Abstract:Multiple PCR (Multiplex polymerase chain reaction, MPCR) is a technology to simultaneously amplify multiple targets through a single reaction and to detect the amplification products by reliable detection means so as to realize the diagnosis of multiple targets. MPCR has been well studied for its high efficiency, high throughput and low cost. At present, MPCR has been widely used in scientific research, disease diagnosis and other fields. Here, we summarize the development and application of MPCR from amplification and detection, and discuss the advantages and existing problems of MPCR. We propose that separate the reaction mixture into droplets or combined MPCR with the capillary convective PCR is expect to further improve the amplification efficiency of the surface of the solid phase carrier, so as to provide reference for the development of multiple PCR with high amplification efficiency, good consistency, good stability and multiplex detection.

Abstract:Tumor development is usually related to the genetic mutation and abnormal expression of multiple genes. Comprehensive analysis of tumor genome, transcriptome and epigenetics is very important for the rapid identification of disease-specific gene clusters and modification sites. Previously, the next-generation sequencing technology was mainly used to explore the information of genomes, however, it cannot meet the requirement of mechanical researches due to several problems such as difficult sequence assembly and leak detection of the low abundance factors. Therefore, single molecule sequencing technology gradually emerged with its unique superiority. This paper reviews the research processes of single molecule sequencing technology in several human tumors, and prospecs its application in clinical diagnosis.

Abstract:Gibberellin is one of the most important plant growth regulators, and widely used in agricultural production. However, the high cost of gibberellins production is restricting its efficient application. In recent years, biotechnological innovations have improved the synthesis of gibberellin. Gibberellin biosynthesis requires various enzymes. Current research focuses on the biosynthetic mechanisms of gibberellin and the metabolic engineering techniques to improve the production of gibberellin. This paper reviews the current research on gibberellin biosynthesis pathway, the key and enzymes environmental factors involved, and the metabolic regulation of gibberellin in Gibberella fujikuroi, summarizes the application of metabolic regulation in gibberellin biosynthesis, to provide the basis for achieving stable gibberellins production.

Abstract:Biomaterials have been widely used as bone grafts for bone tissue repair. The application of biomaterials needs to consider various aspects of material properties such as biocompatibility, mechanical strength and plasticity. It is also necessary for bone repair to consider the degradability of materials. Previous studies have shown that biomaterials can be degraded by physical, chemical and biological ways. Cell-mediated degradation is an important part of the biodegradation process of materials, mainly carried out by the biological behavior of macrophages and osteoclasts and reactive oxygen species, enzymes and acidic metabolites secreted by them. Illustration of cell-mediated degradation of biological materials helps us understand the biological behavior of cells better, to accurately design and manufacture more effective bone repair materials, which is conducive to initial stability during material implantation, in line with the consistence of material degradation and new bone formation, promoting bone regeneration and bone repair.

Abstract:Clostridioes difficile is a Gram-positive, spore-forming, obligate anaerobic bacterium, and the main cause of hospital-associated diarrhea. In recent years, with the presence of virulent strains (i.e., ribosome type 027), the prevalence and mortality events have increased. Thus, studies on physiological and biochemical characteristics, and pathogenic mechanisms of C. difficile have been performed. The development of efficient and stable genome-editing methods for C. difficile is urgent for the dissection of its physiological and pathogenic mechanism. For example, ClosTron technology plays a key role in study of the relationship between C. difficile toxins (Toxin A and Toxin B) and its pathogenicity. This article reviews the history, recent progress and future prospects of C. difficile genome-editing technologies.

Abstract:As a member of the histone lysine-specific demethylase family, KDM1A plays a pivotal role in biological processes including signal transduction, chromatin reprogramming, embryo development, hematopoiesis, glucose and lipid metabolism. Recently, increasing studies and clinical evidences suggest that the expression of KDM1A is related to initiation and development of tumors and plays a key role in regulating of initiation and development of tumors, such as prostate cancer, breast cancer, lung cancer and liver cancer. KDM1A binds to distinct complexes and mediates different downstream signaling pathways. However, KDM1A often plays an oncogenic role in the initiation and development of tumors. Based on the current literatures, we describe the latest research of KDM1A in the initiation and progression of various tumors, and summarize its mechanism of actions, to provide clues for cancer therapy.

Abstract:As one of the emerging industries, feed industry not only effectively utilizes existing agricultural resources but also provides a strong material foundation and protection for animal food. In this article, status and trends of biological feed in the world based on bibliometrics were analyzed. Issues including major institutions, international cooperation, status, trends and frontiers were analyzed. These co-countries map and keywords clustering analysis can reveal co-countries and hot spots in this field. These analyses can help the researchers get an overview of this field quickly and accurately.

