Metformin regulates the effects of IR and IGF-1R methylation on mast cell activation and airway reactivity in diabetic rats with asthma through miR-152-3p/DNMT1 axis.

BACKGROUND/AIM: Metformin is a drug for treating type 2 diabetes mellitus (T2DM). Recently, metformin has been shown to reduce the risks of asthma-associated outcomes and asthma deterioration, thereby holding promise as a superior medicine for diabetic patients with asthma. However, the mechanism by which metformin reduces diabetic asthma is yet to be clarified. This study aimed at ascertaining the downstream molecules underlying the effect of metformin on the activation of mast cells (MCs) and airway reactivity in a concomitant diabetic and asthmatic rat model. METHODS: A T2DM model was induced utilizing a high-fat diet and streptozotocin. Then, 10% ovalbumin was utilized to stimulate asthma-like pathology in the T2DM rats. RBL-2H3 cells were induced by anti-dinitrophenyl-specific immunoglobulin E for constructing an in vitro model. Luciferase assay and RNA immunoprecipitation (IP) assay were conducted to identify the interaction between microRNA-152-3p (miR-152-3p) and DNA methyltransferase 1 (DNMT1), while chromatin IP to identify the binding of DNMT1 to insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) promoters. The effects of metformin on both pathological changes in vivo and biological behaviors of cells were evaluated. Using gain- and loss-of-function approaches, we assessed the role of the two interactions in the metformin-induced effect. RESULTS: It was suggested that metformin could impede the MC activation and airway resistance in the concomitant diabetic and asthmatic rats. Additionally, metformin downregulated IR and IGF-1R through DNMT1-dependent methylation to repress MC activation and airway resistance. DNMT1 was testified to be a target gene of miR-152-3p. Furthermore, miR-152-3p-induced silencing of DNMT1 was blocked by metformin, hence restraining MC activation and airway resistance. CONCLUSION: The findings cumulatively demonstrate that metformin downregulates IR/IGF-1R to block MC activation and airway resistance via impairing the binding affinity between miR-152-3p and DNMT1.

Levan from Leuconostoc citreum BD1707: production optimization and changes in molecular weight distribution during cultivation.

BACKGROUND: Levan is a well-known homopolymer of fructose composed predominantly of ß-(2, 6) fructofuranosyl linkages in the backbone with occasional ß-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated. METHODS: The cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The levan yield of BD1707 reached 34.86 g/L with a corresponding productivity of 7.47 g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26 °C and 200 rpm for 112 h in tomato juice supplemented with 172 g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320 × 107 Da at 6 h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809 × 106 Da after 120 h of cultivation. CONCLUSION: The levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected.

DM-induced Hypermethylation of IR and IGF1R attenuates mast cell activation and airway responsiveness in rats.

Diabetes has been reported to modulate the airway smooth muscle reactivity and lead to attenuation of allergic inflammatory response in the lungs. In this study, we aimed to study the effect of insulin on cell activation and airway responsiveness in patients with diabetes mellitus (DM). The airway contraction in rat model groups including a non-DM group, a non-DM+INDUCTION group, a DM+INDUCTION group and a DM+INDUCTION+INSULIN group was measured to observe the effect of insulin on airway responsiveness. Radioenzymatic assay was conducted to measure the levels of histamine, and ELISA assay was conducted to measure bronchial levels of interleukin (IL)-1b, tumour necrosis factor (TNF)-a, cytokine-induced neutrophil chemoattractant (CINC)-1, P-selectin and ß-hexosaminidase. The tension in the main and intrapulmonary bronchi of DM+INDUCTION rats was lower than that of the non-DM+INDUCTION rats, whereas the treatment of insulin partly restored the normal airway responsiveness to OA in DM rats. The release of histamine was remarkably suppressed in DM+INDUCTION rats but was recovered by the insulin treatment. Also, OA significantly increased the levels of IL-1b, TNF-a, CINC-1 and P-selectin in non-DM rats, whereas insulin treatment in DM+INDUCTION rats partly restored the normal levels of IL-1b, TNF-a, CINC-1 and P-selectin in DM rats. Moreover, the expression of IR and IGF1R was evidently suppressed in DM rats, with the methylation of both IR and IGF1R promoters was aggravated in DM rats. Therefore, we demonstrated that DM-induced hypermethylation inhibited mast cell activation and airway responsiveness, which could be reversed by insulin treatment.

