SmEIL1 transcription factor inhibits tanshinone accumulation in response to ethylene signaling in Salvia miltiorrhiza.

Among the bioactive compounds, lipid-soluble tanshinone is present in Salvia miltiorrhiza, a medicinal plant species. While it is known that ethephon has the ability to inhibit the tanshinones biosynthesis in the S. miltiorrhiza hairy root, however the underlying regulatory mechanism remains obscure. In this study, using the transcriptome dataset of the S. miltiorrhiza hairy root induced by ethephon, an ethylene-responsive transcriptional factor EIN3-like 1 (SmEIL1) was identified. The SmEIL1 protein was found to be localized in the nuclei, and confirmed by the transient transformation observed in tobacco leaves. The overexpression of SmEIL1 was able to inhibit the tanshinones accumulation to a large degree, as well as down-regulate tanshinones biosynthetic genes including SmGGPPS1, SmHMGR1, SmHMGS1, SmCPS1, SmKSL1 and SmCYP76AH1. These are well recognized participants in the tanshinones biosynthesis pathway. Further investigation on the SmEIL1 was observed to inhibit the transcription of the CPS1 gene by the Dual-Luciferase (Dual-LUC) and yeast one-hybrid (Y1H) assays. The data in this work will be of value regarding the involvement of EILs in regulating the biosynthesis of tanshinones and lay the foundation for the metabolic engineering of bioactive ingredients in S. miltiorrhiza.

Transcriptome Analysis Reveals the Molecular Mechanisms of BR Negative Regulatory Factor StBIN2 Maintaining Tuber Dormancy.

Potato is an important food crop. After harvest, these tubers will undergo a period of dormancy. Brassinosteroids (BRs) are a new class of plant hormones that regulate plant growth and seed germination. In this study, 500 nM of BR was able to break the dormancy of tubers. Additionally, exogenous BR also upregulated BR signal transduction genes, except for StBIN2. StBIN2 is a negative regulator of BR, but its specific role in tuber dormancy remains unclear. Transgenic methods were used to regulate the expression level of StBIN2 in tubers. It was demonstrated that the overexpression of StBIN2 significantly prolonged tuber dormancy while silencing StBIN2 led to premature sprouting. To further investigate the effect of StBIN2 on tuber dormancy, RNA-Seq was used to analyze the differentially expressed genes in OE-StBIN2, RNAi-StBIN2, and WT tubers. The results showed that StBIN2 upregulated the expression of ABA signal transduction genes but inhibited the expression of lignin synthesis key genes. Meanwhile, it was also found that StBIN2 physically interacted with StSnRK2.2 and StCCJ9. These results indicate that StBIN2 maintains tuber dormancy by mediating ABA signal transduction and lignin synthesis. The findings of this study will help us better understand the molecular mechanisms underlying potato tuber dormancy and provide theoretical support for the development of new varieties using related genes.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for determination of phytohormones in the medicinal plant saffron.

Saffron (Crocus sativus L.) is a valuable Chinese herb with high medicinal value. Saffron pistils are used as medicine, so increasing the number of flowers can increase the yield. Plant hormones have essential roles in the growth and development of saffron, as well as the response to biotic and abiotic stresses (especially in floral initiation), which may directly affect the number of flowers. Quantitative analysis of plant hormones provides a basis for more efficient research on their synthesis, transportation, metabolism, and action. However, starch (which interferes with extraction) is present in high levels, and hormone levels are extremely low, in saffron corms, thereby hampering accurate determination of plant-hormone levels in saffron. Herein, we screened an efficient and convenient pre-treatment method for plant materials containing abundant amounts of starch. Also, we proposed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of abscisic acid (ABA) and auxin (IAA). Then, the method was applied for the detection of hormone-content differences between flowering and non-flowering top buds, as well as between lateral and top buds. Our method showed high sensitivity, reproducibility, and reliability. Specifically, good linearity in the range 2-100 ng ml-1 was achieved in the determination of ABA and IAA, and the correlation coefficient (R2) was >0.9982. The relative standard deviation was 2.956-14.51% (intraday) and 9.57-18.99% (interday), and the recovery range was 89.04-101.1% (n = 9). The matrix effect was 80.38-90.50% (n = 3). The method was thoroughly assessed employing various "green" chemistry evaluation tools: Blue Applicability Grade Index (BAGI), Complementary Green Analytical Procedure Index (Complex GAPI) and Red Green Blue 12 Algorithm (RGB12). These tools revealed the good greenness, analytical performance, applicability, and overall sustainability alignment of our method. Quantitative results showed that, compared with saffron with a flowering phenotype cultivated at 25 °C, the contents of IAA and ABA in the terminal buds of saffron cultivated at 16 °C decreased significantly. When cultivated at 25 °C, the IAA and ABA contents in the terminal buds of saffron were 1.54- and 4.84-times higher than those in the lateral buds, respectively. A simple, rapid, and accurate UPLC-MS/MS method was established to determine IAA and ABA contents. Using this method, a connection between the contents of IAA and ABA and the flowering phenotype was observed in the quantification results. Our data lay a foundation for studying the flowering mechanism of saffron.

