Abstract:Extracellular matrix (ECM) proteins play an important role in a series of biological processes in the cell, and their abnormal regulation can lead to many diseases. The theoretical ECM reference dataset is the basis for efficient identification of extracellular matrix proteins. Researchers have developed various ECM protein prediction tools based on machine learning methods. In this review, the main strategy of development of ECM protein prediction tools that based on machine learning methods has been introduced. Then, advances and specific characters of the existing ECM protein prediction tools have been summarized. Finally, the challenges and possible improvements of ECM protein prediction tools are discussed.

Abstract:As the main factor leading to foodborne illnesses, foodborne pathogens have been attached great importance by people. The development of simple, rapid, high-sensitivity and low-cost food-borne pathogen detection methods is of great significance in reducing the incidence of foodborne diseases. Biosensor technology is a new micro-analysis technology developed by multi-disciplinary cross-infiltration. It has the characteristics of high sensitivity and fast analysis speed, and is widely used in the detection of food-borne pathogens. This paper introduces the basic principles of biosensors, summarizes the application of common biosensors in the detection of foodborne pathogens, and prospects for future development.

Abstract:Panax ginseng is a traditional Chinese medicine with significant pharmaceutical effects and wide application. Through orientational modification and transformation of ginsenoside glycosyl, rare ginsenosides with high antitumor activities can be generated. Traditional chemical methods cannot be applied in clinic. because of extremely complex preparation technologies and very high cost Transformations using microorganisms and their enzymatic systems provide the most feasible methods for solving the main problems. At present, the key problems in enzymatic synthesis of ginsenosides include low specific enzyme activities, identity of enzymes involved in the enzymatic synthesis, and their catalytic mechanisms, as well as nonsystematic studies on structural bioinformatics; specificity of enzymatic hydrolysis for saponin glycosyl has been rarely studied. Many reviews have been reported on glycosidase molecular recognition, immobilization, and biotransformation in ionic liquids (ILs), whereas ginsenoside transformation and application have not been systematically studied. To evaluate theoretical and applied studies on ginsenoside-oriented biotransformation, by reviewing the latest developments in related fields and evaluating the widely applied biocatalytic strategy, this review aims to evaluate the ginsenoside-oriented transformation method with improved product specificity, increased biocatalytic efficiency, and industrial application prospect based on the designed transformations of enzyme and solvent engineering of ILs. Therefore, useful theoretical and experimental evidence can be obtained for the development of ginsenoside anticancer drugs, large-scale preparation, and clinical applications in cancer therapy.

Abstract:With the rapid development of modern biotechnology, fermentation process is increasingly important in industrial production. To guarantee the stability of products, fermentation process should be elaborately monitored and controlled. Biomass is an important parameter for on-line monitoring in bioprocesses because biomass can reflect cell growth in a bioreactor directly. In-situ microscope, a non-invasive and image-analysis based technology, can real-time monitor cells in biological process. This review summarizes the development and application of in-situ microscopy in biomass monitoring.

Abstract:With the development of mass spectrometry technologies and bioinformatics analysis algorithms, disease research-driven human proteome project (HPP) is advancing rapidly. Protein biomarkers play critical roles in clinical applications and the biomarker discovery strategies and methods have become one of research hotspots. Feature selection and machine learning methods have good effects on solving the "dimensionality" and "sparsity" problems of proteomics data, which have been widely used in the discovery of protein biomarkers. Here, we systematically review the strategy of protein biomarker discovery and the frequently-used machine learning methods. Also, the review illustrates the prospects and limitations of deep learning in this field. It is aimed at providing a valuable reference for corresponding researchers.

Abstract:Melanogenesis is a biosynthetic pathway to produce melanin pigment in melanocyte, involving a series of intricate enzymatic and chemical catalyzed reactions. Melanogenesis involves five signaling pathways that converge on microphthalmia-associated transcription factor. In addition, many cytokines, involved in the regulation of melanogenesis, play an important role in the development, proliferation, differentiation and migration of melanocytes. Polyoxometalate can be used as a potential inhibitor of melanin production. Hence, this paper reviews the signaling pathways of melanogenesis and their regulatory mechanism, to apply polyoxometalates in the melanin production pathway, and briefly introduces the regulatory factors of related pathways.

Abstract:Cerebrospinal fluid surrounds and supports the central nervous system, including the ventricles and subarachnoid spaces. Cerebrospinal fluid should be an important source of biomarkers for central nervous system diseases because it is in direct contact with the central nervous system. Many studies are reported on cerebrospinal fluid proteomics, highlighting many recent progresses. Here, we review recent advances in proteomics technology and clinical application of cerebrospinal fluid.

