Dynamin-related protein 1 mediates the therapeutic effect of isoliquiritigenin in diabetic intimal hyperplasia via regulation of mitochondrial fission.

Phenotypic shift of vascular smooth muscle cells (VSMCs) plays a key role in intimal hyperplasia, especially in patients with diabetes mellitus (DM). This study aimed to investigate the role of dynamin-related protein 1 (DRP1) in mitochondrial fission-mediated VSMC phenotypic shift and to clarify whether DRP1 is the therapeutic target of isoliquiritigenin (ISL). Wire injury of carotid artery or platelet-derived growth factor treatment was performed in DM mice or high-glucose cultured human aortic smooth muscle cells (HASMCs), respectively. The effects of DRP1 silencing on DM-induced intimal hyperplasia were investigated both in vivo and in vitro. Phenotypic shift of HASMCs was evaluated by detection of reactive oxygen species (ROS) generation, cell viability, and related protein expressions. The effects of ISL on DM-induced intimal hyperplasia were evaluated both in vivo and in vitro. DRP1 silencing and ISL treatment attenuated DM-induced intimal hyperplasia with reduced ROS generation, cell viability, and VSMC dedifferentiation. The GTPase domain of DRP1 protein played a critical role in mitochondrial fission in DM-induced VSMC phenotypic shift. Cellular experiments showed that ISL inhibited mitochondrial fission and reduced the GTPase activity of DRP1, which was achieved by the directly binding to K216 of the DRP1 GTPase domain. ISL attenuated mouse intimal hyperplasia by reducing GTPase activity of DRP1 and inhibiting mitochondrial fission in vivo. In conclusion, increased GTPase activity of DRP1 aggregated DM-induced intimal hyperplasia by increasing mitochondrial fission-mediated VSMC phenotypic shift. ISL attenuated mouse intimal hyperplasia by reducing DRP1 GTPase activity and inhibiting mitochondrial fission of VSMCs.

Glucagon-like peptide-1 receptor agonist exendin 4 ameliorates diabetes-associated vascular calcification by regulating mitophagy through the AMPK signaling pathway.

BACKGROUND: Vascular calcification (VC) is a complication in diabetes mellitus (DM) patients. Osteogenic phenotype switching of vascular smooth muscle cells (VSMCs) plays a critical role in diabetes-related VC. Mitophagy can inhibit phenotype switching in VSMCs. This study aimed to investigate the role of the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin 4 (EX4) in mitophagy-induced phenotype switching. MATERIALS AND METHODS: The status of VC in T2DM mice was monitored using Von Kossa and Alizarin Red S (ARS) staining in mouse aortic tissue. Human aortic smooth muscle cells were cultured in high glucose (HG) and ß-glycerophosphate (ß-GP) conditioned medium. Accumulation of LC3B and p62 was detected in the mitochondrial fraction. The effect of EX4 in vitro and in vivo was investigated by knocking down AMPKα1. RESULTS: In diabetic VC mice, EX4 decreased the percentage of von Kossa/ARS positive area. EX4 inhibited osteogenic differentiation of HG/ß-GP-induced VSMCs. In HG/ß-GP-induced VSMCs, the number of mitophagosomes was increased, whereas the addition of EX4 restored mitochondrial function, increased the number of mitophagosome-lysosome fusions, and reduced p62 in mitochondrial frictions. EX4 increased the phosphorylation of AMPKα (Thr172) and ULK1 (Ser555) in HG/ß-GP-induced VSMCs. After knockdown of AMPKα1, ULK1 could not be activated by EX4. The accumulation of LC3B and p62 could not be reduced after AMPKα1 knockdown. Knockdown of AMPKα1 negated the therapeutic effects of EX4 on VC of diabetic mice. CONCLUSION: EX4 could promote mitophagy by activating the AMPK signaling pathway, attenuate insufficient mitophagy, and thus inhibit the osteogenic phenotype switching of VSMCs.

CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

Comprehensive analysis of prognostic value and immunotherapy prospect of brain cytoplasmic RNA1 in hepatocellular carcinoma.