FFAR2 expressing myeloid-derived suppressor cells drive cancer immunoevasion.

BACKGROUND: Emerging evidences suggest that aberrant metabolites contributes to the immunosuppressive microenvironment that leads to cancer immune evasion. Among tumor immunosuppressive cells, myeloid-derived suppressor cells (MDSCs) are pathologically activated and extremely immunosuppressive, which are closely associated with poor clinical outcomes of cancer patients. However, the correlation between MDSCs mediated immunosuppression and particular cancer metabolism remained elusive. METHODS: Spontaneous lung adenocarcinoma and subcutaneous mouse tumor models, gas chromatography-mass spectrometry (GC-MS) and immunofluorescence assay of patient-derived lung adenocarcinoma tissues, and flow cytometry, RNA sequencing and Western blotting of immune cells, were utilized. RESULTS: Metabolite profiling revealed a significant accumulation of acetic acids in tumor tissues from both patients and mouse model, which contribute to immune suppression and cancer progression significantly through free fatty acid receptor 2 (FFAR2). Furthermore, FFAR2 is highly expressed in the myeloid-derived suppressor cells (MDSCs) from the tumor of lung adenocarcinoma (LUAD) patients which is greatly associated with poor prognosis. Surprisingly, whole or myeloid Ffar2 gene deletion markedly inhibited urethane-induced lung carcinogenesis and syngeneic tumor growth with reduced MDSCs and increased CD8+ T cell infiltration. Mechanistically, FFAR2 deficiency in MDSCs significantly reduced the expression of Arg1 through Gαq/Calcium/PPAR-γ axis, which eliminated T cell dysfunction through relieving L-Arginine consumption in tumor microenvironment. Therefore, replenishment of L-Arginine or inhibition to PPAR-γ restored acetic acids/FFAR2 mediated suppression to T cells significantly. Finally, FFAR2 inhibition overcame resistance to immune checkpoint blockade through enhancing the recruitment and cytotoxicity of tumor-infiltrating T cells. CONCLUSION: Altogether, our results demonstrate that the acetic acids/FFAR2 axis enhances MDSCs mediated immunosuppression through Gαq/calcium/PPAR-γ/Arg1 signaling pathway, thus contributing to cancer progression. Therefore, FFAR2 may serve as a potential new target to eliminate pathologically activated MDSCs and reverse immunosuppressive tumor microenvironment, which has great potential in improving clinical outcomes of cancer immunotherapy.

Study on the ameliorative effect of honeysuckle on DSS-induced ulcerative colitis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Honeysuckle, first documented in the Miscellaneous Records of Famous Physicians, is known for its ability to expel toxin and cool blood to stop diarrhea. Modern pharmacological research has shown that honeysuckle has anti-inflammatory, antibacterial, antioxidant, and immune-regulating effects and is widely used in clinical practice. However, the effect of honeysuckle on ulcerative colitis (UC) is still not fully understood, which presents challenges for quality control, research and development. AIM OF THE STUDY: This study aimed to determine the anti-inflammatory properties and mechanism of action of aqueous extracts of honeysuckle in the treatment of ulcerative colitis. MATERIALS AND METHODS: The dextran sodium sulfate (DSS) induced-ulcerative colitis mouse model was established, and the mice were divided into five groups: the control group, the model group, and the low, medium, and high dose honeysuckle treatment groups. RESULTS: All dose groups of honeysuckle were found to significantly reduce IL-6 and TNF-α levels and regulate DSS-induced mRNA levels of CLDN4, COX-2, IL-6, INOS, MUC-2, occludin and NLRP3. The high-dose group displayed the most effective inhibition, and a differentially expressed mRNA detection indicated abnormal mRNA expression. The 16sRNA sequencing revealed that the honeysuckle was able to significantly upregulate the abundance of beneficial bacteria and downregulate the abundance of harmful bacteria. The study of short-chain fatty acids revealed that the levels of acetic, propionic, isobutyric, valeric and isovaleric acids were significantly increased after administering honeysuckle at medium and high doses. CONCLUSION: Honeysuckle reduces the production of pro-inflammatory cytokines, increases the content of short-chain fatty acids and restores the intestinal ecological balance, resulting in better therapeutic effects.

