HBx downregulated decorin and decorin-derived peptides inhibit the proliferation and tumorigenicity of hepatocellular carcinoma cells.

Hepatitis B virus (HBV) is one of the important risk factors in inducing the occurrence and development of liver cancer, while the mechanism has not been fully clarified. In this study, we found decorin (DCN) was significantly reduced in HBV transgenic cell line HepG2-4D14 compared to HepG2. The data from hepatocellular carcinoma (HCC) patients indicated that the level of DCN mRNA was significantly lower in tumor tissues than healthy control and positively correlated with the survival of HCC patients. We revealed that HBV HBx can inhibit the transcription of DCN by blocking p53 activity. Functional analysis demonstrated that overexpression of DCN substantially inhibits the proliferation of HCC cells, while knockdown of DCN enhances the proliferation of HCC cells. It is known that DCN could competitively bind to c-Met to inhibit HGF/c-Met signaling pathway to inhibit the development of HCC. Therefore, we screened the novel antitumor peptides derived from DCN based on the sequence of DCN and the complex structure of HGF/c-Met with virtual screening and identified a set of DCN-derived peptides (DCN-Ps) which may competitively bind to c-Met. We found that 5 of peptides can reduce the proliferation and migration of HepG2 cells significantly. Among them, DCN-P#3 can inhibit the growth of HCC cells both in vitro and in vivo. In conclusion, we discovered that HBV HBx downregulates the expression of DCN, which in turn promotes the proliferation of hepatocytes and the development of HCC. We identified DCN-derived antitumor peptides which provides the candidates for developing novel drugs against HCC.

The effect of AMF combined with biochar on plant growth and soil quality under saline-alkali stress: Insights from microbial community analysis.

Arbuscular mycorrhizal fungi (AMF) and biochar application individually can enhance plant tolerance to saline-alkali stress and promote plant growth efficiency. However, little is known about the potential synergistic effects of their combination on improving plant growth and soil quality under saline-alkali stress. This experiment adopted the potted method to explore the effects of four treatments on switchgrass growth and soil quality: biochar (BC), Rhizophagus irregularis (Ri), biochar + Ri (BR) and a control without biochar or Ri (CK). Compared to the CK treatment, the switchgrass biomass increased by 92.4 %, 148.6 %, and 177.3 % in the BC, Ri, and BR treatment groups, respectively. Similarly, the rhizosphere soil quality index increased by 29.33 %, 22.7 %, and 49.1 % in the respective treatment groups. The BR treatment significantly altered the rhizosphere soil microbial composition and diversity. Notably, compared to the other treatments, the archaeal α-diversity in the BR group showed a significant decrease. BR treatment significantly increased the relative abundance of bacteria, fungi and archaea at the genus level (e.g., Bacillus, Trichome and candidatus_methanopenens). Network analysis showed that the complexity and closeness of interactions between different microbial taxa were stronger in the BC, Ri and BR treatments than in the CK treatment, with BR being the more prominent. In summary, biochar combined with Ri has a better effect on promoting the growth of switchgrass under saline-alkali stress, improving the quality of saline-alkali soil, and increasing soil microbial diversity. This study provides a new approach for the efficient development and utilization of saline-alkali land.

HBx-upregulated MAFG-AS1 promotes cell proliferation and migration of hepatoma cells by enhancing MAFG expression and stabilizing nonmuscle myosin IIA.

