Mechanism of vanadium(IV) resistance of the strains isolated from a vanadium titanomagnetite mining region.

Microbial treatment for vanadium contamination of soils is a favorable and environment-friendly method. However, information of the resistant mechanism of the strains in soils to vanadium, especially to tetravalent vanadium [vanadium(IV)], is still limited. Herein, potential of the vanadium(IV) biosorption and biotransformation of the strains (4K1, 4K2, 4K3 and 4K4) which were capable of tolerating vanadium(IV) was determined. For biosorption, the bioadsorption and the bioabsorption of vanadium(IV) occur on the bacterial cell wall and within the cell, respectively, were taken into consideration. Comparison of the vanadium(IV) adsorbed on the bacterial cell walls and remained in the cells after sorption indicated the major bacterial vanadium(IV) sorption role of the bioadsorption which was at least one order of magnitude higher than the bioabsorption amount. Isotherm study using various isotherm models revealed a monolayer and a multilayer vanadium(IV) biosorption by 4K2 and the others (4K1, 4K3 and 4K4), respectively. Higher biosorption was observed in acidic conditions than in alkaline conditions, and the maximum biosorption was 2.41, 9.35, 7.76 and 8.44 mg g-1 observed at pH 6 for 4K1, at pH 3 for 4K2, and at pH 4 for 4K3 and 4K4, respectively. At the present experimental range of the initial vanadium(IV) concentration, optimal biosorption capacity of the bacteria was observed at the vanadium(IV) level of 100-250 mg L-1. Different biotransformation level of vanadium(IV) in soils by the stains was observed during a 28-d pot incubation of the soils mixed with the strains, which can be attributed to the discrepancy of both soil properties and bacterial species. Present study can help to fill up the gaps of the insufficient knowledge of the vanadium(IV) resistant mechanism of the strains in soils.

Spleen contributes to restraint stress induced hepatocellular carcinoma progression.

The spleen is the largest secondary immune organ and plays a critical role in the progression of tumor. Psychological stress promotes tumor progression through inhibiting antitumor immune. However, the role of spleen in tumor progression induced by stress is unclear. Here, we showed that restraint stress promoted tumor growth, increased the percentage of CD11b+Gr-1+ MDSC while decreased the percentages of CD3-NK1.1+ NK and CD3+NK1.1+ NKT in the tumor tissues. Restraint stress decreased the percentages of CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes while increased the percentage of CD11b+Gr-1+ MDSC in the blood of tumor-bearing mice. Restraint stress increased the percentages of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, CD4+PD1+ T lymphocytes and CD8+PD1+ T lymphocytes while decreased the percentage of CD11b+Gr-1+ MDSC in the spleen of tumor-bearing mice. Interestingly, splenectomy inhibited tumor growth and attenuated the changes of CD3+CD4+ T lymphocytes, CD3+CD8+ T lymphocytes, and CD11b+Gr-1+ MDSC in blood induced by chronic restraint stress. Finally, splenectomy blocked the increases of CD11b+Gr-1+ MDSC but did not attenuate the decreases of CD3-NK1.1+ NK and CD3+NK1.1+ NKT in tumor tissue induced by chronic stress. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth and suppresses the antitumor immunity of tumor-bearing mice. Splenectomy could inhibit tumor growth and partly block the decrease of antitumor immune activity induced by stress.

Chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells through activating ß-adrenergic signaling.