Umbilical cord MSC-derived exosomes improve alveolar macrophage function and reduce LPS-induced acute lung injury.

Acute lung injury (ALI) is a severe condition that can progress to acute respiratory distress syndrome (ARDS), with a high mortality rate. Currently, no specific and compelling drug treatment plan exists. Mesenchymal stem cells (MSCs) have shown promising results in preclinical and clinical studies as a potential treatment for ALI and other lung-related conditions due to their immunomodulatory properties and ability to regenerate various cell types. The present study focuses on analyzing the role of umbilical cord MSC (UC-MSC))-derived exosomes in reducing lipopolysaccharide-induced ALI and investigating the mechanism involved. The study demonstrates that UC-MSC-derived exosomes effectively improved the metabolic function of alveolar macrophages and promoted their shift to an anti-inflammatory phenotype, leading to a reduction in ALI. The findings also suggest that creating three-dimensional microspheres from the MSCs first can enhance the effectiveness of the exosomes. Further research is needed to fully understand the mechanism of action and optimize the therapeutic potential of MSCs and their secretome in ALI and other lung-related conditions.

Environmental levels of azoxystrobin disturb male zebrafish behavior: Possible roles of oxidative stress, cholinergic system, and dopaminergic system.

A widely applied pesticide of azoxystrobin, is increasingly detected in the water environment. Concern has been raised against its potential detriment to aquatic ecosystems. It has been shown that exposure to azoxystrobin interfere with the locomotor behavior of zebrafish larvae. This study aims to investigate whether exposure to environmental levels of azoxystrobin (2 µg/L, 20 µg/L, and 200 µg/L) changes the behavior of male adult zebrafish. Herein, we evaluated behavioral response (locomotor, anxiety-like, and exploratory behaviors), histopathology, biochemical indicators, and gene expression in male adult zebrafish upon azoxystrobin exposure. The study showed that exposure to azoxystrobin for 42 days remarkably increased the locomotor ability of male zebrafish, resulted in anxiety-like behavior, and inhibited exploratory behavior. After treatment with 200 µg/L azoxystrobin, vasodilatation, and congestion were observed in male zebrafish brains. Exposure to 200 µg/L azoxystrobin notably elevated ROS level, MDA concentration, CAT activity, and AChE activity, while inhibiting SOD activity, GPx activity, ACh concentration, and DA concentration in male zebrafish brains. Moreover, the expression levels of genes related to the antioxidant, cholinergic, and dopaminergic systems were significantly changed. This suggests that azoxystrobin may interfere with the homeostasis of neurotransmitters by causing oxidative stress in male zebrafish brains, thus affecting the behavioral response of male zebrafish.

The adsorption effects of biochar on carbofuran in water and the mixture toxicity of biochar-carbofuran in rats.

Carbofuran, a widely used carbamate insecticide, is frequently detected in water. In this study, a high-performance adsorbent (WAB4) for carbofuran was obtained from laboratory-synthesized biochars. The maximum adsorption of carbofuran by WAB4 reaches 113.7 mg/g approximately. The adsorption of carbofuran by biochar was a multi-molecular layer and the adsorption process conforms to the pseudo-second-order kinetic model (R2 = 0.9984) and Freundlich isotherm model (R2 = 0.99). Importantly, an in vivo rat model was used to assess the combined toxicological effects of biochar-carbofuran complexes. The toxicity of the complexes (LD50 > 12 mg/kg) is lower than that of carbofuran (LD50 = 7.9 mg/kg) alone. The damage of biochar-carbofuran complex on rat liver and lung is significantly less than that of carbofuran. The Cmax and bioavailability of carbofuran were found to be reduced by 64% and 68%, respectively, when biochar was present, by UPLC-MS/MS analysis of carbofuran in rat plasma. Furthermore, it was confirmed that the biochar-carbofuran complex is relatively stable in the gastrointestinal tract, by performing a carbofuran release assay in artificial gastrointestinal fluids in vitro. Collectively, biochar is a bio-friendly material for the removal of carbofuran from water.

