Salt-tolerant and thermostable mechanisms of an endoglucanase from marine Aspergillus niger.

The cellulase cocktail of marine Aspergillus niger exhibited salt-tolerant and thermostable properties, which is of great potential in industrial application. In order to excavate the single tolerant cellulase components from complex cellulase cocktail, constitutive homologous expression was employed for direct obtainment of the endoglucanase (AnEGL). Enzymatic property study revealed that AnEGL exhibited a property of salt tolerance and a strong thermostability in high salinity environment. Significantly, its activity increased to 129% and the half-life at 65 °C increased to 27.7-fold with the presence of 4.5 M NaCl. Molecular dynamics simulation revealed that Na+ and Cl- could form salt bridges with charged residues, and then influenced the activity of loops and the stability of substrate binding pocket, which accounted for the salt tolerance and thermostability. Further, site-specific mutagenesis study proved that the residues Asp95 and Asp99 in the pocket were of great concern for the tolerant properties. The salt-tolerant and thermostable AnEGL was of great value in lignocellulosic utilization and the conjectural mechanisms were of referential significance for other tolerant enzymes.

Interleukin-1ß-Treated Mesenchymal Stem Cells Inhibit Inflammation in Hippocampal Astrocytes Through Exosome-Activated Nrf-2 Signaling.

BACKGROUND: Interleukin-1ß (IL-1)-treated mesenchymal stem cells (MSCs) and IL-1-MSCs-conditioned medium (CM) exert anti-inflammatory roles. Astrocytes are essential for the modulation of synaptic activity and neuronal homeostasis in the brain. Exosomes are the critical mediators in intercellular communication. However, the mechanism underlying the anti-inflammatory effect of IL-1-treated MSCs remains unknown. METHODS: In this study, exosomes (IL-1-Exo) were isolated from IL-1-treated MSCs. In addition, lipopolysaccharide (LPS)-treated hippocampal astrocytes and status epilepticus (SE) mice were treated with IL-1-Exo. Inflammatory activity, astrogliosis, and cognitive performance were measured to determine the effect of IL-1-Exo on inflammation. RESULTS: The results revealed that IL-1-Exo significantly inhibited LPS-induced astrogliosis and inflammatory responses of astrocytes. Also, IL-1-Exo reversed the LPS-induced effect on calcium signaling. The Nrf2 signaling pathway was associated with the effect of IL-1-Exo in LPS-treated astrocytes. Furthermore, IL-1-Exo reduced the inflammatory response and improved the cognitive performance of SE mice. CONCLUSION: The results suggest that IL-1-Exo inhibited LPS-induced inflammatory responses in astrocytes and SE mice and that the effect of IL-1-Exo was primarily mediated through the Nrf-2 signaling pathway. This study provides a new understanding of the molecular mechanism of inflammation-associated brain diseases and an avenue to develop nanotherapeutic agents for the treatment of inflammatory conditions in the brain.

Cross electro-nape-acupuncture ameliorates cerebral hemorrhage-induced brain damage by inhibiting necroptosis.

BACKGROUND: Receptor interacting protein kinase 1 (RIPK1)-mediated cell death, including apoptosis and necroptosis, belongs to programmed cell death. It has been reported that RIPK1-mediated necroptosis exists in lesions of cerebral hemorrhage (CH). Electroacupuncture, a treatment derived from traditional Chinese medicine, could improve neurological impairment in patients with brain injury. AIM: To investigate the protective role of cross electro-nape acupuncture (CENA) in CH, and clarify the potential mechanism. METHODS: CH rat models were established, and CENA was applied to the experimental rats. Neurological functions and encephaledema were then measured. Necrotic cells in the brain of rats with CH were evaluated by propidium iodide staining. Necroptosis was assessed by immunofluorescence. Activation of the necroptosis-related pathway was detected by western blot. Extraction of brain tissue, cerebrospinal fluid and serum samples was conducted to measure the expression and secretion of inflammatory cytokines by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: The necroptotic marker p-MLKL was detectable in the brains of rats with CH. Next, we found that CENA could ameliorate neurological functions in rat models of CH. Moreover, the upregulation of RIPK1-mediated necroptosis-related molecules in the brains of rats with CH were inhibited by CENA. Further investigation revealed that CENA partially blocked the interaction between RIPK1 and RIPK3. Finally, in vivo assays showed that CENA decreased the expression of the inflammatory cytokines tumor necrosis factor-α, interleukin-6 and interleukin-8 in CH rat models. CONCLUSION: These findings revealed that CENA exerts a protective role in CH models by inhibiting RIPK1-mediated necroptosis.