BACKGROUND: The expression of brain cytoplasmic RNA1 (BCYRN1) is linked to the clinicopathology and prognosis of several types of cancers, among which hepatocellular carcinoma (HCC) is one of the most frequent types of cancer worldwide. AIM: To explore the prognostic value and immunotherapeutic potential of BCYRN1 in HCC by bioinformatics and meta-analysis. METHODS: Information was obtained from the Cancer Genome Atlas database. First, the correlation between BCYRN1 expression and prognosis and clinicopathologic characteristics of HCC patients was explored. Univariate and multivariate regression analyses were employed to examine the relationship between BCYRN1 and HCC prognosis. Secondly, potential functions and pathways were explored by means of enrichment analysis of differentially-expressed genes. The relationships between BCYRN1 expression and tumor microenvironment, immune cell infiltration, immune checkpoint, drug sensitivity and immunotherapy effect were also investigated. Finally, three major databases were searched and used to conduct a meta-analysis on the relationship between BCYRN1 expression and patient prognosis. RESULTS: BCYRN1 expression was significantly higher in HCC compared to normal tissues and was linked to a poor prognosis and clinicopathological characteristics. Enrichment analysis showed that BCYRN1 regulates the extracellular matrix and transmission of signaling molecules, participates in the metabolism of nutrients, such as proteins, and participates in tumor-related pathways. BCYRN1 expression was linked to the tumor microenvironment, immune cell infiltration, drug sensitivity and the efficacy of immunotherapy. Furthermore, the meta-analysis in this study showed that BCYRN1 overexpression was related to a worse outcome in HCC patients. CONCLUSION: Overexpression of BCYRN1 relates to poor prognosis and may be a potential prognostic factor and immunotherapeutic target in HCC.

M6A methylation-mediated elevation of SM22α inhibits the proliferation and migration of vascular smooth muscle cells and ameliorates intimal hyperplasia in type 2 diabetes mellitus.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by insulin resistance facilitates intimal hyperplasia of type 2 diabetes mellitus (T2DM) and N6-methyladenosine (m6A) methylation modification mediates the VSMC proliferation. This study aimed to reveal the m6A methylation modification regulatory mechanism. In this study, m6A demethylase FTO was elevated in insulin-treated VSMCs and T2DM mice with intimal injury. Functionally, FTO knockdown elevated m6A methylation level and further restrained VSMC proliferation and migration induced by insulin. Mechanistically, FTO knockdown elevated Smooth muscle 22 alpha (SM22α) expression and m6A-binding protein IGF2BP2 enhanced SM22α mRNA stability by recognizing and binding to m6A methylation modified mRNA. In vivo studies confirmed that the elevated m6A modification level of SM22α mRNA mitigated intimal hyperplasia in T2DM mice. Conclusively, m6A methylation-mediated elevation of SM22α restrained VSMC proliferation and migration and ameliorated intimal hyperplasia in T2DM.

Downregulation of ACSM3 promotes metastasis and predicts poor prognosis in hepatocellular carcinoma.

Understanding mechanisms of cancer metastasis is crucial for reduction of cancer mortality. Acyl-CoA medium-chain synthetase 3 (ACSM3) is an acyl-CoA synthetase which takes part in the first step of fatty acid metabolism. However, the expression, clinical significance and biological function of ACSM3 remain unknown in hepatocellular carcinoma (HCC). In this study, the expression and prognostic relevance of ACSM3 were investigated by tissue microarray and HCC clinical samples. Migration and invasion assays were carried out for functional analysis in vitro and a xenograft model was used to analyze the effects of ACSM3 on cancer metastasis in vivo. Furthermore, human phospho-kinase array assays were performed to explore molecular mechanisms of ACSM3 in HCC. The results showed ACSM3 was downregulated in HCC tissues. HCC patients with low expression of ACSM3 exhibited poor prognosis. Overexpression of ACSM3 attenuated migration and invasion of HCC cells in vitro and in vivo and downregulated the phosphorylation of WNK1 and AKT. Our findings indicate ACSM3 is a novel prognostic marker and a potential therapeutic target for HCC.

Nicotine up-regulated 4-1BBL expression by activating Mek-PI3K pathway augments the efficacy of bone marrow-derived dendritic cell vaccination.