Abstract:The objective of this study was to explore the link between long non-coding RNAs (lncRNAs) and resistance mechanism of albendazole in Haemonchus contortus by analyzing the gene expression profile of lncRNA in sensitive and resistant strains. This study provided the base for the resistance mechanism of Haemonchus contortus. In this experiment, the cDNA sequencing libraries were collected constructed for the sensitive and resistant strains of Haemonchus contortus, and the sequencing was performed using Illumina HiSeq 4000 platform, and differentially expressed lncRNAs (DEIncRNAs) were screened. Then the cis-targeted and trans-targeted genes of DElncRNAs were predicted, and the GO enrichment, KEGG pathway enrichment analysis were also made. The results displayed that 6 377 and 6 356 candidate lncRNA transcripts of sensitive and resistive strains were respectively obtained, 168 DElncRNAs of which were selected. Compared with resistive strains, there were 92 up-regulated and 76 down-regulated lncRNAs in sensitive strains. Meanwhile, 416 candidate target genes of DElncRNAs were obtained. The analytical results indicated that these genes participated in 641 GO terms and 92 signal pathways. The pathways involved in drug resistance are drug metabolism-other enzymes, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450 and so on. In conclusion, these findings inferred that some lncRNA-mediated target genes were associated with the resistance of Haemonchus contortus, and lncRNA may play an important role in the resistance of Haemonchus contortus. This study explored the expression profile of lncRNA in the Haemonchus contortus of sensitive and resistant strains. It was found that DElncRNAs in the sensitive and resistant strains, which helped to find out the Haemonchus contortus resistance mechanism of albendazole and provided a scientific basis for exploring the mechanism of resistance to Haemonchus contortus.

Abstract:GTPase immune-associated protein 5 (Gimap5), a key factor in maintaining T cell homeostasis, plays important roles in immune and inflammatory processes. However, its function and characteristics in poultry have not been reported. In this study, AA+ and Sanhuang broilers were used as models. The full-length coding sequence of the Gimap5 gene was cloned by RT-PCR and analyzed using bioinformatic methods. Tissue expression and distribution characteristics of the Gimap5 gene and its functional characteristics in inflammatory response were analyzed by RT-qPCR, respectively. The full-length coding sequences of the Gimap5 gene from AA+ and Sanhuang broilers were 771 bp, encoding 256 amino acids, and presented low conservative among different species. The AIG-1 domain of N-terminal and alpha-helix structure of C-terminal transmembrane sequence of might play important roles in the Gimap5 protein function and cell localization, respectively. The Gimap5 gene was widely distributed and expressed in various tissues and the pattern of its expression change was basically similar between AA+ and Sanhuang broilers. However, there were some differences in expression activity between the two breeds or various tissues of the same breed. In inflammatory response, the expression activities of the Gimap5 gene were down-regulated in blood and liver (P<0.05), but up-regulated in the bursa of Fabricius (P<0.01). It is speculated that Gimap5 is a multifunctional gene involved in the development of the body and inflammatory response, and has potential application value for diagnostic marker of inflammatory response.

Abstract:In order to determine the effect of transposons on the sequence and structural variations of the porcine ktn1 gene and its flanking regions, we obtained 14 ktn1 sequences (including genic region, 5-kb 5′ flank and 3-kb 3′ flanking regions) from the WGS database in NCBI, multiple sequence alignment by ClustalX and transposon annotation by RepeatMasker. Then we analyzed the effect of transposons on ktn1. A SINEA1 insertion polymorphism was detected by PCR, and correlation was analyzed with related traits in the Sujiang pig population. The ktn1 gene and its flanking regions contained at least 77 transposon fragments, the majority (98.70%) of which was SINE insertions. We observed 9 small structural variations and 4 large structural variations caused by transposons, indicating that transposons were an important source of genetic variations. One of the structural variations caused by SINEA1 insertion polymorphism showed rich polymorphism in different breeds, and in Sujiang pigs, the weaned litter weight of non-inserted individuals (SINE-/-) ((64.20±10.6) kg) was lighter than homozygous insertionindividuals (SINE+/+) ((74.14±9.0) kg) and heterozygous insertion individuals (SINE+/-) ((69.71±7.7) kg) (P<0.05). It is feasible to develop molecular marker based on the transposon insertion polymorphism to provide application potential in molecular-assisted breeding.