To identify hepatitis B virus (HBV)-related lncRNA(s), we previously examined the transcription profiles of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells by RNA deep sequencing and identified 38 upregulated long noncoding RNAs (lncRNAs). In the present study, the lncRNA MAFG-AS1 is investigated in detail because its gene is located adjacent to the MAFG gene, which is an important transcription factor involved in cell proliferation. The level of MAFG-AS1 is significantly higher in HCC tissue than in nontumor tissues. TCGA data show that the expression level of MAFG-AS1 is negatively correlated with survival of HCC patients. GEO cohort data show that compared with healthy tissues, the expression level of MAFG-AS1 is significantly higher in HBV-infected liver tissues. Real-time PCR and luciferase reporter assay data show that HBx can enhance the transcription of MAFG-AS1. Gain-of-function and loss-of-function experiments indicate that MAFG-AS1 promotes proliferation, migration, and invasion of HCC cells. Tumor formation assay results demonstrate that knockdown of MAFG-AS1 significantly inhibits cell proliferation in nude mice. Furthermore, MAFG-AS1 enhances the transcription of adjacent MAFG via E2F1. Additionally, MAFG-AS1 interacts with three subunits (MYH9, MYL12B, and MYL6) of nonmuscle myosin IIA (NM IIA). Knockdown of MAFG-AS1 inhibits ATPase activity of MYH9, interaction of NM IIA subunits, and cell cycle progression. Thus, the lncRNA MAFG-AS1 is upregulated by HBV and promotes proliferation and migration of HCC cells. Our findings suggest that MAFG-AS1 is a potential oncogene that may contribute to HBV-related HCC development.

Analysis of m6A RNA Methylation-Related Genes in Liver Hepatocellular Carcinoma and Their Correlation with Survival.

N6-methyladenosine (m6A) modification on RNA plays an important role in tumorigenesis and metastasis, which could change gene expression and even function at multiple levels such as RNA splicing, stability, translocation, and translation. In this study, we aim to conduct a comprehensive analysis on m6A RNA methylation-related genes, including m6A RNA methylation regulators and m6A RNA methylation-modified genes, in liver hepatocellular carcinoma, and their relationship with survival and clinical features. Data, which consist of the expression of widely reported m6A RNA methylation-related genes in liver hepatocellular carcinoma from The Cancer Genome Atlas (TCGA), were analyzed by one-way ANOVA, Univariate Cox regression, a protein-protein interaction network, gene enrichment analysis, feature screening, a risk prognostic model, correlation analysis, and consensus clustering analysis. In total, 405 of the m6A RNA methylation-related genes were found based on one-way ANOVA. Among them, DNA topoisomerase 2-alpha (TOP2A), exodeoxyribonuclease 1 (EXO1), ser-ine/threonine-protein kinase Nek2 (NEK2), baculoviral IAP repeat-containing protein 5 (BIRC5), hyaluronan mediated motility receptor (HMMR), structural maintenance of chromosomes protein 4 (SMC4), bloom syndrome protein (BLM), ca-sein kinase I isoform epsilon (CSNK1E), cytoskeleton-associated protein 5 (CKAP5), and inner centromere protein (INCENP), which were m6A RNA methylation-modified genes, were recognized as the hub genes based on the protein-protein interaction analysis. The risk prognostic model showed that gender, AJCC stage, grade, T, and N were significantly different between the subgroup with the high and low risk groups. The AUC, the evaluation parameter of the prediction model which was built by RandomForest, was 0.7. Furthermore, two subgroups were divided by consensus clustering analysis, in which stage, grade, and T differed. We identified the important genes expressed significantly among two clusters, including uridine-cytidine kinase 2 (UCK2), filensin (BFSP1), tubulin-specific chaperone D (TBCD), histone-lysine N-methyltransferase PRDM16 (PRDM16), phosphorylase b ki-nase regulatory subunit alpha (PHKA2), serine/threonine-protein kinase BRSK2 (BRSK2), Arf-GAP with coiled-coil (ACAP3), general transcription factor 3C polypep-tide 2 (GTF3C2), and guanine nucleotide exchange factor MSS4 (RABIF). In our study, the m6A RNA methylation-related genes in liver hepatocellular carcinoma were analyzed systematically, including the expression, interaction, function, and prognostic values, which provided an important theoretical basis for m6A RNA methylation in liver cancer. The nine important m6A-related genes could be prognostic markers in the survival time of patients.

