Abstract:The increasing atmospheric carbon dioxide levels have been correlated with global warming. Carbonic anhydrases (CA) are the fastest among the known enzymes to improve carbon capture. The capture of carbon dioxide needs high temperature and alkaline condition, which is necessary for CaCO3 precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential, and polyextremophilic microbes are one of the important sources of these enzymes. The current review focuses on both those isolated by thermophilic organisms from the extreme environments and those obtained by protein engineering techniques, and the industrial application of the immobilized CAs is also briefly addressed. To reduce the greenhouse effect and delay global warming, we think further research efforts should be devoted to broadening the scope of searching for carbonic anhydrase, modifying the technology of protein engineering and developing highly efficient immobilization strategies.

Abstract:As a member of the Sirtuins family in mammals, SIRT7 locates in nucleus and is a highly specific H3K18Ac (acetylated lysine 18 of histone H3) deacetylase. Recent studies showed that SIRT7 could participate in the ribosomal RNA transcription, cell metabolism, cell stress and DNA damage repair through various signaling pathways. In addition, SIRT7 is also closely related with aging, heart disease and fatty liver. In particular, SIRT7 plays important roles in the regulation of initiation and development of various tumors, such as liver cancer, gastric cancer, breast cancer, bladder cancer, colorectal cancer, and head/neck squamous cell carcinoma. This review describes the cellular and molecular functions of SIRT7, and systematically summarizes recent progress of SIRT7 in human disease.

Abstract:Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.

Abstract:A novel protein encoded by the open reading frame 4 (ORF4) was recently discovered in porcine circovirus type 2 (PCV2). However, little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2. In the present study, the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST, yielding recombinant plasmid pCMV-N-Flag-GST-ORF4. The recombinant plasmid was transfected into 293T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE, sequentially. The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry (MS). Finally, five candidate ORF4-interacting proteins, including Serine/threonine-protein phosphatase 6 catalytic subunit, alpha cardiac muscle 1, actin, SEC14-like protein 5 and myosin 9 were identified. These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.

Abstract:To evaluate the immunogenicity of HA globular head domain of H5 subtype influenza virus (H5HA), the gene of H5HA was optimized and the recombinant pPICZaA-H5HA expressing vector was constructed and transfected into Pichia pastoris. The expression of the recombinant H5HA was confirmed by SDS-PAGE and Western blotting and the results demonstrated that the recombinant H5HA (37 kDa) was highly expressed in Pichia pastoris with concentration of 0.2 mg/mL in medium. The recombinant H5HA was concentrated and purified using Ni-NTA affinity chromatography. The immunogenicity of H5HA was evaluated by immunizing eight groups of chicken through intranasal or intramuscular injection with different doses of purified H5HA combined with different adjuvants, respectively. The results showed that the recombinant H5HA could induce high level IgG (HI titer was 1:64 and neutralizing antibody titer was 1:218)?and the optimal?dosage of the recombinant H5HA was 50 μg combined with oil. In addition, intramuscular injection was better than nasal immunization. This study provided a theoretical support for subunit vaccine development.

Abstract:Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates (PHA) family and possesses promising properties including biocompatibility and biodegradability. In this study, we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha, the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli. The polymer content was 83.9% (W/W), and the molar percentage of lactate reached 1.6%. On this basis, in order to accumulate lactate, we reduced the activity of respiratory chain by deleting the ubiX gene, which is involved in the synthesis of coenzyme Q8. Moreover, we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation. With these manipulations, the molar percentage of lactate in the polymer was improved to 14.1%, with an 81.7% (W/W) of polymer content. The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer, and it contributed a new approach to change the content of monomer components in the polymer.

Abstract:Translocation ribonucleic acid (tRNA) is one of the important components in protein synthesis. In order to explore the effect of the changes of tRNAs corresponding to rare codons (rarity tRNAs) on the expression of exogenous genes, the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed. The expression of GFP in P. pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene. The expression amount of the repressed GFP could be increased about 4.9% when tRNAPro CCG gene was cointegrated to the 3′ of the repressed GFP gene through pPIC9K to the genome of P. pastoris GS115. Meanwhile, the expression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNAPro CCG gene to the genome of P. pastoris GS115 through pPIC9K and pFLDα, respectively. Using the same method, NFATc3T-GFP fusion gene and tRNAPro CCG gene were co-expressed in P. pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein. In conclusion, tRNAPro CCG gene has been confirmed to be a kind of rare tRNAs in P. pastoris GS115. Through co-expression of tRNAPro CCG gene and heterologous genes which containing the continuous rare codon CCG, the expression of the repressed heterologous genes could be increased significantly. Furthermore, this co-expression system would contribute to screening and determining the other rare tRNAs.

Abstract:Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.

Abstract:Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH+ (with 6×His-tag at 3? end) and CgDH- (without 6×His-tag at 3? end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH+ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH+ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH+ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH+ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH+ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.

Abstract:The biogenic monoamine 5-hydroxytryptamine (5-HT) is an ancient intracellular signaling molecule widely distributed in all animals with nervous systems, and has been implicated in principal behaviors. Tryptophan hydroxylase (TRH) induces a highly specific catalytic reaction that converts L-tryptophan (tryptophan) to 5-hydroxy-L-tryptophan (5-HTP) that is subsequently used as a substrate by aromatic L-amino acid decarboxylase (DDC) to form 5-HT. Five-HT is an ancient intracellular signaling molecule that is widely distributed in the animal kingdom and has been implicated in regulating the behaviors of animals with nervous systems. However, the role of TRH in Lepidoptera is not well understood. In this study, we cloned 1 667 bp cDNAs of Bombyx mori TRH (BmTRH), which contains a 1 632 bp open reading frame (ORF). Homology analysis revealed that BmTRH shared high amino acid identity with Homo sapiens TPH and Drosophila TRH (DmTRH). The high homology (70%) of BmTRH with DmTRH suggested that BmTRH could have a function similar to DmTRH. Gene expression analysis revealed that BmTRH was mainly expressed in head and central nervous (CNS). Moreover, immunohistochemistry and Western blotting analyses showed that BmTRH was detected only in larval nervous tissues. Taken together, our results indicate that BmTRH could likely function in the regulation of neural activities in B. mori. The transcripts of B. mori decarboxylase (BmDDC) and B. mori phenylalanine hydroxylase (BmPAH) whose proteins had TRH activity, were also expressed in the CNS tissues, indicating that unlike in Drosophila, two distinct mechanisms likely regulate 5-HT synthesis in silkworm.