Isolation and Characterization of a New Fusaricidin-Type Antibiotic Produced by Paenibacillus bovis sp. nov BD3526.

Paenibacillus bovis sp. nov BD3526, isolated from raw yak (Bos grunniens) milk, was able to produce antibacterial substances against Micrococcus luteus. The antibacterial substances produced by the strain BD3526 in 3% (w/v) wheat bran broth under aerobic conditions were precipitated from the cultivated broth with ammonium sulfate at 60% saturation. Two antibacterial compounds were obtained by Sephadex LH-20 chromatography and semi-preparative reverse-phase high-performance liquid chromatography (Semi-Pre RP-HPLC). The chemical structures of the two antibacterial compounds were further elucidated by means of ultra high-performance liquid chromatography-mass spectrometer/mass spectrometer (UHPLC-MS/MS). One compound, with a molecular mass of 883.56195 Da (M + H)+, was determined to be identical in chemical structure with that of the well-known compound fusaricidin A. The other antimicrobial compound with a molecular mass of 911.59393 Da (M + H)+ was determined to be a derivative of fusaricidin A by tandem mass spectrometry and amino acid composition analysis and was designed as bovisin. Bovisin possessed the stability against acid/alkali, heat and some proteases treatment, the same with the fusaricidin A. However, the minimal inhibition concentration (MIC) of bovisin on the tested indicator including Staphylococcus aureus, Micrococcus luteus, Listeria monocytogenes and Bacillus subtilis were 50, 50, 50, 50 µg/mL, respectively, slightly higher than those of fusaricidin A (6.25, 6.25, 6.25, 12.5 µg/mL), indicating bovisin with a weaker inhibitory activity.

Prevalence, awareness, treatment and control of hypertension in Henan Province, China.

OBJECTIVE: The present article aimed to explore and evaluate the epidemiology and determine the status of hypertension awareness, treatment and control in Henan province, China. DESIGN: Cross-sectional survey. SETTING: Thirty counties in Henan province of China. PARTICIPANTS: There were 18 772 randomly selected 15-74 years old. MAIN OUTCOME MEASURES: The distribution of blood pressure and prevalence, awareness, treatment and control of hypertension based on automated electronic monitor and questionnaire. RESULTS: The crude prevalence of hypertension was 24.9%, and the standardised rate was 26.6%, meaning about 25 million were hypertensive in Henan province. Of which, 46% were aware of their hypertension diagnosis, 35.7% were having antihypertensive medicine, only 10.4% had their blood pressure controlled less than 140/90 mmHg, and 29.1% were being controlled when they were having antihypertensive medicine. CONCLUSIONS: Hypertension is highly prevalent in Henan province, but the levels of hypertension awareness, treatment and control are low. Therefore, necessary actions including prevention, detection and treatment must be taken to keep the situation from worsening.

The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis.

Freezing stress can seriously affect plant growth and development, but the mechanisms of these effects and plant responses to freezing stress require further exploration. Here, we identified a NAM, ATAF1/2, and CUC2 (NAC)-family transcription factor (TF), NAC056, that can promote freezing tolerance in Arabidopsis. NAC056 mRNA levels are strongly induced by freezing stress in roots, and the nac056 mutant exhibits compromised freezing tolerance. NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor (CBF) pathway genes. Interestingly, we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants; therefore, NAC056-CBF1-NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress. In addition, 35S::NAC056 transgenic plants show enhanced freezing tolerance, which is partially reversed in the cbfs triple mutant. Thus, NAC056 confers freezing tolerance through the CBF pathway, mediating plant responses to balance growth and freezing stress tolerance.

Effects of Bifidobacterium BL21 and Lacticaseibacillus LRa05 on gut microbiota in type 2 diabetes mellitus mice.