Directed expression of halophilic and acidophilic ß-glucosidases by introducing homologous constitutive expression cassettes in marine Aspergillus niger.

The hydrolysis step by ß-glucosidase (BGL) is generally recognized as the major limiting step in cellulose degradation and the BGLs with prominent enzymatic properties are of great importance for efficient utilization of lignocellulosic biomass. In order to identify some salt-tolerant BGLs, two BGL genes were cloned from marine Aspergillus niger ZJUBE-1 genome. Then two bgl expression cassettes driven by gpdA promoter were respectively transformed into marine A. niger for homologous constitutive expression. Directed expression was achieved for the domination of target BGLs in fermentation broth. Conveniently, two BGLs were purified to homogeneity by two separation steps, ultrafiltration and anion exchange chromatography. The purified BGL1 and BGL2 showed maximum activity at pH 3.0-4.0 and 3.5-4.5, respectively, suggesting these two BGLs were relatively acidophilic, especially for BGL1. Besides, BGL1 was stable to most of metal ions, while BGL2 was sensitive to Cu2+, Fe3+ and Ag+. Most specially, BGL2 activity increased by 44% in the presence of 4 M NaCl, suggesting BGL2 was halophilic. Homology modeling revealed that longer loops and linkers as well as polymerous Glu492 may contribute to the halophilism of BGL2. At last, the medium for directed expression was optimized and the content as well as the purity of target protein was improved.

Effects of hemoglobin variants HbJ Bangkok, HbE, HbG Taipei, and HbH on analysis of glycated hemoglobin via ion-exchange high-performance liquid chromatography.

BACKGROUND: To explore the effects of HbJ Bangkok, HbE, HbG Taipei, and α-thalassemia HbH on the results of HbA1c assessment using ion-exchange high-performance liquid chromatography (IE-HPLC). METHODS: We enrolled five patients in which the results of the IE-HPLC HbA1c assay were inconsistent with the average levels of FBG. We performed hemoglobin capillary (Hb) electrophoresis using whole-blood samples. We also sequenced the genes encoding Hb using dideoxy-mediated chain termination and analyzed HbA1c using borate affinity HPLC (BA-HPLC) and turbidimetric inhibition immunoassay (TINIA). RESULTS: Two patients had the HbJ Bangkok variant. Hb genotypes of these patients were ß41-42 /ßJ Bangkok and ßN /ßJ Bangkok , and the content of HbJ Bangkok was 93.9% and 52.4%, respectively. The remaining three patients had the following: HbE (ßN /ßE Hb genotype, 23.6% HbE content), HbG Taipei (ßN /ßG Taipei Hb genotype, 39.4% HbG Taipei content), and α-thalassemia HbH (6.1% HbH content, 2.8% Hb Bart's content). In the patients with ß-thalassemia and HbJ Bangkok variants, the presence of the variants interfered with the results of HbA1c analyses using IE-HPLC and TINIA; in the remaining four patients, there was interference with the results of HbA1c IE-HPLC but not with the TINIA assay. There was no interference with BA-HPLC HbA1c results. CONCLUSIONS: HbJ Bangkok, HbE, HbG Taipei Hb, and α-thalassemia HbH disease cause varying degrees of interference with the analysis of HbA1c using IE-HPLC. In these patients, we suggest using methods free from such interference for the analysis of HbA1c and other indicators to monitor blood glucose levels.

Evaluation of the Interference of Hemoglobin Variant J-Bangkok on Glycated Hemoglobin (HbA1c) Measurement by Five Different Methods.