StSN2 interacts with the brassinosteroid signaling suppressor StBIN2 to maintain tuber dormancy.

After harvest, potato tubers undergo an important period of dormancy, which significantly impacts potato quality and seed vigor. StSN2 has been reported as a key gene for maintaining tuber dormancy; in this study, we explored the molecular mechanism by which StSN2 maintains dormancy. StBIN2 was first identified as a candidate protein that interacts with StSN2 by co-immunoprecipitation/mass spectrometry, and both qPCR and enzyme activity experiments showed that StSN2 can promote the StBIN2 expression and activity. In addition, the interaction between StSN2 and StBIN2 was verified by yeast two-hybrid, luciferase complementation experiments and co-immunoprecipitation. Bioinformatics analysis and site-directed mutagenesis confirmed the critical role of cysteine residues of StBIN2 in its binding to StSN2. Similar to that of StSN2, overexpression of StBIN2 extended the dormancy of potato tuber. Interaction between StSN2 and StBIN2 increased the activity of the StBIN2 enzyme, inhibited the expression of StBZR1, and suppressed BR signaling. On the contrary, this interaction promoted the expression of StSnRK2.2/2.3/2.4/2.6 and StABI5, key genes of ABA signaling, and the phosphorylation of StSnRK2.3, thereby promoting ABA signaling. Altogether, our results indicate that StSN2 interacts with StBIN2 through key cysteine residues and StBIN2 maintains tuber dormancy by affecting ABA and BR signaling. Findings of this research offer new insights into the molecular mechanism by which StSN2 maintains potato tuber dormancy through interaction with StSIN2 and provide guidance for potato improvement.

Nonivamide inhibits proliferation of human corneal epithelial cells by inducing cell cycle arrest and oxidative stress.

Nonivamide, an agonist of transient receptor potential vanilloid type 1 (TRPV1), is widely used as a riot control agent, police incapacitant spray and pesticide. Although generally considered non-fatal, eye discomfort and even ocular injuries caused by such products are common. Little research has been conducted on the effects of nonivamide on corneal epithelial cells. Cell viability, impedance, flow cytometry, western blotting, and real-time fluorescence analyses were performed to investigate the effects of nonivamide on human corneal epithelial cells (HCE-T cells). We found that nonivamide impaired proliferation at subtoxic doses (100 µM and 200 µM) in HCE-T cells. Next, we described the mechanisms of action of nonivamide. Nonivamide caused cell cycle arrest by increasing p21 and decreasing cyclin D1. TRPV1 was activated by nonivamide, leading to an influx of Ca2+. Enhanced Ca2+ influx partially contributed to oxidative stress. Mitochondrial membrane potential (MMP) also decreased. All combined stress resulted in the inhibition of cell proliferation in HCE-T cells. In summary, nonivamide inhibited the proliferation of HCE-T cells at sub-toxic doses by inducing cell cycle arrest and oxidative stress. Our data demonstrate the corneal toxicity of nonivamide and explain the mechanisms underlying nonivamide-induced corneal injury.

StBIN2 Positively Regulates Potato Formation through Hormone and Sugar Signaling.