Abstract:Para-aminobenzoate (PABA) is an important chemical for organic synthesis and extensively used in pharmaceutical and dye industry. In recent years, PABA has received increasing attention as a potential component of high-strength polymer. In Escherichia coli, three genes of pabA, pabB and pabC are responsible for PABA production from chorismate in folate synthetic pathway. However, E. coli does not accumulate or accumulates very few amounts of PABA under normal growth condition. In this study, the tyrosine-producing E. coli TYR002 constructed previously was used as the starting strain for developing PABA-producing strain. First, the activity of bifunctional chorismate mutase/prephenate dehydrogenase TyrA in E. coli TYR002 was weakened to reduce the production of tyrosine. Then, three different constitutive promoters were used to regulate the expression of pabA, pabB and pabC in recombinant plasmid which was transformed into E. coli for improving PABA production. The shake-flask fermentation showed that the different combination of constitutive promoters significantly affected the production of PABA, and the highest shake-flask fermentation titer was 0.67 g/L. After further condition optimization, the engineered E. coli produced 6.4 g/L PABA under 5 L fed-batch fermentation. This study could be a good reference for improving microbial production of PABA.

Abstract:The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.

Abstract:Drought stress affects the growth and development of rice, resulting in severe loss in yield and quality. Ectopic expression of the bacterial RNA chaperone, cold shock protein (Csp), can improve rice drought tolerance. Archaeal TRAM (TRM2 and MiaB) proteins have similar structure and biochemical functions as bacterial Csp. Moreover, DNA replication, transcription and translation of archaea are more similar to those in eukaryotes. To test if archaeal RNA chaperones could confer plant drought tolerance, we selected two TRAM proteins, Mpsy_3066 and Mpsy_0643, from a cold-adaptive methanogenic archaea Methanolobus psychrophilus R15 to study. We overexpressed the TRAM proteins in rice and performed drought treatment at seedling and adult stage. The results showed that overexpression both TRAM proteins could significantly improve the tolerance of rice to drought stress. We further demonstrated in rice protoplasts that the TRAMs could abolish misfolded RNA secondary structure and improve translation efficiency, which might explain how TRAMs improve drought tolerance transgenic rice. Our work supports that ectopic expression of archaeal TRAMs effectively improve drought tolerance in rice.

Abstract:Translationally controlled tumor proteins (TCTP) and SNF1- related protein kinase (SnRK1) are conserved and widely present in eukaryotic cells. TCTP regulates cell division, plant growth and development, and mediates plant resistance against pathogen infection. SnRK1 participates in a range of physiological processes including sugar metabolism and resistance to abiotic and biotic stresses. Previous work in our laboratory demonstrated that wheat TCTP can respond to Puccinia triticina infection and induce host defense responses. In order to further investigate the mechanism of TaTCTP in wheat resistance to Puccinia triticina infection, we used TAP (tandem affinity purification) and mass spectrometry to screen the potential interactants of TaTCTP. A SNF1- related protein kinase (SnRK1) was identified as a potential interacting protein of TaTCTP. The results of yeast two-hybrid assay showed that TCTP could interact with SnRK1 in yeast, and the yeast carrying TCTP and SnRK1 could grow on SD/-Leu/-Trp/-His/-Ade (SD/-LWHA) medium. The fluorescence signal of the interaction between TCTP and SnRK1 was found to be distributed in the cytoplasm in the Bi-fluorescense complementation experiment. Co-IP experiments further showed that TCTP and SnRK1 could interact in plant cells. This study lays an important foundation for further studying the mechanism of TaTCTP in the interaction between wheat and Puccinia triticina, and it play a great influence on further improving the molecular mechanism of wheat resistant to Puccinia triticina.

Abstract:Yuhua91 is a new peanut variety with high oleic acid content bred by Qingdao Agricultural University. The crossing was conducted with Luhua11 as female parent and with Kainong1715, an F435-type variety with high oleic acid content as male parent. The real F1 hybrids were screened by sequencing on PCR amplification products, and those homozygotes with bb genotype in F2 populations were screened by the same sequencing method as above. The content of oleic and linoleic acid was measured on the kernels harvested from F2 single plants by near infrared ray method, and those kernels whose content of oleic was above 80%, oleic and linoleic acid ratio was above 10.0 were obtained and planted into a row, with pedigree method for subsequent selection breeding. Yuhua91 has some characters of small pod, light and obvious pod texture, 148.06 g per 100 pods, 63.31 g per 100 kernels, 75.15% shelling percentage, long elliptic seed kernel, pink seed coat, without crack, white endotesta. Its content of protein, oil, oleic acid, linoleic acid and palmitic acid was 26.57%, 52.72%, 80.40%, 2.50% and 5.57% respectively. Yuhua91 has other characters of strong seedlings, compact pod areas, and moderate resistance to leaf spot disease and bacterial wilt. Average pod yield is 215.79 kg per Mu, 15.27% higher than the control variety Huayu20. Average seed kernels yield is 157.33 kg per Mu, 21.64% higher than the control variety Huayu20. Yuhua 91 has been registered on department of agriculture in 2018, and the registration No. is GPD peanut (2018) 370210, fit for growing in Shandong Province.