Gut dysbiosis causes damage to the intestinal barrier and is associated with type 2 diabetes mellitus (T2DM). We tested the potential protective effects of probiotic BL21 and LRa05 on gut microbiota in type 2 diabetes mellitus mice and determined whether these effects were related to the modulation of gut microbiota.Thirty specific pathogen-free C57BL/6J mice were randomly allocated to three groups-the (CTL) control group, HFD/STZ model (T2DM) group, and HFD/STZ-probiotic intervention (PRO) group-and intragastrically administered strains BL21 and LRa05 for 11 weeks. The administration of strains BL21 and LRa05 significantly regulated blood glucose levels, accompanied by ameliorated oxidative stress in mice. The BL21/LRa05-treated mice were protected from liver, cecal, and colon damage. Microbiota analysis showed that the cecal and fecal microbiota of the mice presented significantly different spatial distributions from one another. Principal coordinate analysis results indicated that both T2DM and the BL21/LRa05 intervention had significant effects on the cecal contents and fecal microbiota structure. In terms of the fecal microbiota, an abundance of Akkermansia and Anaeroplasma was noted in the PRO group. In terms of the cecal content microbiota, enrichment of Akkermansia, Desulfovibrio, Bifidobacterium, Lactobacillus, and Limosilactobacillus was noted in the PRO group. The probiotics BL21 and LRa05 prevent or ameliorate T2DM by regulating the intestinal flora and reducing inflammation and oxidative stress. Our results suggest that BL21 and LRa05 colonize in the cecum. Thus, BL21/LRa05 combined with probiotics having a strong ability to colonize in the colon may achieve better therapeutic effects in T2DM. Our study illustrated the feasibility and benefits of the combined use of probiotics and implied the importance of intervening at multiple intestinal sites in T2DM mice.

Structure elucidation and immunological activity of a novel exopolysaccharide from Paenibacillus bovis sp. nov BD3526.

A novel exopolysaccharide named BD0.4 was purified from the fermentation broth of Paenibacillus bovis sp. nov BD3526 in wheat bran medium via anion exchange column chromatography. Its fine structure was identified by a variety of physical and chemical methods. BD0.4, with the weight average molecular weight of 376 kDa, consisted of glucuronic acid, glucose and fucose in a molar ratio of 1.58:1:1.66. The backbone included 1,3-linked Fuc, 1,3,4-linked Fuc, 1,3-linked Glc and 1,4-linked GlcA residues, with the branching point located at the O4 position of 1,3,4-linked Fuc residues, and the branched chain composed of terminal GlcA residues. BD0.4 could improve the phagocytic ability of macrophages and significantly stimulate the secretion of NO, TNF-α, IL-1ß and IL-6 from RAW264.7 cells in a dose-dependent manner. BD0.4 could promote the expression of NF-кB and cause nuclear translocation of NF-κB p65, indicating that BD0.4 probably exerted immune activity through the NF-κB signaling pathway.

Single-Cell Transcriptomics Reveals That Metabolites Produced by Paenibacillus bovis sp. nov. BD3526 Ameliorate Type 2 Diabetes in GK Rats by Downregulating the Inflammatory Response.

Chronic low-grade inflammation is widely involved in the development and progression of metabolic syndrome, which can lead to type 2 diabetes mellitus (T2DM). Dysregulation of proinflammatory and anti-inflammatory cytokines not only impairs insulin secretion by pancreatic ß-cells but also results in systemic complications in late diabetes. In our previous work, metabolites produced by Paenibacillus bovis sp. nov. BD3526, which were isolated from Tibetan yak milk, demonstrated antidiabetic effects in Goto-Kakizaki (GK) rats. In this work, we used single-cell RNA sequencing (scRNA-seq) to further explore the impact of BD3526 metabolites on the intestinal cell composition of GK rats. Oral administration of the metabolites significantly reduced the number of adipocytes in the colon tissue of GK rats. In addition, cluster analysis of immune cells confirmed that the metabolites reduced the expression of interleukin (IL)-1ß in macrophages in the colon and increased the numbers of dendritic cells (DCs) and regulatory T (Treg) cells. Further mechanistic studies of DCs confirmed that activation of the WNT/ß-catenin pathway in DCs promoted the expression of IL-10 and transforming growth factor (TGF)-ß, thereby increasing the number of Treg cells.