Potato is an important food crop worldwide. Brassinosteroids (BRs) are widely involved in plant growth and development, and BIN2 (brassinosteroid insensitive 2) is the negative regulator of their signal transduction. However, the function of BIN2 in the formation of potato tubers remains unclear. In this study, transgenic methods were used to regulate the expression level of StBIN2 in plants, and tuber related phenotypes were analyzed. The overexpression of StBIN2 significantly increased the number of potatoes formed per plant and the weight of potatoes in transgenic plants. In order to further explore the effect of StBIN2 on the formation of potato tubers, this study analyzed BRs, ABA hormone signal transduction, sucrose starch synthase activity, the expression levels of related genes, and interacting proteins. The results show that the overexpression of StBIN2 enhanced the downstream transmission of ABA signals. At the same time, the enzyme activity of the sugar transporter and the expression of synthetic genes were increased in potato plants overexpressing StBIN2, which also demonstrated the upregulation of sucrose and the expression of the starch synthesis gene. Apparently, StBIN2 affected the conversion and utilization of key substances such as glucose, sucrose, and starch in the process of potato formation so as to provide a material basis and energy preparation for forming potatoes. In addition, StBIN2 also promoted the expression of the tuber formation factors StSP6A and StS6K. Altogether, this investigation enriches the study on the mechanism through which StBIN2 regulates potato tuber formation and provides a theoretical basis for achieving a high and stable yield of potato.

Transcriptomic Profiling of Tetrodotoxin-Induced Neurotoxicity in Human Cerebral Organoids.

Tetrodotoxin (TTX) is an exceedingly toxic non-protein biotoxin that demonstrates remarkable selectivity and affinity for sodium channels on the excitation membrane of nerves. This property allows TTX to effectively obstruct nerve conduction, resulting in nerve paralysis and fatality. Although the mechanistic aspects of its toxicity are well understood, there is a dearth of literature addressing alterations in the neural microenvironment subsequent to TTX poisoning. In this research endeavor, we harnessed human pluripotent induced stem cells to generate cerebral organoids-an innovative model closely mirroring the structural and functional intricacies of the human brain. This model was employed to scrutinize the comprehensive transcriptomic shifts induced by TTX exposure, thereby delving into the neurotoxic properties of TTX and its potential underlying mechanisms. Our findings revealed 455 differentially expressed mRNAs (DEmRNAs), 212 differentially expressed lncRNAs (DElncRNAs), and 18 differentially expressed miRNAs (DEmiRNAs) in the TTX-exposed group when juxtaposed with the control cohort. Through meticulous Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction (PPI) analysis, we ascertained that these differential genes predominantly participate in the regulation of voltage-gated channels and synaptic homeostasis. A comprehensive ceRNA network analysis unveiled that DEmRNAs exert control over the expression of ion channels and neurocytokines, suggesting their potential role in mediating apoptosis.

Multiomics reveal human umbilical cord mesenchymal stem cells improving acute lung injury via the lung-gut axis.

BACKGROUND: Acute lung injury (ALI) and its final severe stage, acute respiratory distress syndrome, are associated with high morbidity and mortality rates in patients due to the lack of effective specific treatments. Gut microbiota homeostasis, including that in ALI, is important for human health. Evidence suggests that the gut microbiota improves lung injury through the lung-gut axis. Human umbilical cord mesenchymal cells (HUC-MSCs) have attractive prospects for ALI treatment. This study hypothesized that HUC-MSCs improve ALI via the lung-gut microflora. AIM: To explore the effects of HUC-MSCs on lipopolysaccharide (LPS)-induced ALI in mice and the involvement of the lung-gut axis in this process. METHODS: C57BL/6 mice were randomly divided into four groups (18 rats per group): Sham, sham + HUC-MSCs, LPS, and LPS + HUC-MSCs. ALI was induced in mice by intraperitoneal injections of LPS (10 mg/kg). After 6 h, mice were intervened with 0.5 mL phosphate buffered saline (PBS) containing 1 × 106 HUC-MSCs by intraperitoneal injections. For the negative control, 100 mL 0.9% NaCl and 0.5 mL PBS were used. Bronchoalveolar lavage fluid (BALF) was obtained from anesthetized mice, and their blood, lungs, ileum, and feces were obtained by an aseptic technique following CO2 euthanasia. Wright's staining, enzyme-linked immunosorbent assay, hematoxylin-eosin staining, Evans blue dye leakage assay, immunohistochemistry, fluorescence in situ hybridization, western blot, 16S rDNA sequencing, and non-targeted metabolomics were used to observe the effect of HUC-MSCs on ALI mice, and the involvement of the lung-gut axis in this process was explored. One-way analysis of variance with post-hoc Tukey's test, independent-sample Student's t-test, Wilcoxon rank-sum test, and Pearson correlation analysis were used for statistical analyses. RESULTS: HUC-MSCs were observed to improve pulmonary edema and lung and ileal injury, and decrease mononuclear cell and neutrophil counts, protein concentrations in BALF and inflammatory cytokine levels in the serum, lung, and ileum of ALI mice. Especially, HUC-MSCs decreased Evans blue concentration and Toll-like receptor 4, myeloid differentiation factor 88, p-nuclear factor kappa-B (NF-κB)/NF-κB, and p-inhibitor α of NF-κB (p-IκBα)/IκBα expression levels in the lung, and raised the pulmonary vascular endothelial-cadherin, zonula occludens-1 (ZO-1), and occludin levels and ileal ZO-1, claudin-1, and occludin expression levels. HUC-MSCs improved gut and BALF microbial homeostases. The number of pathogenic bacteria decreased in the BALF of ALI mice treated with HUC-MSCs. Concurrently, the abundances of Oscillospira and Coprococcus in the feces of HUS-MSC-treated ALI mice were significantly increased. In addition, Lactobacillus, Bacteroides, and unidentified_Rikenellaceae genera appeared in both feces and BALF. Moreover, this study performed metabolomic analysis on the lung tissue and identified five upregulated metabolites and 11 downregulated metabolites in the LPS + MSC group compared to the LPS group, which were related to the purine metabolism and the taste transduction signaling pathways. Therefore, an intrinsic link between lung metabolite levels and BALF flora homeostasis was established. CONCLUSION: This study suggests that HUM-MSCs attenuate ALI by redefining the gut and lung microbiota.

