[Characteristic Analysis of nirS Denitrifying Bacterial Community in Lijiahe Reservoir During Stratification].

To explore the composition of the nirS denitrifying bacterial community during stratification in spring(March to May) in a drinking water reservoir and its relationship with water quality, the water quality and relative abundance and structure of the denitrifying bacterial community were analyzed using in-situ monitoring coupled with Illumina high-throughput sequencing technology in the Lijiahe Reservoir. The results showed that:① through high-throughput sequencing, 4 phyla and 13 genera were identified. The dominant bacterial phylum was Proteobacteria, and its relative abundance was between 52.5% and 70.6%. The overall trend of the relative abundance of Proteobacteria decreased on the time scale (P<0.05), and its relative abundance in the surface and middle layers was higher than that of the bottom layer on the spatial scale (P<0.05). There was no difference in the proportion of Proteobacteria between the surface and middle layers (P>0.05), and the abundance of its bottom layer was relatively stable; eight genera of bacteria with denitrification function were identified, among which the dominant bacterial genera (relative abundance>1%) were Dechloromonas and Pseudomonas. The relative abundance of Dechloromonas showed a trend of first decreasing and then increasing on the time scale, whereas the relative abundance of Pseudomonas showed a trend of increasing first and then decreasing on the time scale. There were no differences on the spatial scale between these two genera (P>0.05); the changes in bacterial diversity and abundance were basically similar, with a trend of first increasing and then decreasing on the time scale. The highest diversity and abundance of the bacterial community gradually increased with increasing depth on the spatial scale. ② ρ(TN) of the reservoir during stratification was 2.35-2.91 mg·L-1, and the nitrogen pollution was more serious. In March and April, ρ(TN) on the vertical scale was basically similar and showed a decreasing trend. In May, the content of total nitrogen was higher than that in March and April, and the highest value of total nitrogen content occurred in the surface layer. ③ Redundancy analysis showed that water temperature, dissolved oxygen, nitrate, and ammonia nitrogen were the main driving factors, and ammonia nitrogen showed a significantly negative correlation with Dechlormonas. In summary, the study of nirS-type denitrification communities and related influencing factors will contribute to analyzing the characteristics of denitrifying bacterial community changes in a micro-polluted drinking water reservoir and provide a theoretical research basis for the biological remediation of nitrogen pollution in such reservoirs in the future.

[Influence of Storm Runoff on the Spectral Characteristics of Dissolved Organic Matter (DOM) in a Drinking Water Reservoir During the Flood Season].

To explore the influence of storm runoff on reservoir organic matter during the flood season, the Lijiahe Reservoir was selected to analyze variations in the content and components of dissolved organic matter (DOM) during four periods (before runoff, flood peak period, 1 week after runoff, and 6 weeks after runoff) using three-dimensional fluorescence spectroscopy parallel factor analysis (EEMs-PARAFAC) and ultraviolet-visible (UV-Vis) spectra. The results showed that:① the turbidity and DOC content of the reservoir increased significantly during the flood peak period (P<0.01) and gradually decreased thereafter; ② the UV-Vis spectrum characteristics showed that a(254) and a(355) were significantly increased in the flood peak period (P<0.01) while E2/E3 and E3/E4 were significantly decreased (P<0.01), indicating that the concentration, relative molecular weight, and degree of DOM humification in the reservoir were increased by storm runoff; ③ four DOM components were identified as terrestrial humus (C1 and C2), microbial humus (C3), and a tryptophan-like component (C4). The fluorescence intensity of the C1-C3 components increased significantly during the flood peak period (P<0.05), indicating that the increase in the DOM humic-like component was caused by the storm runoff. At the same time, a decrease in the fluorescence intensity of the C1-C4 components was observed after the flood peak period, indicating that DOM continuously settled and degraded after runoff; and ④ Pearson's correlation analyses showed that DOM fluorescence intensity and turbidity were significantly correlated (r>0.467, P<0.05), indicating that the observed decrease in DOM content was related to the sedimentation of suspended solids. A principal component analysis (PCA) showed that the water quality in the reservoir reflected the observed characteristics during the different runoff periods. Overall, this study reveals the effects of the storm runoff on DOM content and its components over the short and long term, providing scientific support for the management of drinking water quality.

Mechanism of two novel human GJC3 missense mutations in causing non-syndromic hearing loss.