HBV HBx-Downregulated lncRNA LINC01010 Attenuates Cell Proliferation by Interacting with Vimentin.

Hepatitis B virus (HBV) infection is closely related to hepatocellular carcinoma (HCC) development. To investigate the mechanism of HBV causing HCC, we previously analyzed the transcription of the HBV-transgenic cell line HepG2-4D14 and parental HepG2 cells and identified a subset of long noncoding RNAs (lncRNAs) differentially expressed between them. In this study, we focus on lncRNA LINC01010, as it is significantly downregulated in HepG2-4D14 cells and in liver tissues of HCC patients, and positively correlated with survival. We found that HBV-encoded HBx can reduce the transcription of LINC01010. Functional analysis showed that the overexpression of LINC01010 inhibits proliferation, migration and invasion of HepG2 cells while the knockdown of LINC01010 promotes these processes. By taking the approach of RNA immunoprecipitation (RIP) and mass spectrometry, we identified that LINC01010 can interact with vimentin. Further studies demonstrated that LINC01010 negatively affects the vimentin network extension and causes more rapid subunit exchange and lower stability of vimentin filaments. In addition, LINC01010 can reduce the amount of insoluble vimentin within cells, which suggests that LINC01010 interfers with vimentin polymerization. These data indicate that LINC01010 can inhibit the assembly of vimentin filament. Thus, we revealed that HBV HBx-downregulated LINC01010, which suppresses cell proliferation and migration by negatively regulating the formation of vimentin filament. Taken together, LINC01010 is a potential tumor suppressor that may restrain HBV-related HCC development.

Changes in soil ammonia oxidizers and potential nitrification after clear-cutting of boreal forests in China.

The Korean pine and broad-leaved mixed forests are the most typical and complete ecosystem among the global boreal forests, with extremely important ecological functions. However, few studies on the changes of soil ammonia oxidizers and potential nitrification after clear-cutting of forests are reported. In this study, in contrast to primary Korean pine forests, nitrate (NO3-) was significantly higher in secondary broad-leaved forests, while ammonium (NH4+) was on the contrary. The abundance of ammonia-oxidizing bacteria (AOB) was greatly higher in secondary broad-leaved forests, while levels of ammonia-oxidizing archaea (AOA) were not significantly different between them. The significant differences of community structure of AOA and AOB were observed in different forest types and soil layers. Compared with AOA, community compositions of AOB was more sensitive to forest type. The dominant groups of AOA were Nitrososphaera and Nitrosotalea, and the dominant group of AOB was Nitrosospira, of which Nitrosospira cluster 2 and 4 were functional groups with highly activity. Soil potential nitrification rate (PNR) was higher in secondary broad-leaved forests. Furthermore, PNR and AOB abundance had a significant positive correlation, but no significant correlation with AOA abundance. These results provide insights into the soil nitrogen balance and effects on forest restoration after clear-cutting.

Proteolysis of methylated SOX2 protein is regulated by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.

SOX2 is a dose-dependent master stem cell protein that controls the self-renewal and pluripotency or multipotency of embryonic stem (ES) cells and many adult stem cells. We have previously found that SOX2 protein is monomethylated at lysine residues 42 and 117 by SET7 methyltransferase to promote SOX2 proteolysis, whereas LSD1 and PHF20L1 act on both methylated Lys-42 and Lys-117 to prevent SOX2 proteolysis. However, the mechanism by which the methylated SOX2 protein is degraded remains unclear. Here, we report that L3MBTL3, a protein with the malignant-brain-tumor (MBT) methylation-binding domain, is required for SOX2 proteolysis. Our studies showed that L3MBTL3 preferentially binds to the methylated Lys-42 in SOX2, although mutation of Lys-117 also partially reduces the interaction between SOX2 and L3MBTL3. The direct binding of L3MBTL3 to the methylated SOX2 protein leads to the recruitment of the CRL4DCAF5 ubiquitin E3 ligase to target SOX2 protein for ubiquitin-dependent proteolysis. Whereas loss of either LSD1 or PHF20L1 destabilizes SOX2 protein and impairs the self-renewal and pluripotency of mouse ES cells, knockdown of L3MBTL3 or DCAF5 is sufficient to restore the protein levels of SOX2 and rescue the defects of mouse ES cells caused by LSD1 or PHF20L1 deficiency. We also found that retinoic acid-induced differentiation of mouse ES cells is accompanied by the enhanced degradation of the methylated SOX2 protein at both Lys-42 and Lys-117. Our studies provide novel insights into the mechanism by which the methylation-dependent degradation of SOX2 protein is controlled by the L3MBTL3-CRL4DCAF5 ubiquitin ligase complex.