Psychological stress promotes tumor progression and has a large impact on the immune system, particularly the spleen. The spleen plays an important role in tumor behavior. However, the role and mechanism of the spleen in hepatocellular carcinoma progression induced by stress is unclear. Here, we showed that the spleen plays a critical role in hepatocellular carcinoma growth induced by restraint stress. Our results demonstrated that restraint stress promoted hepatocellular carcinoma growth, changed the spleen structure, and redistributed splenic myeloid cells to tumor tissues. Interestingly, we found that splenectomy could inhibit hepatocellular carcinoma growth and prevent increases in myeloid cells and macrophages in tumor tissues in stressed mice. Restraint stress significantly elevated the concentration of norepinephrine in the spleen, serum and tumor tissues. Meanwhile, propranolol, an inhibitor of ß-adrenergic signaling, could inhibit hepatocellular carcinoma growth and prevent the redistribution of splenic myeloid cells induced by restraint stress, suggesting that restraint stress promotes hepatocellular carcinoma growth and redistributes splenic myeloid cells through ß-adrenergic signaling. Mechanistic studies revealed that restraint stress upregulated the expressions of CXCL2/CXCL3 in tumor tissues and changed the expression of CXCR2 in myeloid cells. SB225002, an inhibitor of CXCR2, could prevent the recruitment of myeloid cells in tumor tissues and inhibit tumor growth in stressed mice. Together, these data indicate that chronic restraint stress promotes hepatocellular carcinoma growth by mobilizing splenic myeloid cells to tumor tissues via activating ß-adrenergic signaling. The CXCR2-CXCL2/CXCL3 axis contributed to the recruitment of myeloid cells in tumor tissues induced by restraint stress.

Correlation analysis between coronary artery lesion and risk factors of coronary heart disease / 心血管康复医学杂志

Objective :To explore correlation between severity of coronary artery lesion and risk factors of coronary heart disease (CHD) in CHD patients .Methods :The 452 patients ,who had clinical complete data and underwent coronary angiography in our hospital from Jan 2012 to Jan 2013 were retrospectively analyzed ,including 64 non‐CHD cases (non‐CHD group) and 388 CHD cases (CHD group).Correlation among number ,length ,size ,stenotic degree of diseased coronary artery vessels and CHD risk factors were explored .Results : (1) Compared with non‐CHD group ,there were significant rise in percentages of men ,hypertension (EH) and diabetes mellitus (DM) in CHD group , P＜0.05 or ＜0.01 ; (2) In CHD group ,compared with single vessel coronary disease group ,there were significant rise in age ,percentages of EH ,DM and patients with low HDL‐C in multi‐vessel coronary disease group , P＜0. 05 or ＜ 0. 01 ;compared with large vessel lesion patients ,there were significant rise in age and DM percentage ,and significant reduction in percentage of smokers in small vessel lesion patients , P＜ 0.01 all ;com‐pared with stenosis ＜90% group ,there was significant reduction in LVEF and significant rise in percentage of pa‐tients with elevated LDL‐C level in stenosis≥90% group , P＜0.05 or ＜0. 01 ;(3) Multivariate linear regression a‐nalysis and linear correlation analysis indicated that size of diseased coronary artery vessels was significant inversely correlated with age ( r＝ －0. 181 , P＝0.001) ;severity of coronary disease was significant inversely correlated with LVEF ( r＝ －0.213 , P＝0.001) ,and significant positively correlated with heart rate ( r＝0. 133 , P＝0.009) and LDL‐C level ( r＝0. 141 , P＝0.001).Conclusion :Risk factors for CHD , including aging , smoking , complicated DM , EH , hyperlipidemia , tachycardia and left ventricular systolic dysfunction etc ., will further aggravate coro‐ nary artery lesion .

Spleen contributes to restraint stress induced changes in blood leukocytes distribution.

Psychological stress has great impacts on the immune system, particularly the leukocytes distribution. Although the impacts of acute stress on blood leukocytes distribution are well studied, however, it remains unclear how chronic stress affects leukocytes distribution in peripheral circulation. Furthermore, there is no report about the role of spleen in the blood leukocytes distribution induced by stress. Here we show that spleen contributes to the alteration of restraint stress induced blood leukocytes distribution. Our data confirmed that restraint stress induced anxiety-like behavior in mice. Furthermore, we found that restraint stress decreased the CD4/CD8 ratio and elevated the percentages of natural killer cells, monocytes and polymorphonuclear myeloid-derived suppressor cell. We demonstrated that activation of hypothalamic-pituitary-adrenal axis (HPA) and sympathetic nervous system (SNS) contributes to restraint stress induced alteration of blood leukocyte distribution. Interestingly, we found that splenectomy could reverse the change of CD4/CD8 ratio induced by restraint stress. Together, our findings suggest that activation of HPA axis and SNS was responsible for the blood leukocyte subsets changes induced by restraint stress. Spleen, at least in part, contributed to the alteration in peripheral circulation induced by restraint stress.

