Objective The effect of serum exosome isolation method on obtained exosomal RNA is not yet clear .The aim of this study was to provide evidences to selected exosome isolation methods .Methods This was a method comparison study to assess three methods .Vein blood samples were collected from 3 healthy donors from Nanfang hospital , 4 ℃ 12 h was taken to make blood natural coagulated and the supernatant was taken as the serum . Exosomes extracted by Ultracentrifugation , ExoQuick and Total Exosome Isolation Reagent ( TEI ) kits from serum were assessed by transmission electron microscopy , nanoparticle tracking analysis and protein analysis to identify morphology , protein expression , concentration and size distribution of particles .ExoRNA extracted by Trizol-LS, SeraMir, Total Exosome RNA Isolation ( TER) and exoRNeasy were assessed by Bioanalyzer 2100 and qPCR to ascertain RNA distribution and miRNA expression.Results For exosomes isolation , two commercial kits ( ExoQuick and TEI ) showed higher exosomes recovery and protein concentration than ultracentrifugation , but exosome isolated by ultracentrifugation expressed abundant protein makers mostly .For exo-RNA isolation, the distribution of RNA and expression of miRNAinfluenced by three methods , but there was no effect on the relative expression trend of miRNA.ExoRNeasy resulted in the highest yield and most narrow size distribution pattern of small RNA with higher level of miRNA expression .Results of TEI with TER kits showed no obvious bands of small RNA and moderate miRNA expression among methods .Ultra with Trizol-LS or ExoQuick with SeraMir showed low concentration measurable bands around 100 nt, with changeable miRNA profiling irregularly . Conclusions Extraction of exosomes using traditional ultracentrifugation method is applicable for proteomics research work .Extraction using ExoQuick and TEI kits is suitable for preparing exosomes from scarce samples such as serum.

This article was aimed to study the immunomodulatory effect of ethanol sediments of the seeds of Descurainia sophia(L.) Webb. ex Prantl. both in vitro and in vivo. The lymphocyte proliferation test in vitro was carried out to explore the effect of the ethanol sediments on the proliferation of T cell and B cell in the spleen of normal mice. And, the carbon clearance test, serum hemolysin test, and delayed-type hypersensitivity test were used to investigate the influence of fraction on non-specific immunity, humoral immunity and cellular immunity in the immunosuppressive mice induced by cyclophosphamide. Besides, the immunosuppressive model was used to evaluate the effect of fraction on immune organs and content of cellular factors in blood serum. The results showed that the ethanol sediments promoted Concanavalin A (Con A) induced T cell and Lipopolysaccharides (LPS) induced B cell (P < 0.01). It increased the carbon clearance index K, phagocytic index α, half value hemolysis (HC50), and swelling degree of auricula (P < 0.05 or P < 0.01). It reduced the body weight and atrophy of thymus and spleen index (P < 0.05 or P < 0.01). It increased the contents of IL-2, IFN-γ and IL-4 in serum in immunosuppressive mice (P < 0.05 or P < 0.01). It was concluded that ethanol sediments of the seeds of D. sophia(L.) Webb. ex Prantl. can boost the lymphocyte proliferation, protect the immune organs, and enhance the non-specific and specific immunity in immunosuppressive mice, which indicated that it had immune-promotion effect.

Since the outbreak of the novel coronavirus pneumonia (COVID-19), it is urgently to develop the effective strategies for its clinical test or treatments world widely. Extracellular vesicles (EV)/exosomes could function as effective carriers for intercellular communication. Increasing evidence revealed that mesenchymal stem cell-derived EV/exosomes could be considered as an alternative cell-free therapy against the acute respiratory distress syndrome. Moreover, the EV-related metabolomics, proteomics, relapsing, deep-vein-thrombosis, myocardial-toxicity, liquid-biopsy and bio-therapeutic researches focusing on the COVID-19 will not only extended our knowledge for the diagnosis, but also provide novel ideas for treatment of this fatal disease. This article will systemically review the progresses of EV/exosomes in the diagnosis and treatment of COVID-19.

Catalytic Domain ; genetics ; Dual Specificity Phosphatase 3 ; chemistry ; genetics ; metabolism ; Genetic Code ; Humans ; Mutation ; Phosphorylation ; Protein Conformation ; Signal Transduction ; genetics ; Structure-Activity Relationship ; Tyrosine ; analogs & derivatives ; chemistry ; genetics

This article was aimed to study immunomodulatory effect of chemical split fractions ofMori Cortex, in order to initially explain effective parts that played a role in immunomodulatory effect ofMori Cortex. The carbon clearance test, serum hemolysin test, E-rosette test, and lymphocyte transformation test were carried out to explore influence of these chemical split fractions ofMori Cortex on immune organs, nonspecific immunity, humoral immunity and cellular immunity. The results showed that in the carbon clearance test, 50% ethanol fraction markedly reduced the thymus index (P<0.01) and the correction indexα (P<0.05). In hemolysin test, the half value hemolysis (HC50) was improved by 30% ethanol fraction and fatty oil fraction (P<0.05). Besides, in the E-rosette test, the E-rosette ration was increased in the 30% ethanol fraction group (P<0.05). In the lymphocyte transformation test, the 30% ethanol fraction can promote the thymus and spleen lymphocytes proliferation (P<0.05 orP<0.01), while the 50% ethanol fraction inhibited the proliferation (P<0.05 orP<0.01). It was concluded that the 30% ethanol fraction can boost both the humoral immunity and cellular immunity; the 50% ethanol fraction can induce the growth of thymus with a suppressive effect on nonspecific immunity and cellular immunity; the fatty oil fraction can improve humoral immunity.