Neddylation of M1 negatively regulates the replication of influenza A virus.

Post-translational modification plays a critical role in viral replication. Previously we reported that neddylation of PB2 of influenza A virus (IAV) can inhibit viral replication. However, we found that NEDD8 overexpression can still inhibit the replication of PB2 K699R mutant viruses, implying that other viral protein(s) can be neddylated. In this study, we revealed that M1 of IAV can also be modified by NEDD8. We found that the E3 ligase HDM2 significantly promotes M1 neddylation. Furthermore, we identified M1 K187 as the major neddylation site. We generated an IAV M1 K187R mutant (WSN-M1 K187R) and compared the growth of wild-type and mutant viruses in Madin-Darby canine kidney (MDCK) cells. The data showed that the replication of WSN-M1 K187R was more efficient than that of wild-type WSN. More importantly, we observed that overexpression of NEDD8 inhibited the replication of the wild-type WSN more effectively than that of WSN-M1 K187R. In addition, we found that the neddylation-deficient M1 mutant (M1 K187R) had a longer half-life than that of wild-type M1, indicating that the neddylation of M1 reduces stability. Then we performed a viral infection assay and found that WSN-M1 K187R exhibited greater virulence in mice than wild-type WSN, suggesting that the neddylation of M1 reduced IAV replication in vivo. In conclusion, we uncovered that neddylation of M1 by HDM2 negatively regulates the stability of M1, which in turn inhibits viral replication.

Long Noncoding RNA Lnc-MxA Inhibits Beta Interferon Transcription by Forming RNA-DNA Triplexes at Its Promoter.

Previously, we identified a set of long noncoding RNAs (lncRNAs) that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the Sendai virus (SeV)- and IAV-induced activation of beta interferon (IFN-ß). A luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-ß reporter upon stimulation with RIG-I, MAVS, TBK1, or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. A chromatin immunoprecipitation (ChIP) assay showed that the enrichment of IRF3 and p65 at the IFN-ß promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-ß promoter. Chromatin isolation by RNA purification (ChIRP), triplex pulldown, and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-ß promoter. Furthermore, an electrophoretic mobility shift assay (EMSA) showed that lnc-MxA can form complexes with the IFN-ß promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-ß promoter to interfere with the activation of IFN-ß transcription. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that lnc-MxA can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene (ISG) that negatively regulates the transcription of IFN-ß by forming an RNA-DNA triplex.IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus, known as a "cytokine storm," can also cause widespread tissue damage (X. Z. J. Guo and P. G. Thomas, Semin Immunopathol 39:541-550, 2017, https://doi.org/10.1007/s00281-017-0636-y; S. Yokota, Nihon Rinsho 61:1953-1958, 2003; I. A. Clark, Immunol Cell Biol 85:271-273, 2007). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-ß transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative-feedback loop involved in maintaining immune homeostasis.

In vitro and in vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast.

BACKGROUND: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway. RESULTS: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologous ß-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11-8.51 folds and 3.0-9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg g-1 DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains. CONCLUSIONS: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

LSD1 demethylase and the methyl-binding protein PHF20L1 prevent SET7 methyltransferase-dependent proteolysis of the stem-cell protein SOX2.