Efficient expression of recombinant human heavy chain ferritin (FTH1) with modified peptides.

Human heavy chain ferritin (FTH1) can self-assemble into a diameter of 12-nm spherical cage with an interior cavity of 8 nm in diameter. FTH1 has great potential as a nanocage in molecular imaging and drug delivery. Different peptides have been fused with FTH1 for targeting delivery; however, the expression of FTH1 modified with peptides in soluble form is not equivalent to natural FTH1. As shown in recent study, a novel scaffold protein --thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx)--exhibits a superior solubilization capacity and thermal stability [19]. Here we report a new construct (FTH1-PfTrx-His) that can be easily expressed and purified in Escherichia coli. Of note, different peptides inserted into FTH1-PfTrx-His did not influence the expression of proteins. Finally, the doxorubicin packaging ability of FTH1-PfTrx-His is comparable to natural FTH1. Our results showed that FTH1-PfTrx-His had a potential role as a novel peptide-modified ferritin carrier for drugs or imaging probes.

[Analysis of Prokaryotic Community Structure in River Waters of the Ningbo Sanjiang Mouth].

The prokaryotic community structure in river waters of the Ningbo Sanjiang Mouth was analyzed for the first time using 16S rRNA gene based-Illumina Miseq high-throughput sequencing. A total of 215 504 high-quality sequences were obtained, and the results of alpha-diversity analysis revealed that Yongjiang River Watershed (YRW) harbored high diversity and richness of prokaryotic communities. Taxonomic assignment analysis indicated that ß-Proteobacterium, Actinobacteria and Bacteroidetes dominated in the river water of YRW, and accounted for 78. 88% of the total prokaryotic communities. Hydrological condition may play an important role in influencing the composition and structure of YRW prokaryotic community. In addition, several kinds of sewer- and fecal-pollution indicator bacterial groups were observed in this area with the highest abundance of pollution indicator bacteria occurring in the water sample of Yuyao River, implying that the Yuyao River might have a high potential risk of sewer- and fecal-pollution. Moreover, a total of 76 species and 18 subspecies of potential pathogenic bacteria, which accounted for 2. 19% and 0. 40% of total sequences respectively, were identified using BLASTN analysis with a local pathogenic bacteria database. Overall, this study provided an important basic data for shedding light on the structure and ecological function of YRW prokaryotic community.

[Reductive Dechlorination of Trichloroethylene by Benzoate-Enriched Anaerobic Cultures].

Gas chromatography was used to monitor the reductive dechlorination of trichloroethylene (TCE) by anaerobic enrichment cultures with benzoate as the sole carbon source. The 454 pyrosequencing technique was used to investigate the microbial community and the real-time quantitative PCR was used to quantify the gene copies of Dehalococcoides spp. (DHC). The results showed that TCE was dechlorinated to vinyl chloride along with the formation of methane in 94 days. The anaerobic enrichment cultures exhibited a high diversity, which were classified into 16 phyla, 33 classes, 52 orders, 88 families and 129 genera, while 51.2% of them belonged to unclassified group, which inferred that there were a large portion of bacteria with unknown functional in this system. Degradation of TCE was accomplished by reductive dechlorinating and other functional populations, and the DHC which carried tceA gene could be the dominant reductive dechlorinating populations in the system.

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

[Preparation and property study of the recombinant Camel anti-SpaA-N single domain antibodies].