Structural elucidation and immunomodulatory activity of a neutral polysaccharide from the Kushui Rose (Rosa setate x Rosa rugosa) waste.

In this present study, the structure and immunomodulatory activity of a novel polysaccharide (WSRP-1b) from Kushui rose (Rosa setate x Rosa rugosa) waste were investigated. Structure characterization demonstrated that WSRP-1b had a weight-average molecular weight of 1.11 × 104 Da and consisted of glucose (42.6 %), mannose (21.4 %), arabinose (9.9 %), xylose (2.2 %), and galactose (23.9 %). Its backbone was composed of 1, 4-linked α-Glcp, 1, 4-linked ß-Glcp, and 1, 4-linked ß-Manp, with branches of 1, 4-linked α-Glcp and 1, 4-linked ß-Manp substituted at C-6 by 1, 6-linked ß-Galp. The branches mainly contained 1, 5-linked Araf, terminal arabinose and terminal glucose. Bioactivity assays showed that WSRP-1b had immunomodulatory activity by enhancing phagocytosis of macrophages, increasing production of ROS, NO, cytokines (IL-6, TNF-α), and activating NF-κB signaling pathway. These results suggested that it could be developed as a potential and safe immunomodulatory agent in fields of pharmacological or functional foods.

Preparation and characterization of arginine-modified chitosan/hydroxypropyl methylcellose antibacterial film.

Chitosan and its derivatives are widely used in medical, cosmetic and food fields. In this study, chitosan-N-arginine (CSA) was synthesized and characterized by Fourier-transform infrared (FT-IR), 1H NMR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). A novel antibacterial composite film consisting of CSA, hydroxypropyl methylcellose (HPMC) and glycerol was then prepared. The transparent and homogeneous film presented good compatibility between CSA and HPMC, confirmed by SEM. The thickness of the film was about 44.8 µm and its moisture content was 23.0%. Antimicrobial evaluation of CSA/HPMC film showed 9.0 mm bacteriostatic diameter zone against E. coli and 10.5 mm one against S. aureus. The film exhibited cell biocompatibility and promoted proliferation with L929 cell cytotoxicity test. Both antibacterial and cytotoxic results showed that the CSA/HPMC film was a promising material for medication, cosmetics and food preservation applications.

Ethylene insensitive3-like2 (OsEIL2) confers stress sensitivity by regulating OsBURP16, the ß subunit of polygalacturonase (PG1ß-like) subfamily gene in rice.

The transcription factors EIN3 (ETHYLENE-INSENSITIVE 3) and EILs (EIN3-Likes) play important roles in plant development and defense responses; however, their mechanism in these processes remain unclear. Here, we report that OsEIL2, an EIN3-like transcription factor from rice (Oryza sativa), plays important roles in abiotic stress and leaf senescence. OsEIL2 is a nuclear-localized protein with transactivation activity in the C-terminus (amino acids 344-583) and can be induced by NaCl, polyethylene glycol (PEG), dark, and abscisic acid (ABA) treatment. Transgenic plants of overexpressing OsEIL2 (OsEIL2-OX) show reduced tolerance to salt and drought stress compared with the controls. While the transgenic plants of overexpressing OsEIL2-RNA interference (OsEIL2-RNAi) exhibit enhanced tolerance to salt and drought stress compared with the controls. Moreover, seedlings of OsEIL2-overexpressing transgenic plants exhibit delayed leaf development and an accelerated dark-induced senescence phenotype, whereas OsEIL2-RNAi plants display the opposite phenotype. We further found that OsEIL2 functions upstream of OsBURP14 and OsBURP16. OsBURP14 and OsBURP16 are the members of the ß subunit of polygalacturonase subfamilies. OsBURP16 overexpression reduced pectin content and cell adhesion and increased abiotic stress sensitivity in rice. OsEIL2 binds directly to the promoter of OsBURP14 and OsBURP16 and activates their transcript levels. We also found that OsEIL2 overexpression decreased the pectin content by increasing polygalacturonase (PG) activity. Taken together, these results revealed a new mechanism of OsEIL2 in abiotic stress responses. These findings provide new insights into plant resistance to abiotic stress.