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.

Phosphatase Cdc25A Negatively Regulates the Antiviral Immune Response by Inhibiting TBK1 Activity.

The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-ß) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-ß stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-ß. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-ß reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity.IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-ß transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-ß. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection.

Lnc-ISG20 Inhibits Influenza A Virus Replication by Enhancing ISG20 Expression.

Long noncoding RNAs (lncRNAs) are involved in many aspects of cellular processes, including the antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA deep sequencing to compare the profiles of lncRNAs in A549 and HEK293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that lnc-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduces ISG20 protein levels in A549 cells induced with poly(I·C) and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as endogenous RNA competing with ISG20 to enhance its translation. Indeed, we identified that microRNA 326 (miR-326) is a mutual microRNA for both ISG20 and lnc-ISG20 that targets the 3' untranslated region of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulated gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a competitive endogenous RNA to bind miR-326 to reduce its inhibition of ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication.IMPORTANCE The replication of influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is upregulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

Valproate ameliorates nitroglycerin-induced migraine in trigeminal nucleus caudalis in rats through inhibition of NF-кB.

BACKGROUND: As a complex nervous system disease, migraine causes severe healthy and social issues worldwide. Valproate (VPA) is a widely used treatment agent against seizures and bipolar disorder, and its function to alleviate damage due to migraine has also been verified in clinical investigations. However, the mechanism underlying the protective effect of VPA against migraine remains poorly revealed. In the current study, the major purpose was to uncover the mechanism which drove VPA to antagonize migraine. METHODS: Nitroglycerin (NTG) was employed to induce a migraine model in rats and the migraine animals were exposed to treatment of VPA of different doses. Thereafter, the levels of indicators related to oxidative stress were measured and used to evaluate the anti-oxidant potential of VPA. The expression of calcitonin gene-related peptide (CGRP) and c-Fos was also quantified with ELISA and immunohistochemistry, respectively. Western blotting and electrophoretic mobility shift assays (EMSA) were conducted to explore the effect of VPA treatment on NF-кB pathway. RESULTS: NTG induced the activation of oxidative stress and led to migraine in model animals, but pre-treatment with VPA attenuated the damage due to migraine attack in brain tissues. The level of lipid peroxidation was significantly reduced while the prodcution of anti-oxidant factors was restored. Furthermore, expressions of CGRP and c-Fos, which represented the neuronal activation, were also down-regulated by VPA. The results of western blotting and EMSA demonstrated that the above mentioned effect of VPA acted through the inhibition of NF-кB pathway. CONCLUSIONS: Although controversies on the effect of VPA on NF-кB pathway existed, our study revealed an alternative mechanism of VPA in protecting against migraine, which would promote the development of therapeutic strategies of migraine.

Transcriptional responses and flavor volatiles biosynthesis in methyl jasmonate-treated tea leaves.