PURPOSE: To explore the role of 4-1BBL in nicotine-treated immature dendritic cells (imDCs) mediated anti-tumor effects. METHODS: Bone marrow-derived imDCs were stimulated with nicotine and 4-1BBL expression was determinated by flow cytometry, Western blot and RT-PCR respectively. Then, the roles of 4-1BBL in nicotine-augmented DCs-dependent T cell proliferation, CTL priming and anti-tumor effects were investigated by BrdU cell proliferation assay, enzyme-linked immunospot assay and in vivo preventive effect on tumor development, respectively. Finally, using relative kinase inhibitors, the mechanism of 4-1BBL up-regulation by nicotine stimulation and the roles of Mek-PI3K signal pathways in nicotine-augmented DCs-dependent T cell proliferation were explored by Western blot and BrdU cell proliferation assay, respectively. RESULTS: Firstly, nicotine could up-regulate 4-1BBL expression in both protein and mRNA levels. Secondly, the effects of nicotine-augmented DCs-dependent T-cell proliferation, CTL priming and anti-tumor effects could be significantly abolished by blocking CD80, CD86 and 4-1BBL activity, respectively. Thirdly, the combined blockages of CD80/CD86, CD80/4-1BBL, CD86/4-1BBL or CD80/CD86/4-1BBL signals could decrease 53.2 %, 29.6 %, 27.9 % and 54.5 % nicotine-enhanced T cell proliferation, respectively. Importantly, nicotine-induced 4-1BBL up-regulation could be decreased by the usage of Mek-PI3K pathway kinase inhibitors. The pre-treatment of Mek-p38-PI3K kinase inhibitors could obviously abolish nicotine-augmented DCs-dependent T cell proliferation. CONCLUSIONS: CD80/CD86 and 4-1BBL are critical for nicotine augmented DCs-mediated anti-tumor effects. 4-1BBL and CD80/CD86 could be considered as potential candidates for preventive and therapeutic tumor vaccination.

Nicotine stimulated bone marrow-derived dendritic cells could augment HBV specific CTL priming by activating PI3K-Akt pathway.

Our previous studies have revealed that nicotine-treated immature dendritic cells (imDCs) have anti-tumor effects in murine lymphoma models. The present study is to explore HBV-specific CTL priming and its cytolytic activities of nicotine-treated murine DCs, the mechanism of α7 nicotinic acetylcholine receptor (nAChR) up-regulation by nicotine and the efficiency of nicotine with other cytokines. To address these hypotheses, bone marrow-derived imDCs were stimulated by nicotine and expression of α7 nAChR was firstly determined by flow cytometry and Western blot. Then, DCs-dependent HBV-specific T cell proliferation and IL-12 secretion were secondly determined by BrdU cell proliferation assay and ELISA, respectively. The HBV-specific CTL priming and its activities were further explored by intraperitoneal transfer of nicotine treated imDCs. The mechanism of nicotine up-regulating α7 nAChR was finally explored by Western blot. The results showed that: first, the maximal activation of PI3K and Akt was reached at 30 and 60-120 min respectively after nicotine stimulation. Nicotine up-regulated the expression of α7 nAChR by activating PI3K-Akt pathway in murine DCs; secondly, nicotine stimulation could enhance DCs' ability of HBV-specific T cell proliferation and IL-12 secretion; thirdly, adoptive transfer of nicotine stimulated DCs could induce HBV specific CTL priming in vivo and those CTL had cytolytic activities; fourthly, nicotine had equal efficiencies to 2 ng/ml IFN-γ in DCs-mediated T cell proliferation. All these data presented here indicated that nicotine treated imDCs might be considered as a potential candidate for HBV immunotherapy.

Lipopolysaccharide and dose of nicotine determine the effects of nicotine on murine bone marrow-derived dendritic cells.

The reported effects of nicotine on dendritic cells (DCs) are controversial. To investigate the factors which determine the effects of nicotine on DCs, immature dendritic cells (imDCs) induced from murine bone marrow were treated with different doses of nicotine with or without lipopolysaccharides (LPS). The morphology and expression of the co-stimulatory molecules CD80, CD86, CD40 and CD54 were observed and determined by microscopy and flow cytometry, respectively. The results showed that, firstly, nicotine treatment promoted the development of DC precursors into imDCs with a semi-mature phenotype revealed by a higher expression of CD11c and more branched projections. Secondly, lower doses of nicotine (16.5 ng/ml), but not higher (200 µg/ml), up-regulated the expression of the co-stimulatory molecules CD80, CD40 and CD54 on imDCs. Co-administration of LPS and nicotine revealed differential effects on co-stimulatory molecule expression on imDCs. Thirdly and importantly, treatment with lower doses of nicotine (16.5 ng/ml) did not augment expression of the CD80, CD86, CD40 and CD54 molecules in mature DCs. Fourthly and interestingly, high doses of nicotine (more than 165 µg/ml) revealed pro-apoptotic activity but lower doses of nicotine (16.5-0.165 ng/ml) achieved an anti-apoptotic effect on imDCs. All data presented here indicate that the controversial effects of nicotine on DCs may be due to the LPS of the nicotinic environment and the dose of nicotine used.