Abstract:Quorum sensing (QS) plays a major role in the outbreak mechanism of foodborne diseases caused by food poisoning and food spoilage. QS affects the formation of cell membrane and pathogenicity ofpathogenic bacteria. Through the in-depth understanding of QS molecules of food-borne pathogens, we describe here the types of signal molecules produced by Gram-negative and Gram-positive bacteria, and the differences in QS molecules. Meanwhile, we introduce the detection of QS molecules by different technologies. According to the influence of QS on food, we propose also future research needs for the control of foodborne pathogenic bacteria.

Abstract:The liver is the metabolic center of mammalian body. Systematic study on liver’s proteome expression under different physiological and pathological conditions helps us understand the functional mechanisms of the liver. With the rapid development of liquid chromatography tandem mass spectrometry technique, numerous studies on liver physiology and pathology features produced a large number of proteomics data. In this paper, 834 proteomics experiments of mouse liver were systematically collected and the mouse liver proteome database (Mouse Liver Portal, http://mouseliver.com) was established. The Mouse Liver Portal contains the liver’s proteomics data under different physiology and pathology conditions, such as different gender, age, circadian rhythm, cell type and different phase of partial hepatectomy, non-alcoholic fatty liver. This portal provides the changes in proteins’ expression in different conditions of the liver, differently expressed proteins and the biological processes which they are involved in, potential signal transduction and regulatory networks. As the most comprehensive mouse liver proteome database, it can provide important resources and clues for liver biology research.

Abstract:To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12–200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%–5.6% and 5.6%–7.2%, respectively, and the fortified recovery was 89%–103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.

Abstract:The development of orally administrated heparin drugs requires a systematic understanding of the interaction between heparin and gut flora. The in vivo distribution of fluorescein-labeled heparin that is orally administrated by mice was observed using fluorescein microscopy. In addition, the stability of heparin in simulated gastric and intestinal fluids, as well as the in vitro degradation of heparin by gut flora were detected by HPLC. The results show that orally administrated heparin was mainly distributed in the gastrointestinal tract of mice, and exerted structural stability under the condition of simulated gastric and intestinal fluids in vitro. However, heparin could be degraded by intestinal flora cultured in medium containing heparin. In order to further study the effect of orally administrated heparin on intestinal flora in mice, the fecal microbiota 16S rRNA fragment of C57BL/6J mice was tested by the Illumina Mi-Seq high-throughput sequencing technology. Compared with the gut flora of mice that orally administrated by saline, the biodiversity of gut flora in mice with orally administrated heparin was decreased. The difference of microflora structure was not significant at the phylum level, and the relative abundance of Alistipes, Parasutterella and Akkermansia was increased at the genus level, and the relative abundance of Bilophila, Enterorhabdus, Ruminiclostridium, Prevotellaceae_UCG_001, Ruminiclostridium-9, Bacteroides, Lachnoclostridium, Candidatus, Saccharimonas, Intestinimonas and Dubosiella was reduced. These findings indicate that heparin could influence the gut flora of mice. In addition, no obvious toxic and side effects were found in mice that orally administrated heparin, suggesting the safety of orally administrated heparin.

Abstract:Due to limited availability of autologous blood vessels (blood vessels from the same recipient used for vascular transplantation materials) and inadequate growth ability of non-autologous blood vessels (artificial blood vessel transplantation materials), more and more attention has been paid to tissue engineering blood vessels. In this study, we constructed an ammonium phosphate zwitterion modified acellular vascular scaffold with highly biocompatible bone marrow-derived endothelial progenitor cells as the inner layer of a new vascular transplantation material. The vascular acellular scaffolds were modified by a simple method—co-precipitation method. The platelet adhesion test, hemolysis test, recalcification test and cytotoxicity of acellular vascular scaffolds in vitro were evaluated. Ammonium phosphate zwitterions modified endothelial progenitor cells on the surface of acellular scaffolds with concave and convex structure on the surface of natural vascular lumen can be effectively promoted by improving anticoagulant activity. Modified acellular scaffolds have similar mechanical properties to natural blood vessels and can effectively construct endothelialization in vitro. The results of this study provide a preliminary exploration for the modification of vascular acellular scaffolds to achieve anti-thrombosis and endothelialization in vitro.

Abstract:Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2–5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.

Abstract:Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.

Abstract:Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.