The arginyl-tRNA synthetase (ArgRS) catalyzes the esterification reaction between L-arginine and its cognate tRNA(Arg). Previously reported structures of ArgRS shed considerable light on the tRNA recognition mechanism, while the aspect of amino acid binding in ArgRS remains largely unexplored. Here we report the first crystal structure of E. coli ArgRS (eArgRS) complexed with L-arginine, and a series of mutational studies using isothermal titration calorimetry (ITC). Combined with previously reported work on ArgRS, our results elucidated the structural and functional roles of a series of important residues in the active site, which furthered our understanding of this unique enzyme.
Arginine ; chemistry ; Arginine-tRNA Ligase ; chemistry ; Binding Sites ; Catalytic Domain ; Crystallography, X-Ray ; Escherichia coli ; Ligands ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Conformation ; RNA, Transfer ; chemistry ; Structure-Activity Relationship

Objective:To investigate the difference and characteristics of autoantibodies expression in patients infected by 2019-nCoV with various severity, and explore the associations between expression profile of autoantibodies and prognosis of COVID-19 patients.Methods:This retrospective study was conducted on patients with COVID-19 admitted to Zhongnan Hospital, Wuhan University from January 30, 2020 to March 16, 2020. Data on medical records, expression of autoantibodies including antinuclear antibody profile (ANA), anticardiolipin antibody (ACA), inflammatory factor and other laboratory indexes were collected and analyzed. The age and sex matched disease controls (cases of pulmonary infection unrelated to 2019-nCoV infection and autoimmune disease) and healthy controls (healthy check-up individuals) were also included. Following groups were established, ANA test groups: 72 cases of COVID-19 group (including 17 critical and severe cases, and 55 mild cases), 37 disease controls and 44 healthy controls; ACA test groups: 111 cases of COVID-19 group (including 37 critical and severe cases, and 74 mild cases), 37 disease controls and 40 healthy controls. The difference of positive rate or expression level of autoantibodies among various groups was analyzed, and the difference of inflammatory biomarkers and other parameters were compared between patients with ANA positive results and negative results. The Spearman correlation test was applied to determine the relationship between ACA and other parameters. Kaplan-Meier estimation was used to plot survival curves, the log-rank analysis was utilized to explore the association between antibodies and outcome of COVID-19 patients.Results:The positive rate of antibodies was significantly higher in the COVID-19 group than disease and healthy control groups, the ANA fluorescence: 22.22% (16/72), 5.41% (2/37), 6.82% (3/44); ANA spectrum:26.39% (19/72), 8.11% (3/37), 9.09% (4/44); and ACA:37.84% (42/111), 8.11% (3/37), 5.00% (2/40); all P<0.05. The positive rate of ANA, ACA-IgM and the expression level of ACA-IgM were significantly higher in severe COVID-19 subgroups (critically and severe COVID-19 patients) than in the mild COVID-19 patients (the ANA fluorescence: 47.06% [8/17] vs. 14.55% [8/55], ANA spectrum:66.67% [9/17] vs. 18.18% [10/55], ACA-IgM:30.43% [10/37] vs. 9.46% [7/74]; all P<0.05). There were significant differences in the number of red blood cells, hemoglobin concentration, hematocrit, activated partial thromboplastin time, C-reactive protein, interleukin-6 and serum amyloid A between COVID-19 ANA-positive group and COVID-19 ANA-negative group (all P<0.05). The level of ACA-IgM was positively correlated with white blood cell count ( r=0.354, P<0.001), neutrophil count ( r=0.344, P<0.001), platelet count ( r=0.198, P=0.038), D-Dimer ( r=0.260, P=0.009), glutamic-pyruvic transaminase ( r=0.214, P=0.024), γ-glutamyl transpeptidase ( r=0.283, P=0.003), blood urea nitrogen ( r=0.223, P=0.019), and negatively correlated with superoxide dismutase ( r=-0.228, P=0.020). Survival analysis showed that cumulative survival rate of event-free survival (EFS) was lower in patients with positive ANA/ACA-IgM results than in patients with negative ANA/ACA-IgM results ( P<0.05). Conclusions:ANA and ACA autoantibodies can be detected in COVID-19 patients. The positive rate and the expression level of ANA and ACA increase in proportion with the severity of COVID-19 patients. ANA and ACA-IgM could be used as risk stratification determinants for predicting survival of COVID-19 patients.

Objective:To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology.Methods:In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 μg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification.Results:After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly ( P<0.05), and the release of lactate dehydrogenase increased significantly ( P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance ( P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed ( P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 μg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 ( P<0.05) . Conclusion:Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.

Objective:To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells.Methods:In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 μg/ml Cd, 100 μg/ml CBNPs, 100 μg/ml CBNPs+2 μg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results:There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined ( P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group ( P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression ( P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group ( P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group ( P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene ( P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 ( P<0.05), but the interaction between the two chemicals had no statistical significance ( P>0.05) . Conclusion:CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

Objective:To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology.Methods:In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 μg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification.Results:After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly ( P<0.05), and the release of lactate dehydrogenase increased significantly ( P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance ( P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed ( P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 μg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 ( P<0.05) . Conclusion:Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.