Alamandine attenuates hepatic fibrosis by regulating autophagy induced by NOX4-dependent ROS.

Angiotensin II (Ang II) has been reported to aggravate hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Alamandine (ALA) protects against fibrosis by counteracting Ang II via the MAS-related G-protein coupled (MrgD) receptor, though the effects of alamandine on hepatic fibrosis remain unknown. Autophagy activated by reactive oxygen species (ROS) is a novel mechanism of hepatic fibrosis. However, whether autophagy is involved in the regulation of Ang II-induced hepatic fibrosis still requires investigation. We explored the effect of alamandine on hepatic fibrosis via regulation of autophagy by redox balance modulation. In vivo, alamandine reduced CCl4-induced hepatic fibrosis, hydrogen peroxide (H2O2) content, protein levels of NOX4 and autophagy impairment. In vitro, Ang II treatment elevated NOX4 protein expression and ROS production along with up-regulation of the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis. These changes resulted in the accumulation of impaired autophagosomes in hepatic stellate cells (HSCs). Treatment with NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine (NAC), and NOX4 small interfering RNA (siRNA) inhibited Ang II-induced autophagy and collagen synthesis. Alamandine shifted the balance of renin-angiotensin system (RAS) toward the angiotensin converting enzyme 2 (ACE2)/alamandine/MrgD axis, and inhibited both Ang II-induced ROS and autophagy activation, leading to attenuation of HSCs migration or collagen synthesis. In summary, alamandine attenuated liver fibrosis by regulating autophagy induced by NOX4-dependent ROS.

In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies.

Previous studies have indicated that heparanase (Hpa) might represent a candidate universal tumor-associated antigen. However, vaccine therapy targeting only one cytotoxic T lymphocyte (CTL) epitope is suboptimal in preventing cancer. In the present study, we designed heparanase multi-epitope vaccines to increase the immune response to standard single heparanase epitopes. The results showed that multi-epitope vaccines Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 induced higher Hpa-specific lysis of various cancer cells from different tissues in a HLA-A2-restricted and heparanase-specific manner compared with the single epitope vaccines Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363, both in vitro and ex vivo. Heparanase multi-epitope vaccines not only induced the heparanase-specific CTL to lyse tumor cells but also increased CTL secretion of interferon-γ. However, these heparanase-specific CTL did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirms the safety of these multi-epitope vaccines. Therefore, the present study provides theoretical evidence for the use of heparanase multi-epitope vaccines for clinical application.

Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations.

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high-efficiency protein refolding. We have herein found that electrostatic repulsion between like-charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion-exchange resins when the protein and ion-exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like-charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies.

[Characteristics and Sources of Water-soluble Ion Pollution in PM2.5 in Winter in Shenyang].

In order to explore the characteristics of PM2.5 and water-soluble ions in Shenyang in winter, the URG-9000D online monitoring system was used to continuously sample PM2.5 and gas components during 2018. The results indicated that the average concentration of PM2.5 in Shenyang during the sampling period was 80.67 µg·m-3, and the total water-soluble ion concentration ranged from 2.68 to 132.79 µg·m-3. Compared with clean days, the proportion of NO3-, SO42-, and NH4+ (SNA) in polluted days increased significantly, reached 43.7% of PM2.5. The rapid accumulation of SO2 in a short period of time made atmospheric PM2.5 explosively increase in Shenyang in winter. A Pearson correlation analysis showed that the correlation coefficients of SNA, Cl-, and PM2.5 were all above 0.78, indicating that the main contribution components of winter PM2.5 in Shenyang were SNA and Cl-. The apportionment of PMF sources indicated that the sources of pollutants in winter in Shenyang mainly included secondary reaction sources, coal and biomass combustion sources, and dust sources.

Modeling of Endothelial Calcium Responses within a Microfluidic Generator of Spatio-Temporal ATP and Shear Stress Signals.