Objective The interference of the hemoglobin variant (Hb J-Bangkok) was evaluated on 4 different glycated hemoglobin assays and compared with a reference immuno assay. Methods An overall test of coincidence of 2 least-squares linear regression lines was performed to determine whether the presence of Hb J-Bangkok caused a statistically significant difference in HbA1c results compared with a reference immuno assay. Statistical analysis was performed on the difference of the estimated average glucose calculated from HbA1c values and fasting plasma glucose in the Hb J-Bangkok variant group using the different detection systems. Deming regression analysis was used to determinate whether Hb J-Bangkok had a significant interference on HbA1c results using an HbA1c±10% relative bias at 6% and 9% HbA1c as evaluation limits. Results Turbidimetric inhibition immunoassay method, and enzymatic methods were not affected by Hb J-Bangkok. However, Hb J-Bangkok showed statistically significant interference to the two ion-exchange high-performance liquid chromatography methods. Conclusion When performing HbA1c tests, clinical laboratory personnel should identify the Hb variant and select the appropriate methods or use alternative indicators.

[The Effect of the Phase on Characteristic Energy of All-Trans-ß-Carotene].

All-trans-ß-carotene has important functions of light collection and light protection, and it is also an important electrooptical material. The Raman spectra of polyenes are a result of the modulation effect of the π electron energy gap on the vibration of CC bonds, which associate with the external field. So it has higher theoretical significance and practical value to study the molecular structure and properties change under the external field. Ultraviolet-visible absorption spectra and resonant Raman spectra of all-trans-ß-carotene in cyclohexanol were measured from 341 to 275 K. The liquid-solid phase transition of the sample appears at 295 K. The characteristic energy describes the conformational change of all-trans-ß-carotene molecule. After the solution phase transition, the characteristic energy ε of all-trans-ß-carotene molecule becomes bigger. And when temperature decreasing, the rate of change of the Huang-Rhys, the wavelength of UV absorption peak, electron-phonon Parameter, RSCSs of the CC bond increase. the Huang-Rhys in solid phase is an order of magnitude higher then liquid phase. The characteristic energy of liquid is 0.206 7 eV. The characteristic energy of liquid is 0.559 6 eV. The increasing of the characteristic energy ε makes the rate of increasing of the effectively conjugated length becomes bigger. The decreasing of the π electric energy gap quickens. The function of moderation from electron energy gap to all-trans-ß-carotene molecule enhances. Electron-phonon Parameter increases. RSCSs of the CC bond substantially increases.

One-pot preparation of a mixed-mode organic-silica hybrid monolithic capillary column and its application in determination of endogenous gibberellins in plant tissues.

A newly improved one-pot method, based on "thiol-ene" click chemistry and sol-gel approach in microemulsion system, was developed for the preparation of C8/PO(OH)2-silica hybrid monolithic capillary column. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The monolithic column was demonstrated to have cation exchange/reversed-phase (CX/RP) mixed-mode retention for analytes on nano-liquid chromatography (nano-LC). On the basis of the developed nano-LC system with MS detector coupled to pipette tip solid phase extraction (PT-SPE) and derivatization process, we then realized simultaneous determination of 10 gibberellins (GAs) with low limits of detection (LODs, 0.003-0.025 ng/mL). Furthermore, 6 endogenous GAs in only 5mg rice leaves (fresh weight) were successfully detected and quantified. The developed PT-SPE-nano-LC-MS strategy may offer promising applications in the determination of low abundant bioactive molecules from complex matrix.

Facile synthesis of polyaniline-coated SiO2 nanofiber and its application in enrichment of fluoroquinolones from honey samples.

