Characterization of a novel Toll-like receptor 13 homologue from a marine fish Nibea albiflora, revealing its immunologic function as PRRs.

Congenital immunity mediated by Toll-like receptor (TLR) family is the first line of defense for disease-resistant immunity of fish and plays a vital role as a bridge between innate immunity and acquired immunity. As a less known member of the TLR family TLR13 can participate in the immune and inflammatory reactions of the body for recognizing the conserved sequence of 23S rRNA in bacteria and induce immune response. In this study, the full-length cDNA of TLR13 from Nibea albiflora (named as NaTLR13) was cloned and was functionally characterized. It was 4210bp (GenBank accession no. MT701899) including an open reading frame (ORF) of 2886bp to encode 962 amino acids with molecular weight of 110.37 kDa and the theoretical isoelectric point of 9.08. There were several conservative structures in NaTLR13 such as 15 leucine-rich repeat sequences (LRRs), a Toll-IL-1 receptor domain (TIR), an LRR-CT terminal domain, two LRR-TYP structures and two transmembrane domains. The multiple sequence alignment and phylogenetic analysis manifested that NaTLR13 had high similarity with Larimichthys crocea and Collichthys lucidus (88.79% and 87.02%, respectively) and they fell into the same branch. The Real-time PCR showed that NaTLR13 was expressed in all selected tissues, with the highest in the spleen, followed by the liver, kidney, gill, heart and muscle. After being challenged by Vibrio alginolyticus, Vibrio parahaemolyticus or Poly (I:C), the expression of NaTLR13 increased firstly, then decreased and finally stabilized with time for its immune defense function. Subcellular localization analysis revealed that NaTLR13 was unevenly distributed in the cytoplasm with green fluorescence and MyD88 was evenly spread in the cytoplasm with red signals. When NaTLR13 and MyD88 were co-transfected, they obviously overlapped and displayed orange-yellow color, which showed that the homologous TLR13 might interact with MyD88 for NFκB signaling pathway transmission. The functional domains of NaTLR13 (named NaTLR13-TIR and NaTLR13-LRR) were expressed in E.coli BL21 (DE3) and purified by Ni-NAT Superflow Resin conforming to the expected molecular weights, and the recombinant proteins could bind to three Vibrios (V.alginolyticus, V.parahaemolyticus and Vibrio harveyi), indicating that NaTLR13 could be bounden to bacteria through its functional domain. These results suggested that NaTLR13 might play an important role in the defense of N.albiflora against bacteria or viral infection and the data would provide some information for further understanding the regulatory mechanism of the innate immune system in fish.

Efficient Removal of Methylene Blue by Bio-Based Sodium Alginate/Lignin Composite Hydrogel Beads.

Dye pollution is a serious issue in current environment protection, and bio-based adsorbents have been receiving much attention in wastewater treatment, due to their low cost, renewable, and environmentally friendly characteristics. Bio-based sodium alginate/lignin composite (SA/Lig) hydrogel beads were fabricated by a facile cross-linking with calcium ion and used for the removal of methylene blue (MB). The obtained SA/Lig microbeads were characterized with SEM, FTIR, and TG, and the effect of lignin content, pH, and temperature on the MB adsorption was investigated. The results indicated that the introduction of aromatic lignin can not only enhance thermal stability but also can improve the adsorption performance. Under optimal conditions, the maximum adsorption capacity (254.3 mg/g) was obtained for the SA/Lig-20% beads, with a removal efficiency of 84.8%. The adsorption process for MB is endothermic, and the rate-limiting step is chemical adsorption. The removal efficiency is higher than 90% after five cycles, revealing that the prepared beads show good regeneration ability.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Nibea albiflora.