Intracellular calcium dynamics play essential roles in the proper functioning of cellular activities. It is a well known important chemosensing and mechanosensing process regulated by the spatio-temporal microenvironment. Nevertheless, how spatio-temporal biochemical and biomechanical stimuli affect calcium dynamics is not fully understood and the underlying regulation mechanism remains missing. Herein, based on a developed microfluidic generator of biochemical and biomechanical signals, we theoretically analyzed the generation of spatio-temporal ATP and shear stress signals within the microfluidic platform and investigated the effect of spatial combination of ATP and shear stress stimuli on the intracellular calcium dynamics. The simulation results demonstrate the capacity and flexibility of the microfluidic system in generating spatio-temporal ATP and shear stress. Along the transverse direction of the microchannel, dynamic ATP signals of distinct amplitudes coupled with identical shear stress are created, which induce the spatio-temporal diversity in calcium responses. Interestingly, to the multiple combinations of stimuli, the intracellular calcium dynamics reveal two main modes: unimodal and oscillatory modes, showing significant dependence on the features of the spatio-temporal ATP and shear stress stimuli. The present study provides essential information for controlling calcium dynamics by regulating spatio-temporal biochemical and biomechanical stimuli, which shows the potential in directing cellular activities and understanding the occurrence and development of disease.

Starch/tea polyphenols nanofibrous films for food packaging application: From facile construction to enhance mechanical, antioxidant and hydrophobic properties.

Starch based food packaging has been receiving increasing attention. However, the inherent poor properties of starch restrict its practical applications in the versatile material science field. In this study, a fast, simple, and environmentally friendly route to construct polyfunctional starch/tea polyphenols nanofibrous films (STNFs) by one-step temperature-assisted electrospinning was developed. The effects of introduction of tea polyphenols (TP) on the mechanical and antioxidant activity of STNFs were comprehensively investigated. Results of ABTS·+ free radical scavenging assay showed that the antioxidant activity of STNFs was endowed by addition of TP with optimum mechanical properties confirmed by tensile test. More interestingly, the hydrophobicity of STNFs was improved dramatically with increasing cross-linking time as indicated by water contact angle (WCA) measurement showing no effect on the antioxidant activity of the films. The results of this work offer a major step forward to promote functional starch-based materials for sustainable application in food packaging.

Promising Rice-Husk-Derived Carbon/Ni(OH)2 Composite Materials as a High-Performing Supercapacitor Electrode.

Improving the electrochemical performance of biomass-derived carbon electrode-active materials for supercapacitor applications has recently attracted considerable attention. Herein, we develop hybrid electrode materials from rice-husk-derived porous carbon (RH-C) materials and ß-Ni(OH)2 via a facile solid-state reaction strategy comprising two steps. The prepared RH-C/Ni(OH)2 (C-Ni) was investigated using scanning electron microscopy (SEM) (energy-dispersive X-ray spectrometer (EDS)), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) to acquire the physical and chemical information, which was used to demonstrate the successful fabrication of C-Ni. Thermogravimetric analysis (TGA) measurement results confirmed that the thermal stability of C-Ni changed due to the presence of Ni(OH)2. As expected, C-Ni possesses a high capacitance of ∼952 F/g at a current density of 1.0 A/g. This result is higher than that of pure biomass-based carbon materials under the three-electrode system. This facile preparation method, which was used to synthesize the electrode-active materials, can extend to the value-added utility of other waste biomass materials as high-performing supercapacitor electrodes for energy storage applications.

[Establishment and Characteristics of an Artificial Ammonia Emissions Inventory in Jiangsu Province from 2013 to 2017].

According to activity level data of various ammonia emission sources in Jiangsu Province, using a reasonable inventory calculation method and emission factor, an ammonia emission inventory in Jiangsu Province from 2013 to 2017 was established, and the trend of anthropogenic ammonia emissions over these years was analyzed. The distribution characteristics of anthropogenic ammonia emissions and emission intensity in Jiangsu Province were analyzed using ArcGIS software. The results showed that the ammonia emissions in Jiangsu Province decreased from 624.84 kt in 2013 to 562.47 kt in 2017 with an average annual rate of decline of approximately 2.6%. Agricultural has always been the most important source of ammonia emissions in Jiangsu Province and accounted for 82.4% of the total ammonia emissions in 2017. Laying hens are the largest source of ammonia emissions from livestock and poultry sources, accounting for 49.3% of the ammonia emissions from livestock and poultry. The average ammonia emission intensity in Jiangsu Province was 5.3 t·km-2 in 2017. Yancheng and Xuzhou are two cities with the largest anthropogenic ammonia emissions and emission intensity in Jiangsu Province. Zhenjiang City has the lowest ammonia emission and emission intensity.