AIM: In order to obtain single domain antibody against surface protective antigen A (SpaA)of Erysipelothrix rhusiopathiae. METHODS: The SpaA-N recombinant protein was used to screen binders from Bactrian camel VHH phage display library. After sequencing, the interested VHH gene fragments were subcloned into pET-30a vector to overexpress the protein in E.coli BL21. The binding specificity of the recombinant VHH with SpaA-N was determined by Western blotting. The thermal stability of single-domain antibody was evaluated by ELISA. RESULTS: By enrichment of screening, 2 clones were selected. Recombinant single domain antibodies purified by Ni-ion affinity chromatography showed a single band at M(r); 29 000, 23 000 on SDS-PAGE. ELISA results showed that VHH can bind its antigen specifically. After thermal denaturation, VHH can restore the antigen binding ability after refolding. Western blotting results showed that the recombinant VHH specific bind surface protective antigen of Erysipelothrix rhusiopathiae at M(r); 66 000. Two VHH single domain antibodies with high thermal stability and good antigen binding specificity were identified by screening Bactrian camel VHH phage display library. CONCLUSION: Two single domain antibodies that specifically aggulated SpaA-N is obtained, which provide the basis for further study in the immune role of single domain antibody against Erysipelothrix rhusiopathiae infection.

[Enhancement of antibodies to protective domain of surface protective antigen A of Erysipelothrix rhusiopathiae by DNA immunization with plasmids expressing spaA-chimeras].

AIM: DNA vaccines expressing protective domain of surface protective antigen A(spaA)of Erysipelothrix rhusiopathiae have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in murine models. METHODS: This paper report using a DNA vaccine expressing a fusion of the spaA protein and various elements, such as a secretion leader sequence from the highly expressed human gene encoding alpha1-antitrypsin (AAT), a highly soluble and stably folded domain from the rat cartilage oligomerization matrix protein (COMP), and three copies of the complement component, C3d3, to enhance the titers of neutralizing spaA-specific antibody. RESULTS: Analysis of titers of the antibody raised in vaccinated mice at different time points indicated that immunizations with the DNA expressing pcDNA3-AAT-COMP-spaAN-3C3d((pcD-ACSC)) had higher titers than pcDNA3-spaA(N)(pcD-S) at weeks 4. Furthermore, the immune protective efficacy of the spaA-chimeras was demonstrated by lethal challenge with a virulent homologous strain 1249 against immunized mice. CONCLUSION: These results suggest that using a plasmid vector containing a strong heterologous signal sequence that mediate efficient antigen secretion in vivo and a fused piece of sequence improving antigens solubility, as well as C3d3, genetic adjuvant, could enhance the antibody responses level. This approach might be an efficient way to improve the antibody level of spaA(N) DNA vaccination.

[Human esophageal carcinoma antigens screened by serologic analysis of recombinant cDNA expression libraries (SEREX)].

BACKGROUND & OBJECTIVE: In malignant transformation, mutant gene products and dysregulated proteins can become tumor antigens and activate immunoreactions. Therefore, auto-antibodies exist in sera of cancer patients. Serologic analysis of recombinant cDNA expression libraries (SEREX) using autologous and allogenic patient sera provides a powerful approach to identify tumor antigens. This study was to identify esophageal cancer antigens with SEREX for serologic diagnosis, gene therapy, and immune therapy. METHODS: Expression library of cDNA from esophageal squamous cell carcinoma was constructed. SEREX screened out 21 positive clones from the 1.6x10(6) clones in the established library. The 21 positive clones were subcloned to monoclonality and submitted to in vivo excision of pBluescript phagemids. The nucleotide sequences of cDNA inserts were analyzed with DNASIS and BLAST software on EMBL and GenBank. According to the bioinformatics analyses, serologic immunoreactions of 4 colons in 10 samples of esophageal cancer serum and 10 samples of normal control serum were further detected by SADA. RESULTS: Of the 21 positive clones, 4 had no homology to any known genes, 17 were known fragments which were defined as antigens of esophageal cancer for the first time. The serologic immunoreaction rates of 4 selected antigens, including Ribosomal protein S4, and so on, were 40%, 60%, 70%, and 30%, respectively, in cancer sera, and 0%, 10%, 20%, and 20%, respectively, in normal sera. CONCLUSIONS: Antigens, such as Ribosomal protein S4, are frequently involved in serologic immunoreactions of esophageal cancer. The 21 antigens identified by the present study can be used as potential targets for gene therapy and serologic biomarkers of esophageal cancer.