Mitochondrial dysfunction following repeated administration of alprazolam causes attenuation of hippocampus-dependent memory consolidation in mice.

The frequently repeated administration of alprazolam (Alp), a highly effective benzodiazepine sedative-hypnotic agent, in anxiety, insomnia, and other diseases is closely related to many negative adverse reactions that are mainly manifested as memory impairment. However, the exact molecular mechanisms underlying these events are poorly understood. Therefore, we conducted a proteomic analysis on the hippocampus in mice that received repeated administration of Alp for 24 days. A total of 439 significantly differentially expressed proteins (DEPs) were identified in mice with repeated administration of Alp compared to the control group, and the GO and KEGG analysis revealed the enrichment of terms related to mitochondrial function, cycle, mitophagy and cognition. In vitro experiments have shown that Alp may affect the cell cycle, reduce the mitochondrial membrane potential (MMP) to induce apoptosis in HT22 cells, and affect the progress of mitochondrial energy metabolism and morphology in the hippocampal neurons. Furthermore, in vivo behavioral experiments including IntelliCage System (ICS) and nover object recognition (NOR), hippocampal neuronal pathological changes with HE staining, and the expression levels of brain-deprived neuron factor (BDNF) with immunohistochemistry showed a significant decrease in memory consolidation in mice with repeated administration of Alp, which could be rescued by the co-administration of the mitochondrial protector NSI-189. To the best of our knowledge, this is the first study to identify a link between repeated administration of Alp and mitochondrial dysfunction and that mitochondrial impairment directly causes the attenuation of memory consolidation in mice.

Fecal microbiota transplantation from HUC-MSC-treated mice alleviates acute lung injury in mice through anti-inflammation and gut microbiota modulation.

