels of CS (citrate synthase) genes were consistent with the continued accumulation of citric acid. Overall, this study demonstrates the metabolic changes that occur during the development of hawthorn fruit, explores the molecular mechanisms that underlie metabolite changes during fruit development, and lays a strong theoretical foundation for the improvement of hawthorn fruit quality and the development of functional components.

Bisphenol A analogues induce a feed-forward estrogenic response in zebrafish.

Because exposure to bisphenol A (BPA) has been linked to health problems in humans and wildlife, BPA analogues have been synthesized to be considered as replacement molecules. We here have examined estrogenic activity of BPA and five of its analogues, BPAF, BPE, BPC, BPC-Cl, and BPS by a combination of zebrafish-based in vivo and in vitro assays. We used transgenic estrogen reporter (5xERE:GFP) fish to study agonistic effects of bisphenols. Exposures to BPA, BPAF, BPE, and BPC, induced GFP expression in estrogen reporter fish at low exposure concentrations in the heart valves and at higher concentrations in the liver, whereas BPC-Cl activated GFP expression mainly in the liver, and BPS faintly in the heart only. The in vivo response was compared to in vitro estrogenicity of bisphenol exposure using reporter cells that express the zebrafish estrogen receptors driving expression of an estrogen response element (ERE)-luciferase reporter. In these cells, BPA, BPAF, BPC, BPE and BPS preferentially activated Esr1, whereas BPC-Cl preferentially activated Esr2a. By quantitative PCR we found that exposure to BPAF induced expression of the classical estrogen target genes vtg1, esr1, and cyp19a1b in a concentration response manner, but the most responsive target gene was f13a1a. Exposure to BPC-Cl resulted in a different expression pattern of vtg1 and f13a1a with an activation at low concentrations, followed by a declining expression at higher concentrations. Because expression of f13a1a was strongly activated by all compounds tested, we suggest including this mRNA as a biomarker for estrogenicity in larval fish. We further showed that exposure to BPAF and BPC-Cl increased E2 levels in zebrafish larvae, indicating that bisphenol exposures result in a feed-forward response that can further augment the estrogenic activity of these compounds.

Enhancement of fluorescence and anti-tumor effect of ZnO QDs by La doping.

ZnO quantum dots (QDs) have received much attention as biomarkers and drug delivery systems in cancer treatment, due to their low cost, ease of preparation, and pH-responsive degradation. However, its applications are limited by the low quantum yield and light absorption. In this work, a lanthanum-doped zinc oxide (La-ZnO) QDs-based drug delivery platform was constructed. The results show that 4% La doping is the most beneficial for improving the fluorescent properties of the ZnO QDs. After loading the drug, the cell activity was 15% at ZnO@DOX and 12% at La-ZnO@DOX. According to in vitro and in vivo experiment results, the La-ZnO QDs show enhancement of the antitumor effect. Dual enhancement of fluorescence and anti-tumor effects make La-ZnO QDs promising as a drug delivery system in cancer treatment.

Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model.

In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for the identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative concentration-response information on estrogenic activity of chemical compounds.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

Demodifying RNA for Transcriptomic Analyses of Archival Formalin-Fixed Paraffin-Embedded Samples.

Archival formalin-fixed paraffin-embedded (FFPE) tissue samples offer a vast but largely untapped resource for genomic research. The primary technical issues limiting use of FFPE samples are RNA yield and quality. In this study, we evaluated methods to demodify RNA highly fragmented and crosslinked by formalin fixation. Primary endpoints were RNA recovery, RNA-sequencing quality metrics, and transcriptional responses to a reference chemical (phenobarbital, PB). Frozen mouse liver samples from control and PB groups (n = 6/group) were divided and preserved for 3 months as follows: frozen (FR); 70% ethanol (OH); 10% buffered formalin for 18 h followed by ethanol (18F); or 10% buffered formalin (3F). Samples from OH, 18F, and 3F groups were processed to FFPE blocks and sectioned for RNA isolation. Additional sections from 3F received the following demodification protocols to mitigate RNA damage: short heated incubation with Tris-Acetate-EDTA buffer; overnight heated incubation with an organocatalyst using 2 different isolation kits; or overnight heated incubation without organocatalyst. Ribo-depleted, stranded, total RNA libraries were built and sequenced using the Illumina HiSeq 2500 platform. Overnight incubation (± organocatalyst) increased RNA yield >3-fold and RNA integrity numbers and fragment analysis values by > 1.5- and >3.0-fold, respectively, versus 3F. Postsequencing metrics also showed reduced bias in gene coverage and deletion rates for overnight incubation groups. All demodification groups had increased overlap for differentially expressed genes (77%-84%) and enriched pathways (91%-97%) with FR, with the highest overlap in the organocatalyst groups. These results demonstrate simple changes in RNA isolation methods that can enhance genomic analyses of FFPE samples.