Myeloid differentiation factor 88 (MyD88), composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-IR homology domain, is a key connector protein in the TLR signal transduction pathway. In this study a novel isoform of MyD88 in Nibea albiflora (named as NaMyD88) was identified and functionally characterized (GenBank accession no. MN384261.1). Its complete cDNA sequence was 1672 bp and contained an open reading frame of 879 bp encoding 292 amino acid residues, which was similar to its teleost fish counterparts in the length. The theoretical molecular mass was 33.63 kDa and the isoelectric point was 5.24. BLASTp analysis suggested that the deduced amino acids sequence of NaMyD88 shared high identity to the known MyD88, for instance, 94.77% identity with Collichthys lucidus. Sequence analysis showed that NaMyD88 protein was consistent with MyD88 protein of other species at three conserved domains, N-terminal DD, short middle domain and C-terminal TIR, and the TIR domain contained three highly conserved motifs: Box1, Box2, and Box3. NaMyD88 and red fluorescent protein (Dsred) were fused and expressed in the cytoplasm of the epithelioma papulosum cyprini (EPC cells). The NaTLR9-TIR-EGFP fusion protein, which was obtained in our previous studies, showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection, NaMyD88-Dsred and NaTLR9-TIR-EGFP obviously overlapped and displayed orange-yellow color. The results showed that the homologous MyD88-Dsred could interact with NaTLR9-TIR-EGFP. Based on this result pcMV-NaMyD88-TIR-Myc plasmids and the pcDNA3.1-NaTLR9-TIR-flag were constructed and co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, the protein eluted by Flag-beads could be detected by anti-Flag-tag antibody and anti-Myc tag antibody respectively, while the protein without NaTLR9-TIR could not be found, which further proved that TLR and MyD88 could interact each other. The prokaryotic plasmid of MyD88-TIR domain was constructed, expressed in BL21 (DE3) and purified by Ni-NAT super flow resin conforming to the expected molecular weight of 27 kDa with the corresponding active sites for its conferring protein-protein interaction functions. Real-time fluorescence quantitative PCR showed that NaMyD88 could be expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the kidney, and it was up-regulated after being infected with Polyinosinic:polycytidylic acid - Poly (I:C) and Pseudomonas plecoglossicida, which showed that NaMyD88 was involved in the immune response of N.albiflora. These data afforded a basis for understanding the role of NaMyD88 in the TLR signaling pathway of N.albiflora.

Molecular insights of a novel fish Toll-like receptor 9 homologue in Nibea albiflora to reveal its function as PRRs.

Toll-like receptors (TLRs) are an important class of molecules involved in non-specific immunity, and they are also the bridge connecting between non-specific immunity and specific immunity. As a vital member of TLR family TLR9 can be activated by bacterial DNA and induce the production of inflammatory cytokines. In this study, a full length of TLR9 homologue of 3677 bp in Nibea albiflora (named as NaTLR9, GenBank accession no: MN125017.1) was characterized, and its ORF was 3180 bp encoding 1059 amino acid residues with a calculated molecular weight of 121.334 kDa (pI = 6.29). Several leucine-rich repeated sequences (LRR domain) and conservative TIR domain were found in NaTLR9, which was mainly expressed in dendritic cells and macrophages. The phylogenetic and synteny analysis further revealed high sequence identity of NaTLR9 with its counterparts of other teleost, confirming their correct nomenclature and conservative during evolution as an important pattern recognition receptor. The NaTLR9-TIR-pEGFP-N1 fusion protein showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection of NaTLR9-TIR-pEGFP-N1 and NaMyD88-pDsRED-Monomer-N1, green fluorescence obviously overlapped with red and changed into yellowish-green, which suggested that there might be the interaction between homologous NaTLR9-TIR and MyD88. Based on this result the pCDNA3.1-NaTLR9-TIR-flag and pcMV-NaMyD88-TIR-Myc plasmids were co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, TLR9 and MyD88 protein could interact with each other. Furthermore, NaTLR9 was ubiquitously expressed in all the investigated tissues, most abundantly in head kidney, followed by stomach, spleen, liver and gill, but lower in muscle. The vitro immune stimulation experiments revealed that Pseudomonas plecoglossicida and polyinosinic-polycytidylic acid [Poly (I:C)] induced higher levels of NaTLR9 mRNA expression with the peaks of 9.52 times at 2 h and 39.91 times at 24 h compared with the control group respectively. The functional domains (LRRs and TIR, named NaTLR9-TIR and NaTLR9-LRR respectively) of NaTLR9 were expressed and purified, the recombinant proteins both could bind three kinds of typical aquatic pathogenic bacteria (Vibrio. parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi), which showed that NaTLR9 could couple to bacteria by its function domains. The aforementioned results indicated that NaTLR9 played a significant role in the defense against pathogenic bacteria infection in innate immune response of sciaenidae fish, which may provide some further understandings of the regulatory mechanisms in the teleostean innate immune system.

Effects of traditional Chinese exercises on blood glucose and hemoglobin A1c levels in patients with prediabetes: A systematic review and meta-analysis.