BACKGROUND: Tea (Camellia sinensis) has long been consumed worldwide for its amazing flavor and aroma. Methyl jasmonate (MeJA), which acts as an effective elicitor among the plant kingdom, could mostly improve the quality of tea aroma by promoting flavor volatiles in tea leaves. Although a variety of volatile secondary metabolites that contribute to aroma quality have been identified, our understanding of the biosynthetic pathways of these compounds has remained largely incomplete. Therefore, information aboaut the transcriptome of tea leaves and, specifically, details of any changes in gene expression in response to MeJA, is required for a better understanding of the biological mechanisms of MeJA-mediated volatiles biosynthesis. Moreover, MeJA treatment could exaggerate the responses of secondary metabolites and some gene expression which offer a better chance to figure out the mechanism. RESULTS: The results of two-dimensional gas-chromatograph mass-spectrometry showed that the terpenoids content in MeJA-treated tea leaves increased, especially linalool, geraniol, and phenylethyl alcohol. More importantly, we carried out RNA-seq to identify the differentially expressed genes (DEGs) related to volatiles biosynthesis pathways induced by MeJA treatment (0 h, 12 h, 24 h and 48 h) in tea leaves. We identified 19245, 18614, 11890 DEGs respectively in the MeJA_12h, MeJA_24 h and MeJA_48 h samples. The α-Lenolenic acid degradation pathway was firstly responded resulting in activating the JA-pathway inner tea leaves, and the MEP/DOXP pathway significantly exaggerated. Notably, the expression level of jasmonate O-methyltransferase, which is associated with the central JA biosynthesis pathway, was increased by 7.52-fold in MeJA_24 h tea leaves. Moreover, the genes related to the terpenoid backbone biosynthesis pathway showed different expression patterns compared with the untreated leaves. The expression levels of 1-deoxy-D-xylulose-phosphate synthase (DXS), all-trans-nonaprenyl-diphosphate synthase, geranylgeranyl reductase, geranylgeranyl diphosphate synthase (type II), hydroxymethylglutaryl-CoA reductase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase increased by approximately 2-4-fold. CONCLUSIONS: The results of two-dimension gas-chromatography mass-spectrometry analysis suggested that exogenous application of MeJA could induce the levels of volatile components in tea leaves, especially the geraniol, linalool and its oxides. Moreover, the transcriptome analysis showed increased expression of genes in α-Lenolenic acid degradation pathway which produced massive jasmonic acid and quickly activated holistic JA-pathway inner tea leaves, also the terpenoid backbones biosynthesis pathway was significantly affected after MeJA treatment. In general, MeJA could greatly activate secondary metabolism pathways, especially volatiles. The results will deeply increase our understanding of the volatile metabolites biosynthesis pathways of tea leaves in response to MeJA.

Applications of pain-related evoked potentials and short-latency somatosensory evoked potentials in acupuncture research: a narrative review.

OBJECTIVE: To review and discuss the Chinese and English literature on the use of pain-related evoked potentials (PREP) and short-latency somatosensory EP (SLSEP) in acupuncture research. METHODS: China National Knowledge Infrastructure Database and MEDLINE were searched for the following key words: acupuncture and PREP or SLSEP. RESULTS: Thirty-seven articles were included in the review. Researchers usually use PREPs to study the analgesic effect of acupuncture, observe influential factors, or for mechanistic exploration. In the SLSEP studies, researchers focused on response characteristics of acupuncture, acupoint specificity, and influential factors of the treatment. There were some problems with the study design and conclusions. CONCLUSION: Researchers could use PREP and SLSEP to objectively validate the effects of acupuncture and explore its mechanisms using nerve electrophysiology. Further studies can benefit from observing more acupoints' effects using PREPs or SLSEPs and investigating the placebo effect of acupuncture.

Apoptosis, necrosis, and autophagy in mouse intestinal damage after 15-Gy whole body irradiation.

Enterocytes die during high-dose radiation exposure in radiation accidents. The modality of cell death has a profound effect on the therapeutic response. The ilea from mice with 15 Gy total body irradiation (TBI) were drawn, morphological features observed by hematoxylin and eosin staining and transmission electron micrographs. The biochemical features of mouse ileum presented with the structure were cleaved Caspase-3 (apoptosis marker), Light Chain 3 (LC3)-I's conversion to LC3-II (autophagy marker) and high mobility group box chromosomal protein 1's secretion (necrosis marker). Then, the autophagy inhibitor (3-methyladenine), caspase inhibitor (Z-VAD-FMK) or necrosis inhibitor (necrostatin) was used to prevent death. Apoptosis, autophagy and necrosis were all appeared in the ileum, but necrosis had the biggest size; the use of 3-methyladenine and Z-VAD-FMK prolong one day's life of the mice after 15 Gy TBI, necrostatin significantly extended the lifespan of 15 Gy irradiated mice (p < 0.05). The results suggest that the death of enterocytes could not be classified into one type of cell death but rather as 'mixed death.'

