Abstract:Like Yellow fever virus, Dengue virus, Japanese encephalitis virus and West Nile virus, Zika virus is also a mosquito-borne flavivirus. Since it was isolated in 1947, there has been little concern over Zika virus due to its limited distribution and mild symptoms. In recent years, especially since 2015, Zika virus has become a global concern because of its outbreak in Brazil and associated microcephaly. Vaccines against Zika virus, regarded as the effective measures to control Zika fever epidemic, are being developed in nearly thirty institutions worldwide. In this paper, biology, epidemiology and clinical features of Zika virus were reviewed along with current research and development of different types of Zika vaccines. In addition, several other flavivirus vaccines approved or in clinical trials were briefly introduced, to provide valuable reference for Zika vaccines researchers.

Abstract:Cofactor balance plays an important role in producing enzymes, pharmaceuticals and chemicals. To meet the demand of industrial production, microbes should maintain a maximal carbon flux towards target metabolites without fluctuations in cofactor. We reviewed the physiological function of cofactor and discussed detailed strategies to manipulate cofactor balance through biochemical engineering and metabolic engineering. Furthermore, we indicated future research needs to further regulate cofactor balance.

Abstract:Cathelicidins play critical roles in mammalian innate immune defense against invasive bacterial infection. In addition to their broad-spectrum bactericidal effect, cathelicidins are interesting peptide-based drug templates because they have multiple functions including anti-inflammatory, wound healing, and angiogenesis promotion. This article summarizes the aim and method of cathelicidin molecular designs. Residue mutation, fragment assembly, chemical modification, and construction of conjugates and dimers are usually used to increase the biological activities. Addition or deletion of certain residues, disruption of leucine zipper and phenylalanine zipper are used to reduce the hemolysis and cytotoxicity. By substituting L-amino acids with D-amino acids, circular constructions and immobilization, cathelicidins’ in vitro and in vivo stability could be greatly enhanced, especially their proteinase resistance.

Abstract:IL-6 is an important cytokine that plays an important role in the pathogenesis of rheumatoid arthritis. We summarized the clinical efficacy and safety of tocilizumab, the IL-6 receptor monoclonal antibody in rheumatoid arthritis, and compared tocilizumab with TNF-α blocking mAbs. The efficiency of tocilizuamb is equivalent to that of TNF-α blockers, and each of the drugs has its advantages and disadvantages. We also summarized the clinical trials of the mAbs blocking IL-6 pathway in development. According to the results of recent studies by several research teams including our research group, IL-6 is another key target for the treatment of rheumatoid arthritis after TNF-α. The listing of the IL-6 blockers provides more choices for personalized treatment of rheumatoid arthritis in the future.

Abstract:To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10–9, 1.61×10–9, and 2.36×10–9, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1–40, 80–112, and 40–80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.

Abstract:Biosynthesis of vitamin B12 (VB12) requires the methylation at positions C-2 and C-7 of the precursor uroporphyrinogen Ⅲ (urogen Ⅲ) to precorrin-2 by S-adenosyl-L-methionine uroporphyrinogen Ⅲ methyltransferase (SUMT), which is a potential bottleneck step. Most of SUMTs are inhibited by urogenⅢand by-product S-adenosyl-L-homocysteine (SAH). In order to mine an SUMT that lacks such an inhibitory property allowing greater flux through the VB12 biosynthetic pathway, we cloned two SUMT genes (RCcobA1, RCcobA2) from Rhodobacter capsulatus SB1003 and expressed them in Escherichia coli BL21 (DE3). Thereafter, the two enzymes were purified and their specific activity of 27.3 U/mg, 68.9 U/mg were determined respectively. The latter was 2.4 times higher than PDcobA (27.9 U/mg) from Pseudomonas denitrifican. Additionally, RCcobA2 could tolerate over 70 μmol/L of urogen Ⅲ, which has never been reported before. Hence, RCcobA2 can be used as an efficient enzyme to regulate the VB12 metabolic pathway and enhance VB12 production in industrial strains.

Abstract:Biomass is an important parameter reflecting the fermentation dynamics. Real-time monitoring of biomass can be used to control and optimize a fermentation process. To overcome the deficiencies of measurement delay and manual errors from offline measurement, we designed an experimental platform for online monitoring the biomass during a 1,3-propanediol fermentation process, based on using the fourier-transformed near-infrared (FT-NIR) spectra analysis. By pre-processing the real-time sampled spectra and analyzing the sensitive spectra bands, a partial least-squares algorithm was proposed to establish a dynamic prediction model for the biomass change during a 1,3-propanediol fermentation process. The fermentation processes with substrate glycerol concentrations of 60 g/L and 40 g/L were used as the external validation experiments. The root mean square error of prediction (RMSEP) obtained by analyzing experimental data was 0.341 6 and 0.274 3, respectively. These results showed that the established model gave good prediction and could be effectively used for on-line monitoring the biomass during a 1,3-propanediol fermentation process.