Paenibacillus sophorae sp. nov., a nitrogen-fixing species isolated from the rhizosphere of Sophora japonica.

A nitrogen-fixing bacterium, designated strain S27(T), was isolated from rhizosphere soil of Sophora japonica. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain S27(T) is a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain S27(T) and Paenibacillus durus DSM 1735(T) (97.3 %), Paenibacillus sabinae DSM 17841(T) (96.9 %), Paenibacillus forsythiae DSM 17842(T) (96.7 %) and Paenibacillus zanthoxyli DSM 18202(T) (96.6 %). However, DNA-DNA hybridization values between strain S27(T) and the four type strains were 37.64 %, 23.12 %, 25.6 % and 34.99 %, respectively. Levels of 16S rRNA gene sequence similarity between strain S27(T) and the type strains of other recognized members of the genus Paenibacillus were below 96.5 %. The DNA G+C content of strain S27(T) was 46.0 mol%. The major fatty acids were anteiso-C(15 : 0), C(16 : 0) and iso-C(16 : 0). The major isoprenoid quinone was MK-7. On the basis of its phenotypic characteristics and DNA-DNA hybridization results, strain S27(T) is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sophorae sp. nov. is proposed. The type strain is S27(T) ( = CGMCC 1.10238(T)  = DSM 23020(T)).

Paenibacillus jilunlii sp. nov., a nitrogen-fixing species isolated from the rhizosphere of Begonia semperflorens.

A nitrogen-fixing bacterium, designated strain Be17(T), was isolated from rhizosphere soil of Begonia semperflorens planted in Beijing Botanical Garden, PR China. Phylogenetic analyses based on a segment of the nifH gene sequence and a full-length 16S rRNA gene sequence revealed that strain Be17(T) was a member of the genus Paenibacillus. High levels of 16S rRNA gene sequence similarity were found between strain Be17(T) and Paenibacillus graminis RSA19(T) (97.9 %), Paenibacillus sonchi LMG 24727(T) (97.8 %), Paenibacillus riograndensis CECT 7330(T) (96.2 %) and Paenibacillus borealis DSM 13188(T) (96.1 %), respectively. Levels of 16S rRNA gene sequence similarity between strain Be17(T) and the type strains of other recognized members of the genus Paenibacillus were below 96.0 %. However, the DNA-DNA hybridization values between strain Be17(T) and P. graminis RSA19(T), P. sonchi LMG 24727(T) and P. riograndensis CECT 7330(T) were 47.9 %, 38.7 % and 37.5 %, respectively. The DNA G+C content of strain Be17(T) was 52.9 mol%. The major fatty acid component of strain Be17(T) was anteiso-branched C(15 : 0) (30.92 %). The major isoprenoid quinone was MK-7. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of its phenotypic characteristics, 16S rRNA gene sequences, DNA G+C content, DNA-DNA relatedness, chemotaxonomic properties and nifH gene sequence, strain Be17(T) represents a nitrogen-fixing strain of a novel species of the genus Paenibacillus, for which the name Paenibacillus jilunlii sp. nov. is proposed. The type strain is Be17(T) ( = CGMCC 1.10239(T) = DSM 23019(T)).

Metabolic engineering for ethylene production by inserting the ethylene-forming enzyme gene (efe) at the 16S rDNA sites of Pseudomonas putida KT2440.

The ethylene-forming enzyme gene (efe) from Pseudomonas syringae pv. glycinea was transferred into Pseudomonas putida KT2440 by recombination at five of the seven 16S rDNA sites. PCR analysis demonstrated that strains DC1, DC2 and DC3 contained three, four and five copies of efe, respectively. In contrast to the parent strain which produced ethylene at 14.7 mg h(-1) g(-1) dry weight, strains DC1, DC2 and DC3 produced ethylene at 36.2, 47.2 and 53.8 mg h(-1) g(-1) dry weight, respectively. Quantitative PCR showed that efe mRNA levels increased with increasing efe copy numbers. When additional copies of efe were introduced into strain DC3 via the broad-host-range plasmid pBBR1MCS2 an ethylene production rate of 80.2 mg h(-1) g(-1) dry weight was obtained and 489 mg ethylene was produced in 24h corresponding to a conversion rate of 21.7 mg ethylene g(-1) glucose. Our results indicated that P. putida KT2440 could be genetically modified to be a promising biomaterial producer.