Introduction: Acute lung injury (ALI) is a severe respiratory tract disorder facilitated by dysregulated inflammation, oxidative stress and intestinal ecosystem. Fecal microbiota transplantation (FMT) is a rapid method for gut microbiota (GM) reconstruction. Furthermore, our previous studies have confirmed that human umbilical cord mesenchymal stromal cells (HUC-MSCs) can alleviate ALI by improving GM composition. Therefore, we aimed to explore the efficacy and mechanism of FMT from HUC-MSCs-treated mice on ALI. Methods: In brief, fresh feces from HUC-MSCs-treated mice were collected for FMT, and the mice were randomly assigned into NC, FMT, LPS, ABX-LPS, and ABX-LPS-FMT groups (n = 12/group). Subsequently, the mice were administrated with antibiotic mixtures to deplete GM, and given lipopolysaccharide and FMT to induce ALI and rebuild GM. Next, the therapeutic effect was evaluated by bronchoalveolar lavage fluid (BALF) and histopathology. Immune cells in peripheral blood and apoptosis in lung tissues were measured. Furthermore, oxidative stress- and inflammation-related parameter levels were tested in BALF, serum, lung and ileal tissues. The expressions of apoptosis-associated, TLR4/NF-κB pathway-associated, Nrf2/HO-1 pathway related and tightly linked proteins in the lung and ileal tissues were assessed. Moreover, 16S rRNA was conducted to assess GM composition and distribution. Results: Our results revealed that FMT obviously improved the pathological damage of lung and ileum, recovered the immune system of peripheral blood, decreased the cell apoptosis of lung, and inhibited inflammation and oxidative stress in BALF, serum, lung and ileum tissues. Moreover, FMT also elevated ZO-1, claudin-1, and occludin protein expressions, activating the Nrf2/HO-1 pathway but hindering the TLR4/NF-κB pathway. Of note, the relative abundances of Bacteroides, Christensenella, Coprococcus, and Roseburia were decreased, while the relative abundances of Xenorhabdus, Sutterella, and Acinetobacter were increased in the ABX-LPS-FMT group. Conclusion: FMT from HUC-MSCs-treated mice may alleviate ALI by inhibiting inflammation and reconstructing GM, additionally, we also found that the TLR4/NF-κB and Nrf2/HO-1 pathways may involve in the improvement of FMT on ALI, which offers novel insights for the functions and mechanisms of FMT from HUC-MSCs-treated mice on ALI.

Dual functionalized hyaluronic acid micelles loading paclitaxel for the therapy of breast cancer.

Although many carriers for the delivery of chemotherapeutic drugs have been investigated, the disadvantages of passive targeting and uncontrolled drug release limit their utility. Herein, hyaluronic acid (HA) was hydrophobically modified to serve as a carrier for binding to cluster determinant 44 (CD44) overexpressed on tumor cell surfaces. Specifically, after deacetylation, HA was grafted to dodecylamine or tetradecylamine to afford amphiphilic zwitterionic polymer micelles, designated dHAD and dHAT, respectively, for the delivery of paclitaxel (PTX). The micelles were negatively charged at pH 7.4 and positively charged at pH 5.6, and this pH sensitivity facilitated PTX release under acidic conditions. The cell uptake efficiencies of the dHAD-PTX and dHAT-PTX micelles by MCF-7 cells after 4 h of incubation were 96.9% and 95.4%, respectively, and their affinities for CD44 were twice that of HA. Furthermore, the micelles markedly inhibited tumor growth both in vitro and in vivo, with IC50 values of 1.943 µg/mL for dHAD-PTX and 1.874 µg/mL for dHAT-PTX for MCF-7 cells; the tumor inhibition rate of dHAD-PTX (92.96%) was higher than that of dHAT-PTX (78.65%). Importantly, dHAD and dHAT micelles showed negligible systemic toxicity. Our findings suggest that these micelles are promising delivery vehicles for antitumor drugs.

Study of Huperzine A derivatives with extended protection against soman intoxication.

Pre-administration of huperzine A (Hup A) was validated to prevent poisoning from exposure to nerve agents (NAs) by reversibly inhibiting acetylcholinesterase (AChE). However, like the currently commonly used reversible inhibitors, Hup A has a short half-life and is unable to produce a long-term preventative effect. To extend the protective time of Hup A against NAs, 42 derivatives with a CN bond were designed based on the structure of Hup A in this study. All designed derivatives showed good binding capability with AChE via molecular docking. Six compounds (H3, H4, H11, H14, H16, and H25) with representative structures were selected for synthesis by Schiff base reaction, and their structures were stable. The modified Ellman's method showed the six compounds concentration-dependently inhibited AChE, and the half maximal inhibitory concentration (IC50) were higher than that of Hup A. Pretreatment of AChE with the derivatives significantly increased the IC50 of soman. In vivo experiments demonstrated H3, H4, H14, H16, and H25 had longer protective capacities against 1 × LD95 soman-induced death in mice than Hup A. The 12 h protective index showed that the protective ratios of H3, H4, H14 and H16 were 2.31, 1.85, 2.23 and 1.99 respectively, better than that of Hup A. The extended protection of the derivatives against soman may be explained by their transformation to Hup A in vivo. Furthermore, all six compounds showed lower acute oral toxicity than Hup A. Overall, our study provided an optional strategy to acquire pretreatment agents for NAs with extended action and low toxicity.

Synthesis, Bioactivity and Molecular Docking of Nereistoxin Derivatives Containing Phosphonate.