Abstract:Serine elastic chymotrypsin Pr1 is an enzyme that efficiently degrades insect body wall protein through its connection with the virulence of entomogenous fungi. Therefore, it is important to explore the relationship between the Pr1 protease activity, the Pr1 gene expression and the virulence of different strains of entomogenous fungi. Specific peptide substrate Suc-Ala-Ala-Pro-Phe-pNA and fluorogenic quantitative PCR were used for detecting Pr1 protease activity and Pr1 gene expression, and the slope spray method was used for evaluating the virulence of the fungi on the Myzus persicae. The results indicated that the linear regression equation of the Pr1 protease activity and the virulence of different strains were: y=3.64x+0.62, R2=0.432. It was shown that there is a positive correlation between the Pr1 protease activity and virulence of different strains. Moreover, the result of the multiple linear regression analysis between Pr1 protease activity, Pr1 gene expression and the virulence of different strains was: y=0.236+10.833x1–0.039x2 (x1 represents Pr1 protease activity while x2 represents Pr1 gene expression), R2=0.568, which suggested that the raw data could be represented by a linear fitting equation. The serial correlation coefficient was high (D-W was 2.444), indicating that Pr1 protease activity and Pr1 gene expression have great effect on the virulence of the fungi. Additionally, VIF=12.705, which shows that moderate multiple collinear exists between Pr1 protease activity and Pr1 gene expression. Therefore, Pr1 protease activity and Pr1 gene expression could be recommended as important indicators for strain virulence selection.

Abstract:Isomalto-oligosaccharides (IMO) have good physiochemical properties and excellent physiological functions to make it widely used in food, medicine, feed, cosmetics and other industries. However, the procedures for industrial production of IMO are complicated. Therefore, it is necessary to develop an economical and easy-to-operate method. The genes encoding for β-amylase and α-transglucosidase were fused and co-displayed on the yeast cell surface of Yarrowia lipolytica which can convert liquefied starch to IMO in one step. The highest IMO purity of 75.3% was obtained using the displayed fusion-enzyme at 50 °C. This method showed potential application in IMO production.

Abstract:Cilia and flagella on eukaryotic cells are polarized organelles extending from the surfaces of cells, which participate not only in cell motility, but also in signal transduction and other processes. Structural or functional abnormalities of cilia can cause various human diseases, termed ciliopathies. Bardet-Biedl syndrome (BBS) is a ciliopathic human genetic disorder, and the pathogenesis is that mutated BBS genes result in abnormal cilia function. In order to study the pathogenic genes BBS8, we screened bbs8 mutant in Chlamydomonas reinhardtii and did a lot of physiology and biochemistry experiments. We affirmed that BBS8 protein was a cilia protein and had specific localization in the basal body by immunofluorescence (IF). The bbs8 mutant lost photokinesis, and it was defective in flagella shortening with drug induction. The results of silver staining and mass spectrometric analysis showed aberrant accumulation of flagellar proteins in the mutant flagella. We concluded that the BBS8 protein plays a significant role in flagellar membrane proteins transport, and the BBS8 protein might mediate retrograde transport to exert physiological function in the process.

Abstract:Tet2 (member 2 of the Tet family) plays an important role in DNA demethylation modification, epigenetic regulation, and hematopoiesis. In our previous study, we found that Tet2 knockout mice progressively developed lymphocytic leukemia and myeloid leukemia with aging. However，the role of Tet2 in bone marrow microenvironment is unclear. Here in this study, we found that more Tet2-/- mesenchymal stem cells (MSCs) from bone marrow were in the G2/M cell cycle stages. The division time of Tet2-/- MSCs was shorter than that of the control cells. The growth rate of Tet2-/- MSCs was accelerated. The cobblestone area-forming cells assay (CAFC) showed that Tet2 knockout MSCs supported the expansion of hematopoietic stem cells (HSCs) and the differentiation of HSCs was skewed towards myeloid cells. Through the dot blotting experiment, we found that the total methylation level was increased in Tet2-/- bone marrow cells (BM). We used the methylation-chip to analyze the methylation level of Tet2-/- bone marrow cells and found that the level of methylation was increased in the transcriptional starting area (TSS), exons (EXONS) and 3? untranslated region (3? UTR). Moreover, we found that the cytokines secreted by Tet2-/- MSCs, such as IL-8 and IL-18, were decreased. While the expressions of GM-CSF and CCL-3, which supported hematopoietic stem cells to differentiate to myeloid cells, were increased in Tet2-/- MSCs. Our results demonstrated that Tet2 regulates MSCs to support hematopoiesis.

Abstract:Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10–9 mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 103/mL and 100% when the sperm cells number was equal to or more than 104/mL. When the number of sperm cells was 103/mL, 104/mL and 105/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

Abstract:Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe2+ had an activating effect on the ribonuclease activities of two isoforms while Ca2+, Mg2+, Zn2+, Mn2+, Ag+, Cu2+, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.