Lnc-RPS6P3 Inhibits Influenza A Virus Replication and Attenuates the Inhibitory Effect of NS1 on Innate Immune Response.

Host factors play important roles in influenza A virus (IAV) replication. In order to identify novel host factors involved in IAV replication, we compared the differentially expressed genes in A549 cells after IAV infection. We found that lncRNA lnc-RPS6P3 was up-regulated upon viral infection and poly(I:C) and IFN-ß treatment, indicating it was an interferon-stimulated gene. Functional analysis demonstrated that overexpression of lnc-RPS6P3 inhibited IAV replication while knockdown of lnc-RPS6P3 promoted viral infection in A549 cells. Lnc-RPS6P3 inhibited both transcription and replication of IAV. Further study showed that lnc-RPS6P3 interacted with viral NP and interfered with NP self-oligomerization and, consequently, inhibited vRNP activity. In addition, lnc-RPS6P3 interacted with viral NS1 and reduced the interaction of NS1 and RIG-I; it also attenuated the inhibitory effect of NS1 on IFN-ß stimulation. In conclusion, we revealed that lnc-RPS6P3 is an interferon-stimulated gene that inhibits IAV replication and attenuates the inhibitory effect of NS1 on innate immune response.

Effective reduction on flame soot via plasma coupled with carbon dioxide.

This study explored the impact of non-thermal plasma and CO2 on the flame soot characteristics within the diffusion flames. We analyzed on flame structures that were diluted with either CO2 or N2, temperature distributions, and soot characteristics, both in the presence and absence of plasma. Due to the higher specific heat capacity of CO2 compared to N2, the optical observations consistently showed lower temperatures in flames diluted with CO2 as compared to those diluted with N2. The inclusion of plasma and carbon dioxide resulted in the lowest soot concentration, indicating that plasma coupled with CO2 has a synergistic inhibitory effect on soot emissions. The findings revealed that when CO2 was used to dilute the flames and the oxygen concentration was low, the soot nanostructure appeared amorphous. Raman results showed that the level of graphitization observed in soot particles from CO2 dilution flames was lower than that from N2 dilution flames. In the presence of plasma and CO2, the soot obtained exhibited the shortest fringe length and the highest fringe tortuosity. Significant correlations were observed between the nanostructure of soot and its reactivity. The combined application of plasma and CO2 proved to be effective in reducing the soot carbonization degree.

Calcium transferring from ER to mitochondria via miR-129/ITPR2 axis controls cellular senescence in vitro and in vivo.

Senescent cells are known to be accumulated in aged organisms. Although the two main characteristics, cell cycle arrest (for dividing cells) and secretion of senescence-associated secretory phenotype (SASP) factors, have been well described, the lack of sufficient senescent markers and incomplete understanding of mechanisms have limited the progress of the anti-senescence field. Calcium transferred from the endoplasmic reticulum (ER) via inositol 1, 4, 5-trisphosphate receptor type 2 (ITPR2) to mitochondria has emerged as a key player during cellular senescence and aging. However, the internal regulatory mechanisms, particularly those of endogenous molecules, remain only partially understood. Here we identified miRNA-129 (miR-129) as a direct repressor of ITPR2. Interestingly, miR-129 controlled a cascade of intracellular calcium signaling, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and consequently cellular senescence through ITPR2 and mitochondrial calcium uniporter (MCU). In addition, miR-129 was repressed in different senescence models and delayed bleomycin-induced cellular senescence. Importantly, intraperitoneal injection of miR-129 partly postponed bleomycin-accelerated lung aging and natural aging markers as well as reduced immunosenescence markers in mice. Altogether, these findings demonstrated that miR-129 regulated cellular senescence and aging markers via intracellular calcium signaling by directly targeting ITPR2.