Abstract:The lignocellulolytic filamentous fungus Neurospora crassa is able to assimilate various mono- and oligo-saccharides. However, more than half of predicted sugar transporters in the genome are still waiting for functional elucidation. In this study, system analysis of substrate spectra of predicted sugar transporters in N. crassa was performed at genome-wide level. NCU01868 and NCU08152 have the capability of uptaking various hexose, which are named as NcHXT-1 and NcHXT-2 respectively. Their transport activities for glucose were further confirmed by fluorescence resonance energy transfer analysis. Over-expression of either NcHXT-1 or NcHXT-2 in the null-hexose-transporter yeast EBY.VW4000 restored the growth and ethanol fermentation under submerged fermentation with glucose, galactose, or mannose as the sole carbon source. NcHXT-1/-2 homologues were found in a variety of cellulolytic fungi. Functional identification of two filamentous fungal-conserved hexose transporters NcHXT-1/-2 via genome scanning would represent novel targets for ongoing efforts in engineering cellulolytic fungi and hexose fermentation in yeast.

Abstract:Aminopeptidase N (APN) belonging to zinc-dependent metalloproteinase, not only catalyzes protein proteolytic process, but also is involved in the pathogenic process as the receptor of pathogenic toxin. In Bombyx mori, APN gene family consists of 16 members, of which BmAPN4 binds trypsin-activated parasporal crystal (PC) toxin isolated from Bacillus bombysepticus (Bb). In order to verify whether or not other APNs interact with PC toxin during the pathogenesis of Bb, we cloned BmAPN5, a member of aminopeptidase family, from the silkworm midgut. The full length of BmAPN5 is 3313 bp, encoding 953 amino acids, containing a zinc peptidase_M1 and ERAP1_C domains. A recombinant GST-BmAPN5 was purified by a prokaryotic expression system. Far-Western blotting, co-immunoprecipitation and ELISA. Binding saturation assays demonstrated that PC after activated by trypsin could be bound by BmAPN5. Additionally, cytotoxic activity of trypsin-activated PC in Sf9 cells transfected with BmAPN5 showed that cells exhibited dramatic cytological changes, including swelling and lysis, revealing BmAPN5 serves as a functional receptor that participates in Bb and PC pathogenicity. These provide some clues for further exploring the pathogenesis relationships of Bb and host.

Abstract:Molting fluid, a liquid between the old epidermis and new epidermis, plays an important role in the process of ecdysis and metamorphosis for insect. In order to explore the function of molting fluid, we used two-dimensional electrophoresis to study the molting fluid during the prepupal stage and pre-eclosion stage. More than 200 protein spots were found in the molting fluid of the 2 stages, which distributed in the 4?9 of pI and 10?180 kDa of molecular weight. We selected 42 spots to be analyzed by the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF) from the molting fluid of pre-eclosion stage, of which 34 proteins were identified successfully, including apolipoprotein, protease and protease inhibitors, chitin-binding protein and protein involved in immunity. Some of the proteins demonstrated differential expression between the two stages of metamorphosis. In order to validate the result from proteomics analysis, we studied expression of the apolipoprotein D by Q-PCR from the developmental stages. The results showed that the gene encoding apolipoprotein D had the high expression from the 1st day to the 4th day of the pupa stage, which indicated they could be involved in eclosion due to the abundant accumulation in the late pupa. Our results offered more clues for understanding the mechanism of ecdysis and metamorphosis in insect and could give reference for further study of molting fluid.

Abstract:Studies have shown that some plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factors may increase plants resistance to stress. We screened the genes differentially expressed in transgenic SlNAC1 Arabidopsis compared to the wild type by cDNA microarry, to provide scientific basis for studying the genes related to abiotic stress responses in transgenic Arabidopsis. There were 3 046 genes differentially expressed more than twice in the total 43 604 genes of transgenic SlNAC1 Arabidopsis. Gene ontology analysis was used on genes differentially expressed more than five-fold. Genes relevant to cellular components occupied 33.05%, genes correlated with molecular function accounted for 33.95% and genes pertinent to biological process constituted a 33.00% portion. The genes differentially expressed more than twice were processed through kyoto encyclopedia of genes and genomes pathways enrichment (KEGG) analysis. The total 2 431 genes were involved in 88 different signaling pathways. The screened genes related to abiotic stress responses provide direction and theoretical support for the following research on the downstream genes regulated by NAC and construction of the regulatory networks.

Abstract:We analyzed the binding of P.rβ2-GPI-DV with ox-LDL by fluorescence, molecular simulation and circular dichroism. We used SDS-PAGE and Western blotting to identify the purity of P.rβ2-GPI-DV, fluorescence, circular dichroism spectroscopy and molecular docking simulation to analyze the binding between P.rβ2-GPI-DV and oxLDL. P.rβ2-GPI-DV was specifically recognized by anti-His antibody at 12 kDa position. The chromophoric groups, the changes of secondary structure and the molecular docking simulations revealed that the active pocket formed by Cys281-Lys-Asn-Lys-Glu-Lys-Lys287 and Leu313-Ala-Phe-Trp316 of P.rβ2-GPI-DV and the -COOH carboxyl of oxLig-1 were the key for binding. P.rβ2-GPI combined with ox-LDL via the fifth functional domain and the -COOH group. Our findings provide theoretical basis to further study the binding between β2-GPI and ox-LDL in serum.

Abstract:To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Abstract:The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche’s CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche’s was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche’s.