In this study, polyaniline coated SiO2 nanofibers (PANI/SiO2) was prepared by combining electrospinning technique with in-situ polymerization. The proposed strategy for the preparation of PANI/SiO2 can eliminate the aggregation of PANI and the yield of PANI/SiO2 was high. Scanning electron microscopy (SEM) images showed that PANI nanoparticles were uniformly coated on the surface of SiO2 nanofibers. The as-prepared PANI/SiO2 nanofibers were then applied as the sorbent for in-syringe dispersive solid-phase extraction (dSPE) for the extraction of fluoroquinolones (FQs) from honey samples. The influence of SiO2 amount on the formation of PANI/SiO2 and several parameters that affect the extraction efficiency were investigated. Under optimized conditions, a rapid, simple and effective method for the determination of FQs in honey sample was developed by coupling with liquid chromatography-fluorescence detector (LC-FLD) analysis. Due to the fast extraction equilibrium, the whole sample pretreatment process could be accomplished within 4 min. The limits of detection (LODs) for the target FQs were found to be 0.1-1.3 ng/g. The recoveries in honey sample were in the range of 81.4-118.1% with the RSDs of 0.8-14.4% (intra-day) and 1.4-14.9% (inter-day). This study offers a new strategy for the preparation of functional SiO2 nanofibers using post-electrospinning modification by in-situ polymerization, which could be generally applied in the preparation of various separation materials with electrospun nanofibers.

[Resonance Raman Spectral Properties Studies of Beta-carotene in Solution].

Beta-carotene is an important kind of polyene biomolecules, which has significant applications on researching optoelectronic and functional materials. In-situ high pressure Raman spectra of beta-carotene are measured in CS2 solution and water respectively at pressure range from 0-0.60 GPa. Then we compared both of them the Raman shift and CC bond of the full width at half maximum (FWHM) of the Raman spectra. It is therefore concluded that both of the samples' Raman shift moved to the high wave number and the full width at half maximum increased depending of the pressure. The experiment phenomena were interpreted by the theory of "coherent weakly damped electronic-lattice vibration model" and "effective conjugation length model". The mechanism is that the beta-carotene is compressed and has the lower structure order, shorte the effective conjugation length, decreased Raman active, weaker the coherent weakly damping CC bond vibration in high pressure. Because of the CC bond length become short, so the Raman spectra are found to blueshift. The CC bond of the full width at half maximum (FWHM) of the Raman spectra increased is attributed to the increase of difference in C--C and C==C bond lengths. Moreover, due to dissolving in non-polar CS2 solvent, the beta-carotene encounters the interaction of the surrounding solvent molecules. So the dispersion force interaction between solute and solvent is more sensitive to pressure. Then it makes that the slop of Raman shift and the full width at half maximum in the CS2 solution are faster than dissolved in water with increasing pressure. This paper provides an application value for research on molecular structure change under the external field and the presence form of polyenes biomolecules in the solvent.

Sodium ferulate lowers portal pressure in rats with secondary biliary cirrhosis through the RhoA/Rho-kinase signaling pathway: a preliminary study.

Cirrhotic rats show higher expression levels of hepatic RhoA and Rho-kinase than normal healthy rats, and the activation of this signaling pathway leads to portal hypertension. Sodium ferulate (SF) has been shown to decrease the production of geranylgeranyl pyrophosphate (GGPP), a substance essential for RhoA activation. In the present study, to investigate the effects of SF on fibrosis, portal hypertension and the RhoA/Rho-kinase pathway, hepatic cirrhosis was induced in rats by bile duct ligation. Liver function and fibrogenesis-related biochemical parameters, the hepatic hydroxyproline content, the pathological characteristics of the liver sections and the levels of hepatic α-smooth muscle actin (α-SMA; by immunohistochemistry) were analyzed to assess effects of SF on hepatic fibrosis. In addition, hepatic RhoA, Rho-kinase and endothelial nitric oxide synthase (eNOS) expression was examined by immunohistochemistry. Apoptosis in the SF-treated and SF + GGPP-treated rat primary hepatic stellate cells (HSCs) and a human stellate cell line (LX-2) was examined by flow cytometry. Intrahepatic resistance and responsiveness to the α1-adrenoceptor agonist, methoxamine, were investigated by in situ liver perfusion. Treatment with SF did not affect fibrosis-related biochemical parameters or the hydroxyproline content; however, SF reduced the histological evidence of fibrosis and hepatocyte damage. The SF-treated rats had a significantly lower expression of α-SMA and Rho-kinase, as well as an increased hepatic eNOS content; however, SF did not affect RhoA expression. The SF-treated HSCs had a significantly increased apoptotic rate compared to the untreated rats. Following the addition of GGPP, the rate apoptotic rate decreased. SF reduced basal intrahepatic resistance and the responsiveness of hepatic vascular smooth muscle to methoxamine. Therefore, our data demonstrate that SF reduces fibrogenesis, decreases portal pressure in cirrhotic rats and inhibits the activation of the RhoA/Rho-kinase signaling pathway.