Farnesoid X Receptor Signaling Shapes the Gut Microbiota and Controls Hepatic Lipid Metabolism.

The gut microbiota modulates obesity and associated metabolic phenotypes in part through intestinal farnesoid X receptor (FXR) signaling. Glycine-ß-muricholic acid (Gly-MCA), an intestinal FXR antagonist, has been reported to prevent or reverse high-fat diet (HFD)-induced and genetic obesity, insulin resistance, and fatty liver; however, the mechanism by which these phenotypes are improved is not fully understood. The current study investigated the influence of FXR activity on the gut microbiota community structure and function and its impact on hepatic lipid metabolism. Predictions about the metabolic contribution of the gut microbiota to the host were made using 16S rRNA-based PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), then validated using 1H nuclear magnetic resonance-based metabolomics, and results were summarized by using genome-scale metabolic models. Oral Gly-MCA administration altered the gut microbial community structure, notably reducing the ratio of Firmicutes to Bacteroidetes and its PICRUSt-predicted metabolic function, including reduced production of short-chain fatty acids (substrates for hepatic gluconeogenesis and de novo lipogenesis) in the ceca of HFD-fed mice. Metabolic improvement was intestinal FXR dependent, as revealed by the lack of changes in HFD-fed intestine-specific Fxr-null (FxrΔIE) mice treated with Gly-MCA. Integrative analyses based on genome-scale metabolic models demonstrated an important link between Lactobacillus and Clostridia bile salt hydrolase activity and bacterial fermentation. Hepatic metabolite levels after Gly-MCA treatment correlated with altered levels of gut bacterial species. In conclusion, modulation of the gut microbiota by inhibition of intestinal FXR signaling alters host liver lipid metabolism and improves obesity-related metabolic dysfunction. IMPORTANCE The farnesoid X receptor (FXR) plays an important role in mediating the dialog between the host and gut microbiota, particularly through modulation of enterohepatic circulation of bile acids. Mounting evidence suggests that genetic ablation of Fxr in the gut or gut-restricted chemical antagonism of the FXR promotes beneficial health effects, including the prevention of nonalcoholic fatty liver disease in rodent models. However, questions remain unanswered, including whether modulation of FXR activity plays a role in shaping the gut microbiota community structure and function and what metabolic pathways of the gut microbiota contribute in an FXR-dependent manner to the host phenotype. In this report, new insights are gained into the metabolic contribution of the gut microbiota to the metabolic phenotypes, including establishing a link between FXR antagonism, bacterial bile salt hydrolase activity, and fermentation. Multiple approaches, including unique mouse models as well as metabolomics and genome-scale metabolic models, were employed to confirm these results.

Bioinformatic analysis of microRNA networks following the activation of the constitutive androstane receptor (CAR) in mouse liver.

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily that functions as a xenosensor, serving to regulate xenobiotic detoxification, lipid homeostasis and energy metabolism. CAR activation is also a key contributor to the development of chemical hepatocarcinogenesis in mice. The underlying pathways affected by CAR in these processes are complex and not fully elucidated. MicroRNAs (miRNAs) have emerged as critical modulators of gene expression and appear to impact many cellular pathways, including those involved in chemical detoxification and liver tumor development. In this study, we used deep sequencing approaches with an Illumina HiSeq platform to differentially profile microRNA expression patterns in livers from wild type C57BL/6J mice following CAR activation with the mouse CAR-specific ligand activator, 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP). Bioinformatic analyses and pathway evaluations were performed leading to the identification of 51 miRNAs whose expression levels were significantly altered by TCPOBOP treatment, including mmu-miR-802-5p and miR-485-3p. Ingenuity Pathway Analysis of the differentially expressed microRNAs revealed altered effector pathways, including those involved in liver cell growth and proliferation. A functional network among CAR targeted genes and the affected microRNAs was constructed to illustrate how CAR modulation of microRNA expression may potentially mediate its biological role in mouse hepatocyte proliferation. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model.