Deletion of Smad4 reduces hepatic inflammation and fibrogenesis during nonalcoholic steatohepatitis progression.

OBJECTIVE: To explore the effects of mothers against decapentaplegic homolog family member 4 (Smad4) deletion on inflammation and fibrogenesis in nonalcoholic steatohepatitis (NASH). METHODS: Biopsied liver samples from NASH patients and normal liver tissue samples from patients who had received liver resection for trauma were collected. Smad4Co/Co and wild-type (WT) mice were used to construct the NASH model using a high-fat diet (HFD) or methionine- and choline-deficient diet (MCD). HE staining and TUNEL assay were used to observe the pathological changes and cell apoptosis, respectively. Quantitative real-time polymerase chain reaction was used to detect the expression of inflammatory, fibrogenesis and apoptosis-related genes, and immunohistochemistry to determine the protein expression of SMAD4, MCP-1 and α-SMA. RESULTS: SMAD4 protein expression significantly increased in NASH patients than in the control group. Compared with WT mice, HFD- and MCD-fed Smad4Co/Co mice showed decreased hepatic steatosis, inflammation, liver cell apoptosis and nonalcoholic fatty liver activity score, reduced plasma glucose, triglyceride, free fatty acids, alanine aminotransferase and aspartate aminotransferase levels but increased adiponectin. Moreover, Smad4Co/Co decreased the expression of inflammatory markers (TNF-α, MCP-1, IFN-γ), fibrogenetic markers (COL1A1, α-SMA and TGF-ß1), lipogenic (Srebp1c, Fas and Acc) and proapoptotic genes (Bax and caspase-3), but increased the expression of ß-oxidation (Ppar-α, Cpt1 and Aco) and antiapoptotic genes (Bcl-2). CONCLUSION: Smad4 deletion may inhibit lipogenesis, stimulate ß-oxidation, improve lipid metabolism and liver function, alleviate inflammation and fibrosis, and reduce cell apoptosis in NASH.

MicroRNA-21 Mediates Angiotensin II-Induced Liver Fibrosis by Activating NLRP3 Inflammasome/IL-1ß Axis via Targeting Smad7 and Spry1.

AIMS: Angiotensin II (AngII), a vasoconstrictive peptide of the renin-angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1-7) [Ang-(1-7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1ß axis in liver fibrosis. RESULTS: In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1-7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1-7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects. Innovation and Conclusions: Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1-7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1-20.

[Assessment of the capability of the fluid model of automatic blood cell analyzer in white blood cell count and classification].

OBJECTIVE: To evaluate the application of XT-4000i automatic blood cell analyzer in white cell count and classification of cerebrospinal fluid (CSF). METHODS: The fluid model of XT-4000i automatic blood cell analyzer was directly used to detect the white cell count and classification in 200 samples of CSF, and compared with the results obtained by manually microscopic counting method. White blood cell classification was performed by manual method under microscope with Wright's staining,and instrumental method with fluorescence staining and flow cytometry. RESULTS: There is no statistical difference between instrumental and manual method in detecting the absolute counting of WBC, RBC, mononuclear cell and multinucleate cells (P>0.05), and there is a good correlation between the two methods (r=0.987, 0.999, 0.981 and 0.983 for WBC, RBC, mononuclear cell and multinucleate cell counts). Tumor cells in the samples with high fluorescent staining by instrumental method can be identified with Wright's staining by microscope method, which were consistent with the clinical diagnosis. CONCLUSION: The fluid model of XT-4000i automatic blood cell analyzer was rapid and accurate in CSF white cell count and classification,and it also can provide preliminary information for tumor cells screening. The fluid model of XT-4000i automatic blood cell analyzer in white cell count and classification of CSF has the value of popularization in clinical application.

Multiple antigenic peptides based on H-2K(b)-restricted CTL epitopes from murine heparanase induce a potent antitumor immune response in vivo.