[Effect of pressure on electron-phonon coupling constants of all-trans-beta-carotene].

The present paper cited that R Tubino and other people introduced a kind of electron-phonon coupling constants with dimension, which can establish the relation with the Huang-Rhys factor and calculate the electron-phonon coupling constants of every C-C bond vibration mode. There are many reports about the visible absorption and Raman spectra of all-trans-beta-carotene with pressure. But the study about the Raman scattering cross section and the Huang-Rhys factor with pressure have not been reported now. Visible absorption and Raman spectra of all-trans-beta-carotene were measured in carbon disulfide in the pressure range from 0. 04 to 0. 60 GPa. The results indicated that the visible absorption spectra of beta-carotene in nonpolar solvent carbon disulfide are red-shifted with pressure increasing, but the frequency shifts towards higher frequencies in the Raman spectra, the Raman scattering cross section decreases, Huang-Rhys factor increases, and the electron-phonon coupling constants of CC bond vibration modes increase. The mechanism is that all-trans-beta-carotene caused by compression and a decrease in the structurally ordered properties of the molecules leads to narrow energy gap of the pi, shortens effective conjugation length, hinders delocalization of pi-electron, decreases the Raman scattering cross section, and increases the Huang-Rhys factor and the electron-phonon coupling constants.

Response gene to complement-32 enhances metastatic phenotype by mediating transforming growth factor beta-induced epithelial-mesenchymal transition in human pancreatic cancer cell line BxPC-3.

BACKGROUND: Response gene to complement-32 (RGC-32) is comprehensively expressed in many kinds of tissues and has been reported to be expressed abnormally in different kinds of human tumors. However, the role of RGC-32 in cancer remains controversial and no reports have described the effect of RGC-32 in pancreatic cancer. The present study investigated the expression of RGC-32 in pancreatic cancer tissues and explored the role of RGC-32 in transforming growth factor-beta (TGF-ß)-induced epithelial-mesenchymal transition (EMT) in human pancreatic cancer cell line BxPC-3. METHODS: Immunohistochemical staining of RGC-32 and E-cadherin was performed on specimens from 42 patients with pancreatic cancer, 12 with chronic pancreatitis and 8 with normal pancreas. To evaluate the role of RGC-32 in TGF-ß-induced EMT in pancreatic cancer cells, BxPC-3 cells were treated with TGF-ß1, and RGC-32 siRNA silencing and gene overexpression were performed as well. The mRNA expression and protein expression of RGC-32 and EMT markers such E-cadherin and vimentin were determined by quantitative reverse transcription-PCR (qRT-PCR) and western blot respectively. Finally, migration ability of BxPC-3 cells treated with TGF-ß and RGC-32 siRNA transfection was examined by transwell cell migration assay. RESULTS: We found stronger expression of RGC-32 and higher abnormal expression rate of E-cadherin in pancreatic cancer tissues than those in chronic pancreatitis tissues and normal pancreatic tissues. Immunohistochemical analysis revealed that both RGC-32 positive expression and E-cadherin abnormal expression in pancreatic cancer were correlated with lymph node metastasis and TNM staging. In addition, a significant and positive correlation was found between positive expression of RGC-32 and abnormal expression of E-cadherin. Furthermore, in vitro, we found sustained TGF-ß stimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of siRNA silencing and gene overexpression, we further demonstrated that RGC-32 mediated TGF-ß-induced EMT and migration in BxPC-3 cells. CONCLUSIONS: The results above indicated that RGC-32 might be a novel metastasis promoting gene in pancreatic cancer and it enhances metastatic phenotype by mediating TGF-ß-induced EMT in human pancreatic cancer cell line BxPC-3.