BACKGROUND: Traditional Chinese exercises (TCEs) have a positive effect on glycemic control and hemoglobin A1c (HbA1c), but there is no consensus on the benefits of TCEs for patients with prediabetes. OBJECTIVE: The objective of this study was to systematically investigate the effects of TCEs on blood glucose control in patients with prediabetes. SEARCH STRATEGY: Comprehensive retrieval of randomized controlled trials (RCTs) was carried out using PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure, VIP Database for Chinese Technical Periodicals, Wanfang Data Knowledge Service Platform, China Biology Medicine disc, Google Scholar and Baidu academic databases. The retrieval window ranged from the establishment of the database to December 2018, and references related to the included trials were searched without language restrictions. INCLUSION CRITERIA: The study included RCTs with a clinical diagnosis of prediabetes that was also treated with TCEs. DATA EXTRACTION AND ANALYSIS: Literature screening, data extraction and literature quality assessment were performed independently by two researchers. In the case of disagreement, a third party was invited to negotiate and make a decision. Standardized mean difference (SMD) was used to estimate the therapeutic effect. Meta-analysis was performed using Review Manager 5.3.5 and Stata 15.0. Heterogeneity was assessed using Q test and I2, and the source of heterogeneity was determined using Galbraith diagram and sensitivity analysis. A Q test resulting in P < 0.1 and I2 > 50% indicated significant difference and random effect model analysis was performed. Otherwise, a fixed effect model was applied. Begg's and Egger's tests were used to assess publication bias. RESULTS: Nine RCTs involving 485 participants were included in this study. The results showed that TCEs could reduce fasting blood glucose (FBG), 2 h blood glucose (2hPBG) and HbA1c in patients with prediabetes. The treatment subgroup showed that an intervention of 6 months had better results, while the Gongfa subgroup showed that the TCE Baduanjin yielded better results. (1) FBG: SMD = -0.73, 95% confidence interval (CI) [-0.97, -0.50], P < 0.00001; Baduanjin: SMD = -0.83, 95% CI [-1.13, -0.53], P < 0.00001; 6 month treatment: SMD = -0.73, 95% CI [-1.20, -0.26], P = 0.002. (2) 2hPBG: SMD = -0.75, 95% CI [-0.94, -0.57], P < 0.00001; Baduanjin: SMD = -0.62, 95% CI [-0.91, -0.32], P < 0.00001; 6 month treatment: SMD = -0.91, 95% CI [-1.39, -0.44], P = 0.0002. (3) HbA1c: SMD = -0.56, 95% CI [-0.89, -0.23], P = 0.00008; Baduanjin: SMD = -0.46, 95% CI [-0.83, -0.08], P = 0.02; 6 month treatment: SMD = -0.77, 95% CI [-1.24, -0.29], P = 0.002. CONCLUSION: TCEs had positive effects in improving blood glucose levels in patients with prediabetes. Hence, TCEs may be of potential therapeutic value for patients with prediabetes, as an adjuvant therapy along with other treatments. Although the evidence suggests that the intervention is effective for 6 months, the mechanism of TCEs on glycemic control, the minimum exercise dose and their safety remain to be further studied.

Correlation between microstructure and properties of graphene oxide/waterborne polyurethane composites investigated by positron annihilation spectroscopy.

A quantitative description of microstructure is highly desirable for the precise construction of high performance graphene based polymer composites. In this paper, the effects of doping graphene oxide (GO) on the microstructure, thermal and mechanical properties of the obtained graphene oxide/waterborne polyurethane (GO/WPU) composites were systematically investigated. In order to give a deep insight into the microstructure of GO/WPU composites, especially for the relative free volume fraction (f r) and interfacial interaction intensity (ß), positron annihilation lifetime spectroscopy (PALS) was employed for its quantitative characterization. With the increase of GO content, the f r decreased first and then increased, the lowest value was observed for the composites containing 0.5 wt% GO. This can be ascribed to the change in the dispersed state of GO and interfacial interactions, which agree well with the results of SEM. The correlation between microstructure and properties was established with the PALS results, the values of f r and ß give a good explanation of the variation in glass transition temperature and tensile strength, respectively.

Cyclosporin A Protected Cardiomyocytes Against Oxidative Stress Injury by Inhibition of NF-κB Signaling Pathway.

PURPOSE: This study aims to investigate the effects and the molecular mechanism of cyclosporin A (CsA) against oxidative stress injury in cultured neonatal rat cardiomyocytes. METHODS: Bax/Bcl-2, cl-casp-9/casp-9, cl-casp-3/casp-3, and iNOS/ß-actin ratios and p-IκB and IκB levels were analyzed by western blot. IL-1ß and TNF-α levels were analyzed by ELISA. RESULTS: CsA effectively improved the cell viability and reduced the extracellular lactate dehydrogenase release in cardiomyocytes after H2O2-induced oxidative damage. CsA significantly increased the superoxide dismutase activity, glutathione production, and catalase activity but decreased the malonaldehyde level. CsA treatment considerably reduced the H2O2-induced intracellular generation of reactive oxygen species, mitochondrial dysfunction, and release of cytochrome c. CsA could act against H2O2-induced ATP reduction, TCA cycle enzymes, mitochondrial complex I enzyme, and complex V enzyme in cardiomyocytes. CsA significantly decreased the Bax/Bcl-2 ratio, cl-casp-9/casp-9, and cl-casp-3/casp-3 in a concentration-dependent manner. CsA also remarkably reduced the cleaved PARP level and DNA fragmentation. NF-κB was closely related to oxidative stress injury. CsA inhibited NF-κB activation, thereby preventing the upregulation of IL-1ß, TNF-α, iNOS, and intracellular NO release. CONCLUSIONS: CsA protected cardiomyocytes against H2O2-induced cell injury. Hence, CsA may be developed as a candidate drug to prevent or treat myocardial ischemia reperfusion injury.