Novel nereistoxin derivatives containing phosphonate were synthesized and characterized via 31P, 1H and 13C NMR and HRMS. The anticholinesterase activity of the synthesized compounds was evaluated on human acetylcholinesterase (AChE) using the in vitro Ellman method. Most of the compounds exhibited good inhibition of acetylcholinesterase. All of these compounds were selected to assess their insecticidal activity (in vivo) against Mythimna separata Walker, Myzus persicae Sulzer and Rhopalosiphum padi. Most of the tested compounds displayed potent insecticidal activity against these three species. Compound 7f displayed good activity against all three insect species, showing LC50 values of 136.86 µg/mL for M. separata, 138.37 µg/mL for M. persicae and 131.64 µg/mL for R. padi. Compound 7b had the highest activity against M. persicae and R. padi, with LC50 values of 42.93 µg/mL and 58.19 µg/mL, respectively. Docking studies were performed to speculate the possible binding sites of the compounds and explain the reasons for the activity of the compounds. The results showed that the compounds had lower binding energies with AChE than with the acetylcholine receptor (AchR), suggesting that compounds are more easily bound with AChE.

Anaerobic purinolytic enzymes enable dietary purine clearance by engineered gut bacteria.

Uric acid, the end product of purine degradation, causes hyperuricemia and gout, afflicting hundreds of millions of people. The debilitating effects of gout are exacerbated by dietary purine intake, and thus a potential therapeutic strategy is to enhance purine degradation in the gut microbiome. Aerobic purine degradation involves oxidative dearomatization of uric acid catalyzed by the O2-dependent uricase. The enzymes involved in purine degradation in strictly anaerobic bacteria remain unknown. Here we report the identification and characterization of these enzymes, which include four hydrolases belonging to different enzyme families, and a prenyl-flavin mononucleotide-dependent decarboxylase. Introduction of the first two hydrolases to Escherichia coli Nissle 1917 enabled its anaerobic growth on xanthine as the sole nitrogen source. Oral supplementation of these engineered probiotics ameliorated hyperuricemia in a Drosophila melanogaster model, including the formation of renal uric acid stones and a shortened lifespan, providing a route toward the development of purinolytic probiotics.

A Stable Cell Line Co-expressing hTRPV1 and GCaMP6s: A Novel Cell-based Assay For High-throughput screening of hTRPV1 agonists.

BACKGROUND: Transient receptor potential vanilloid-1 (TRPV1) is a non-selective cation channel capable of integrating various noxious chemical and physical stimuli. Recently, human TRPV1 (hTRPV1) has attracted wide attention from researchers because it is closely related to pain, inflammation, temperature perception, and tumors. Our study was aimed at generating a stable cell line co-expressing hTRPV1 receptor and GCaMP6s calcium indicator protein and, based on this, developing high-throughput screening methods for targeting hTRPV1 agonists. METHODS: The CHO-hTRPV1-GCaMP6s cell line stably expressing hTRPV1 and GCaMP6s was generated by co-transfection of hTRPV1 and GCaMP6s into Chinese hamster ovary (CHO) cells. The high-throughput screening methods were developed based on detecting the concentration of intracellular calcium ions ([Ca2+]i) by using chemically synthesized dyes and genetically encoded calcium indicator (GECI). Meanwhile, the sensitivity and adaptability of these methods in the evaluation of capsaicinoids were also compared. RESULTS: A stable cell line co-expressing hTRPV1 and GCaMP6s was generated and used to establish a functional high-throughput screening assay based on the measurement of [Ca2+]i by fluorometric imaging plate reader (FLIPR). The GECI exhibited a higher sensitivity and applicability than that of chemically synthesized dyes in detecting the changes in [Ca2+]i induced by capsaicin. The CHO-hTRPV1-GCaMP6s cell line was further used to detect the dose-dependent relationships of various hTRPV1 agonists (comparison of EC50 values: capsaicin (39 ± 1.67 nM) < nonivamide (67 ± 3.05 nM) < piperine (9222 ± 1851 nM)), and this order is consistent with the pharmacological properties of hTRPV1 activation by these agonists. CONCLUSION: The successful establishment of the CHO-hTRPV1-GCaMP6s cell lines and their application in high-throughput screening of hTRPV1 agonists.