Connexins (CXs), as a component of gap junction channel, are homologous four transmembrane-domain proteins, with numerous studies confirming their auditory functions. Among a cohort of patients having incurred non-syndromic hearing loss, we identified two novel missense mutations, p.R15G and p.L23H, in the GJC3 gene encoding CX30.2/CX31.3, as causally related to hearing loss in previous study. However, the functional alteration of CX30.2/CX31.3 caused by the mutant GJC3 gene remains unknown. In this study, we compared the intracellular distribution of mutant CX30.2/CX31.3 (p.R15G and p.L23H) with the wild-type (WT) protein in HeLa cells and the effect of the mutant protein had on those cells. Analytical results indicated that p.R15G and p.L23H mutant exhibited continuous staining along apposed cell membranes in the fluorescent localization assay, which is the same with the WT. Moreover, ATP release (hemichannel function) is less in HeLa cells carrying mutant GJC3 genes than those of WT expressing cells. We believe that although p.R15G and p.L23H mutants do not decrease the trafficking of CX proteins, mutations in GJC3 genes result in a loss of hemichannel function of CX30.2/CX31.3 protein, possibly causing hearing loss. Results of this study provide a novel molecular explanation for the role of GJC3 in hearing loss.

Suppression of the invasion and migration of cancer cells by SERPINB family genes and their derived peptides.

Apart from SERPINB2 and SERPINB5, the roles of the remaining 13 members of the human SERPINB family in cancer metastasis are still unknown. In the present study, we demonstrated that most of these genes are differentially expressed in tumor tissues compared to matched normal tissues from lung or breast cancer patients. Overexpression of each SERPINB gene effectively suppressed the invasiveness and motility of malignant cancer cells. Among all of the genes, the SERPINB1, SERPINB5 and SERPINB7 genes were more potent, and the inhibitory effect was further enhanced by co-expression of any two of them. In addition, single treatment of the synthetic peptides corresponding to the P5-P5' sequences of the reactive center loop (RCL) of SERPINB1, SERPINB5 or SERPINB7 markedly suppressed the invasive and migratory properties of the cancer cells in a dose-dependent manner. More significantly, combination treatment of these peptides in cancer cells further improved the suppressive effect by 20-40%. Here, we determined the expression of all SERPINB family members in lung and breast cancer patients, and identified those members with potent inhibitory ability toward invasion and migration, and designed RCL-derived peptides to suppress the malignancy of cancer cells. Forced re-expression of these anti-invasive SERPINB genes or application of the SERPINB RCL-peptides may provide a reasonable strategy against lethal cancer metastasis.

Human connexin30.2/31.3 (GJC3) does not form functional gap junction channels but causes enhanced ATP release in HeLa cells.

Gap junctional intercellular communication has numerous functions, each of which meets the particular needs of organs, tissues, or groups of cells. Connexins (CXs) are homologous four-transmembrane-domain proteins that are the major components of gap junctions. CX30.2/CX31.3 (GJC3) is a relatively new member of the CX protein family. Until now, however, the functional characteristics of CX30.2/CX31.3 have been unclear. To elucidate the properties of CX30.2/CX31.3 channels, their subcellular localization in HeLa cells, their effectiveness in dye transfer, and function on channels were investigated. In the immunofluorescent assay, cells that express CX30.2/CX31.3-GFP exhibited continuous fluorescence along the apposed cell membranes, rather than punctated fluorescence in contacting membranes between two cells. Surprisingly, dyes that can be capable of being permeated by CX26 GJ, according to a scrape loading dye transfer assay in previous studies, are impermeated by CX30.2/CX31.3 GJ, suggesting a difference between the characteristics of CX30.2/CX31.3 GJ and CX26 GJ. Furthermore, a significant amount of ATP was released from the HeLa cells that stably expressed CX30.2/CX31.3, in a medium with low calcium ion concentration, suggesting a hemichannel-based function for CX30.2/CX31.3. Based on these findings, we suggest that CX30.2/CX31.3 shares functional properties with pannexin (hemi) channels rather than gap junction channels of other CXs.

Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor.