In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative dose-response information on estrogenic activity of chemical compounds.

Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice.

Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) was assessed using global (1)H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolite profiling of extracts obtained from serum and liver. (1)H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an Ahr-dependent manner. Targeted analyses using gas chromatography coupled with mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of stearoyl coenzyme A desaturase 1 was observed. In addition, TCDF exposure resulted in inhibition of de novo fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA, and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from Ahr-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure.

Estrogen receptor signaling during vertebrate development.

Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affecting both the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17ß-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

ERbeta1 represses basal breast cancer epithelial to mesenchymal transition by destabilizing EGFR.

INTRODUCTION: Epithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. 60% of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERbeta1). However, it is still unclear whether the ERbeta expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERbeta1 through regulating EMT can influence invasion and metastasis in basal-like cancers. METHODS: Basal-like breast cancer cells (MDA-MB-231 and Hs578T) in which ERbeta1 was either overexpressed or downregulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Coimmunoprecipitation and ubiquitylation assays were employed to examine whether ERbeta1 alters EGFR protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERbeta1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERbeta1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry. RESULTS: Here we show that ERbeta1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERbeta1-mediated upregulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERbeta1 and E-cadherin expression was additionally observed in breast tumor samples. Downregulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERbeta1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERbeta1 to sustain the epithelial phenotype. CONCLUSIONS: Taken together, the results of our study strengthen the association of ERbeta1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.

High-dose siRNAs upregulate mouse Eri-1 at both transcription and posttranscription levels.

The eri-1 gene encodes a 3' exonuclease that can negatively regulate RNA interference via siRNase activity. High-dose siRNAs (hd-siRNAs) can enhance Eri-1 expression, which in return degrade siRNAs and greatly reduces RNAi efficiency. Here we report that hd-siRNAs induce mouse Eri-1 (meri-1) expression through the recruitment of Sp1, Ets-1, and STAT3 to the meri-1 promoter and the formation of an Sp1-Ets-1-STAT3 complex. In addition, hd-siRNAs also abolish the 3' untranslated region (UTR) mediated posttranscriptional repression of meri-1. Our findings demonstrate the molecular mechanism underlying the upregulation of meri-1 by hd-siRNA.

RNA interference against hepatitis B virus with endoribonuclease-prepared siRNA despite of the target sequence variations.

RNA interference (RNAi) has proven to be very powerful in inhibiting hepatitis B virus (HBV) replication by cell culture and mouse model studies. We have previously reported that endoribonuclease-prepared short interfering RNAs (esiRNAs) were able to inhibit HBV replication more efficiently than synthesized siRNAs. Here we tested the hypothesis that esiRNAs are able to inhibit gene expression with limited mutations within the target region. Target sequences with different similarities to esiHBVP (esiRNA targeting the DNA polymerase and S antigen of Hepatitis B virus) were amplified and cloned into the 3' untranslated region of HBsAg, respectively. When the obtained expression vectors were co-transfected with esiHBVP into CHO cells, HBsAg expression was suppressed with same efficiency regardless of the target sequence similarities. In HepG2 cells, esiHP9 based on one of the amplified sequence that sharing 87% similarity to the target region suppressed HBsAg expression effectively and dose dependently. In vivo experiment showed that a single dose of 5 microg esiHP9 was able to reduce HBsAg and HBeAg level in the mouse sera by 88 and 77% despite of its 87% similarity to the target sequence, which was as good as esiHBVP that is 100% similar to the target sequence. All the data suggest that esiRNA can tolerate limited target sequence variations without losing its inhibitory capacity. It would be very helpful to suppress virus replication by RNAi despite of their high mutation rate.