Accumulating research suggests that heparanase may be a universal tumor-associated antigen (TAA). Several heparanase T-cell epitopes from humans and mice have already been identified. However, because of low immunogenicity, polypeptide vaccines usually have difficulty inducing effective antitumor immune responses in vivo. In this study, to increase the immunogenicity of polypeptide vaccines, we designed and synthesized two four-branch multiple antigenic peptides (MAP) on the basis of mouse heparanase (mHpa) T-cell epitopes (mHpa398 and mHpa519). The dendritic cells (DC) from mice bone marrow loaded with above MAP vaccines from heparanase were used to evaluate immune response against various tumor cell lines, compared with immune response to their corresponding linear peptides, ex vivo and in vivo. We further assessed IFN-γ release both in CD4(+) T-cell-depleted and nondepleted mice. The results showed that effectors generated from DCs, loaded with MAP-vaccinated mice splenocytes, induced a stronger immune response against target cells expressing both heparanase and H-2K(b) than did effectors generated from mice vaccinated with their corresponding linear peptides. Heparanase-specific CD8(+) T-cell responses induced by MAP and linear peptide vaccination required synergy of CD4(+) T cells. In addition, heparanse-derived MAP vaccines significantly inhibited the growth of B16 murine melanoma in C57BL/6 mice, while also increasing the survival rate of tumor-bearing mice. Our data suggest that MAP vaccines based on T-cell epitopes from heparanase are efficient immunogens for tumor immunotherapy.

Acyl cystamine: small-molecular foldase mimics accelerating oxidative refolding of disulfide-containing proteins.

Based on the structural characteristic of Protein disulfide isomerases and DsbA that have hydrophobic regions around the active sites, hydrophobic alkyl tails are linked to cystamine to create new small molecular foldase mimics, acyl cystamine. Both the oxidizing power and oxidation specificity of cystamine are enhanced by n-octanoyl or n-hexanoyl tail. N-octanoyl and n-hexanoyl cystamine are very effective to facilitate oxidative protein refolding at strong reducing environments. In the presence of 0.42 mM DTT, the activity recovery of lysozyme is over 90% by 90-min refolding with 0.1 mM n-octanoyl cystamine and 0.1 mM cystamine as oxidant, while almost no activity is recovered with 0.2 mM GSSG by 160-min refolding. For the refolding of 0.2 mg/mL lysozyme, with 0.6 mM n-hexanoyl cystamine and 1.12 mM residual DTT as redox agents, the activity recovery reaches as high as 93% after refolding for only 20 min. For ribonuclease A (RNase A) refolding, with 0.4 mM n-hexanoyl cystamine and 1.30 mM DTT, the recovery of activity reaches as high as 90% within 3 h. Thus, with n-octanoyl or n-hexanoyl cystamine as the oxidants, the necessity to remove excess DTT in the reduced and denatured protein solutions can be greatly alleviated. With a moderate hydrophobicity, n-hexanoyl cystamine is promising for application in oxidative protein refolding at an extensive concentration range. It is observed that in the oxidative refolding of 0.2 mg/mL lysozyme and RNase A, only about half of n-hexanoyl cystamine is needed when compared to cystamine to achieve the same kinetic effect.

Multiple antigenic peptides of human heparanase elicit a much more potent immune response against tumors.

Peptide vaccination for cancer immunotherapy requires an ideal immune response induced by epitope peptides derived from tumor-associated antigens (TAA). Heparanase is broadly expressed in various advanced tumors. Accumulating evidence suggests that heparanase can serve as a universal TAA for tumor immunotherapy. However, due to the low immunogenicity of peptide vaccines, an ideal immune response against tumors usually cannot be elicited in patients. To increase the immunogenicity of peptide vaccines, we designed three 4-branched multiple antigenic peptides (MAP) on the basis of the human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitopes of human heparanase that we identified previously as antigen carriers. Our results show that MAP vaccines based on the HLA-A2-restricted CLT epitopes of human heparanase were capable of inducing HLA-A2-restricted and heparanase-specific CTL in vitro and in mice. Moreover, compared with their corresponding linear peptides, heparanase MAP vaccines elicited much stronger lysis of tumor cells by activating CD8(+) T lymphocytes and increasing the releasing of IFN-γ. However, these heparanase-specific CTLs did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirm the safety of these MAP vaccines. Therefore, our findings indicate that MAP vaccines based on CTL epitopes of human heparanase can be used as potent immunogens for tumor immunotherapy because of advantages such as broad spectrum, high effectiveness, high specificity, and safety.