Human serine proteinase inhibitor Kazal-type 2 (SPINK2) functions as a trypsin/acrosin inhibitor and is synthesized mainly in the testis and seminal vesicle where its activity is engaged in fertility. The SPINK2 protein contains a typical Kazal domain composed by six cysteine residues forming three disulfide bridges. The expression of SPINK2 is closely related to cancer such as lymphomas, in that a high transcript level of SPINK2 in patients with primary cutaneous follicle center cell lymphomas have better prognosis with lower mortality. To clarify the role of SPINK2 in cancer, we performed quantitative real-time PCR and showed that the expression level of SPINK2 is significantly elevated in most leukemia cell lines except B-lymphoblast TK-6 cells. The molecular function and structural features of SPINK2 were also investigated by employing the recombinant active and mutant inactive SPINK2 proteins to determine its key P2-P2' (Pro(23)-Arg(24)-His(25)-Phe(26)) active site. The inhibition assay results demonstrated that Arg(24) at the P1 site is crucial for the specificity of SPINK2 on target enzyme. Although His(25) at the P1' and Phe(26) at the P2' residues are also involved in trypsin-SPINK2 interaction, Pro(23) at the P2 site may not be directly participated in interacting with trypsin. In addition, we determined the 3D solution structure of SPINK2 and used this structure to predict the SPINK2-proteinase complex structure and binding properties. These studies not only provide critical information about the structural properties and biophysical features of the SPINK2 proteinase inhibitor, but also suggest its important role in tumor progression and response to treatment.

DBBF modified human placenta hemoglobin through the alpha chains.

Hemoglobin-based blood substitute is widely studied. As the starting materials, hemoglobin (Hb) is mainly supplied from outdated human blood and animal sources. But there are many disadvantages. As the alternative source, human placenta hemoglobin (PHb) has its own advantages. We take the lead in being occupied in the research of blood substitutes based on the placenta blood. Purified PHb readily undergoes dissociation to form two dimers that are easily filtered by the kidneys and have high 02 affinity (low P50), failing to transport 02 to tissues. As a first step, to enhance PHb stability and P50, we first adopted DBBF to modify deoxyPHb leading to DBBF-alpha PHb. According to detection, we knew that the PHb stability was improved, 02 affinity of the PHb solution was reduced, DBBF-alpha PHb had the appropriate capacity of oxygen-carrying and oxygen-unloading, and DBBF-alpha PHb kept the biological activity well. On the whole DBBF-alpha PHb could be considered as a first step toward the synthesis of a potential blood substitute.

A method for purification and viral inactivation of human placenta hemoglobin.

For pilot-scale manufacturing of hemoglobin-based oxygen carrying drugs, we should get highly pure and viral inactivated hemoglobin (Hb) at high recovery. In our method, placenta hemoglobin (PHb) solutions were purified by heating in the presence of reducing agent and deoxygenating conditions so that heat-sensitive proteins were selectively precipitated and virus was inactivated. The optimum preparative condition resulted in highly purified PHb solution (>99% pure) with approximate 90% recovery and less than 2% of MetHb content, maintained oxygen carrying capacity, residual phospholipids less than 1 ppm, free of endotoxin, bacteria, type A&B antigens and virus. Finally, we compared the efficacy of blood exchange on rat with poly-PHb and poly-Hb from adult blood. The results showed no significant difference between two products. Therefore, the placenta Hb obtained from this method could be supplied as materials for oxygen carrying drugs.

IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha.

IL-10 is an immunosuppressive cytokine in the immune system. It was in clinical trial as an anti-inflammatory therapy for inflammatory bowel disease and various autoimmune diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiation-associated gene (MDA-7, IL-24), and AK155 (IL-26). Despite a partial homology in their amino acid sequences, they are dissimilar in their biologic functions. Little is known about the biologic function and gene regulation of IL-19. To understand the gene regulation of human IL-19, we identified a human IL-19 genomic clone and analyzed its promoter region. Five fusion genes containing different regions upstream of exon 1 linked to a luciferase reporter gene were expressed in the canine kidney epithelial-like Madin-Darby canine kidney cells. A fusion gene containing 394 bp showed luciferase activity 7- to 8-fold higher than the negative control of the promoterless fusion gene. We also isolated a full-length mouse cDNA clone. Mouse IL-19 shared 71% amino acid identity with human IL-19. Treatment of monocytes with mouse IL-19 induced the production of IL-6 and TNF-alpha. It also induced mouse monocyte apoptosis and the production of reactive oxygen species. Taken together, our results indicate that mouse IL-19 may play some important roles in inflammatory responses because it up-regulates IL-6 and TNF-alpha and induces apoptosis.