[Pathological changes of benign hyperplastic prostate after removal of innervation of cholinergic pelvic nerve: experiment with spontaneous hypertension rats].

OBJECTIVE: To study the pathological change of benign hyperplastic prostate after removal of the innervation of cholinergic parasympathetic pelvic nerve. METHODS: Sixty-five male spontaneous hypertension rats (SHRs) were randomly assigned into 3 groups: operation group (n = 30) undergoing truncation of bilateral originating branches of parasympathetic pelvic nerve of major pelvic ganglion (MPG) followed by cystostomy, sham operation group (operation control group, n = 30) undergoing cystostomy, and normal control group (n = 5) not undergoing operation. 3, 7, 11, 15 and > or = 21 days after operation 6 rats from the 2 operation groups and 1 from the control group were sacrificed to observe the gross morphology and histological and cellular changes of the prostate glands. RESULTS: The prostate of the operation group on post-operational day 7 showed mild granular solidification and such change progressed gradually over time, the ratio of prostate wet weight/rat body weight was (0.4764 +/- 0.0125) mg/g on day 3, then gradually decreased, and became (0.2749 +/- 0.0197) mg/g > or = 21 days post-operationally; while the ratio of prostate tissue dry weight/wet weight on day 3 was (0.1966 +/- 0.0062), then gradually increased, and became (0.2596 +/- 0.0035) > or = 21 days post-operationally. HE staining showed that the glandular structure gradually became dilated and rounded, with accumulation of prostatic fluid. The glandular epithelial cells showed gradual degeneration, necrosis, and detachment. The glandular epithelium became progressively thinner, the smooth muscles elongated and thinned progressively, and the stromal components showed mild to moderate overgrowth. Electron microscopy showed that the glandular cells gradually underwent vacuolar degeneration and the structures of the basement membrane became fuzzy. The smooth muscle cells degenerated mildly, and the fibroblasts and collagenous fibers in the stroma overgrew slowly. All these histological changes were not found in the sham operation control and normal control groups. CONCLUSION: Remarkable atrophy occurs in benign hyperplastic prostatic gland after radical removal of the innervation of cholinergic parasympathetic pelvic nerve. Such operation may represent a novel therapy for BPH.

[The pathological change of rats' benign hyperplastic prostate after radical denervation].

OBJECTIVE: To study the pathological change of rats' benign hyperplastic prostate (BHP) after radical denervation. METHODS: A total of 65 male spontaneous hypertension rats (SHR) at 30 weeks age were randomly assigned into treatment group, sham surgery control group and normal control group. In surgery group, all the axonal branches of the major pelvic ganglion (MPG) supplying the bilateral prostate were truncated, followed performing of cystostomy; In sham surgery control group, only cystostomy was performed; In normal control group, no procedure was performed. The rats were sacrificed at 3, 7, 11, 15 and >or= 21 d post-operation respectively. The gross morphological changes of prostate in all animals were observed. RESULTS: In treatment group, the prostate in 3 d post-operation showed granular solidification and shrunken volume and the changes occurred gradually over time. The glandular epithelial cells showed gradual degeneration, necrosis and detachment. The glandular epithelium became progressively thinner, the smooth muscles elongated and thinned progressively and the stromal components showed mild to moderate overgrowth. At the later stage, the glandular epithelium, glandular lumen and smooth muscles gradually disappeared and the prostate was largely replaced by connective tissues. Electron microscopic study showed that the glandular cells gradually underwent vacuolar degeneration and the structures of basement membrane became fuzzy. The smooth muscles cells degenerated overtime and the fibroblasts and collagenous fibers in the stroma overgrew slowly. At the late stage, most of the glandular cells became necrotic, the basal membrane and smooth muscle cells disappeared and collagenous fibers were highly hyperplasic. In surgery group in 3 d post-operation, the S-100 staining of nerve fiber was diffuse and disappeared after 11 d while it persisted normally in other groups. The two values in sham surgery control group showed no significant changes post-operatively. CONCLUSIONS: After radical denervation, the rat prostate with benign hyperplasia (gland and smooth muscles) undergoes dramatic atrophic changes and the volume decreases significantly. It suggests that this treatment may represent a novel therapy for BPH.