Fermentation Products of Paenibacillus bovis sp. nov. BD3526 Alleviates the Symptoms of Type 2 Diabetes Mellitus in GK Rats.

Gut microbiota is closely related to type 2 diabetes mellitus (T2DM). The gut microbiota of patients with T2DM is significantly different from that of healthy subjects in terms of bacterial composition and diversity. Here, we used the fermentation products of Paenibacillus bovis sp. nov. BD3526 to study the disease progression of T2DM in Goto-kakisaki (GK) rats. We found that the symptoms in GK rats fed the fermentation products of BD3526 were significantly improved. The 16S rRNA sequencing showed that the fermentation products of BD3526 had strong effects on the gut microbiota by increasing the content of Akkermansia. In addition, the interaction of the genus in the gut of the BD3526 group also significantly changed. Additional cytokine detection revealed that the fermentation products of BD3526 can reduce the inflammatory factors in the intestinal mucus of GK rats and thereby inhibit the inflammatory response and ameliorate the symptoms of T2DM.

Quaternized chitosan oligomers as novel elicitors inducing protection against B. cinerea in Arabidopsis.

Chitosan oligomers prepared from enzyme hydrolysis of chitosan have for many years been recognized as potent elicitors of plant innate immunity, but their efficacy is limited by the degree of polymerization and the degree of acetylation. In this study, we presented a new type of chitosan oligomers (COS), with the name of quaternized chitosan oligomers (QCOS) that were prepared by reacting COS with glycidyltrimethylammonium chloride and overcome these problems. First, QCOS clearly induced hydrogen peroxide accumulation and callose deposition in Arabidopsis seedlings. Second, we found that PAD3 expression was significantly upregulated more than 5-fold by QCOS as compared to COS. Further, PAD3 expression activated by QCOS was required for inducing Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, independent of salicylic acid signaling. These results demonstrate that quaternized modifications of COS possess better elicitor properties than the original COS and that QCOS stimulate plant protection against B. cinerea attack in Arabidopsis. Importantly, our work provides a novel and valuable strategy for enhancing elicitor activities of other types of oligosaccharides for plant innate immunity.

OsGA2ox5, a gibberellin metabolism enzyme, is involved in plant growth, the root gravity response and salt stress.

Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2ß-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C19-GA2oxs and a smaller class of C20-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C20-GA2oxs subfamily, a subfamily of GA2oxs acting on C20-GAs (GA12, GA53). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA3 at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.

Fabrication of biocompatible and mechanically reinforced graphene oxide-chitosan nanocomposite films.

BACKGROUND: Graphene oxide (GO)can be dispersed through functionalization, or chemically converted to make different graphene-based nanocomposites with excellent mechanical and thermal properties. Chitosan, a partially deacetylated derivative of chitin, is extensively used for food packaging, biosensors, water treatment, and drug delivery. GO can be evenly dispersed in chitosan matrix through the formation of amide linkages between them, which is different from previous reports focusing on preparing GO/chitosan nanocomposites through physical mixing. RESULTS: In this study, free-standing graphene oxide-chitosan (GO-chitosan) nanocomposite films have been prepared. The GO-chitosan films are biologically compatible and mechanically reinforced. Through the formation of amide linkages between GO's carboxylic acid groups and chitosan's amine groups, GO could be evenly dispersed within the chitosan matrix. We also characterized the GO-chitosan composite films using element analysis, Fourier transform infrared spectroscopy, X-ray photo electron spectroscopy, differential scanning calorimetry, and thermo gravimetric analysis. Compared to pristine chitosan film, the tensile strength of GO-chitosan film is improved by 2.5 folds and Young's modulus increases by nearly 4.6 folds. The glass transition temperature of GO-chitosan composite film shifts from 118°C to 158°C compared to the pristine chitosan, indicating its enhanced thermal stability. GO-chitosan composite film was also evaluated for its biocompatibility with C3H10T1/2 cells by in vitro fluorescent staining. The graphene oxide-reinforced chitosan composite films could have applications in functional biomaterials. CONCLUSION: The present study describes a useful and simple method to chemically attach biocompatible chitosan onto graphene oxide. We envision that the GO-chitosan film will open avenues for next-generation graphene applications in the realm of functional biomaterial.