Abstract:The aim of this study is to obtain bacterial perhydrolases with chlorination activity, expands the resources of perhydrolases, and lays a foundation for it’s industrial applications. We constructed a metagenomic library using environmental DNA isolated from sludge samples of a paper mill of Tanghe county, and identified a per822 gene encoding a bacteria perhydrolase via activity-based functional screening. Then, we overexpressed Per822 heterologously in Escherichia coli, and characterized the recombinant enzyme after purification. Finally, we further investigated the ability of Per822 to produce peracetic acid (PAA). Sequence analysis revealed that per822 encoded a protein of 273 amino acids. The recombinant Per822 had the activity of peroxidase, esterase and halogenase respectively, and thus was regarded as a typical representative of multifunctional enzymes. The purified Per822 exhibited maximal chlorination activity (hyperhydrolysis) at 55 °C and pH 4.5 with monochlorodimedone as substrate, and the enzyme was stable in the pH range of 3.5–8.0 and below 70 °C. Also, the chlorination activity of this enzyme could be activated by Fe2+. In addition, the enzyme displayed high ability to generate PAA using ethyl acetate as cosubstrate. The highly soluble expression, catalytic versatility and good PAA production capacity of Per822 make it a potential candidate in organic synthesis, wastewater treatment, disinfection and biomass pretreatment, etc.

Abstract:China is now the largest and only producer of avermectin in the world. However, its current yield is still lower than other similar antibiotics. Therefore, we studied the effect of nitrogen on the growth and the synthesis ability of B1a to improve the overall yield. Nitrogen had significant effects on the cell activity, PMV of Streptomyces avermitilis and the synthesis of B1a in the middle and later phase of fermentation. Additional feeding yeast powder based on carbon-dioxide evolution rate in a 100-L bioreactor significantly improved the synthesis of B1a. The production of avermectin reached 8 697 mg/L, 26.9% higher than the original process. In short, this study will serve in production enhancement of avermectin at industrial production.

Abstract:γ-polyglutamic acid (γ-PGA) is widely used in food processing, cosmetic production, medicinal industry, etc. Currently, the production strains used in fermentation process are commonly glutamic acid-dependent, which results in extra cost. In this study, a de novo way of producing γ-PGA from sugars was reported. To this end, the γ-polyglutamate synthase gene cluster pgsBCA was cloned from the natural γ-PGA-producing strain Bacillus subtilis (ATCC 6051-U), and was constitutively and inducibly expressed in Corynebacterium glutamicum ATCC 13032. Only inducible expression of pgsBCA can lead to the generation of γ-PGA with a titer of 1.43 g/L from glucose, without any supplementation of glutamic acid. The production was further elevated to 1.98 g/L upon optimization of the induction conditions with the induction time at 2 h post-inoculation and the IPTG concentration of 0.8 mmol/L. Moreover, to achieve a higher titer of γ-PGA, pgsBCA was inducibly expressed in C. glutamicum F343, which shows a paramount glutamate production capacity. The final γ-PGA production reached 10.23 g/L in shake flasks and 20.08 g/L in a 5-L fermentor using glucose as the substrate. The weight-average molecular weight (Mw) of γ-PGA from recombinant strain F343 showed 34.77% higher than that produced by B. subtilis. This study provides a novel way of producing γ-PGA from sugars directly and potentiates new applications of γ-PGA in the future.

Abstract:The combination of high-quality mutagenesis and effective screening can improve the efficiency of enzyme directed evolution. In this study, a high efficiency cloning construction method of Multi-points Combinational Mutagenesis (MCM) was developed. Efficient multi-point combination mutations were performed in this MCM method by introducing DNA assembly, fusion PCR and hybridization techniques. After optimization, the efficiency of MCM was tested by directed evolution of benzoylformate decarboxylase. The obtained number of Colony Forming Units (CFUs) by electroporation to competent cells E. coli TreliefTM 5α exceeded 106 CFUs/μg DNA. Test results show that 90/100 clones were precisely assembled. The efficiency of simultaneous mutation at 5 sites (L109, L110, H281, Q282 and A460) was up to 88%. Finally, a mutant enzyme (L109Y, L110D, H281G, Q282V and A460M) with a 10-fold increase in kcat/Km was obtained. Therefore, this method can effectively create diverse mutant libraries and promote the rapid development of enzyme directed evolution.

Abstract:Gastric cancer is the fourth most common cancer in the world and the second leading cause of death in cancer worldwide. In order to reduce the mortality rate of gastric cancer, the problem that needs to be solved at present is to find new specific markers of early gastric cancer and increase the detection rate of early gastric cancer, thus fundamentally solving the problem of high mortality of gastric cancer. Previous studies in our laboratory found that Peroxiredoxin 4 (PRDX4) has the potential for early gastric cancer markers. In this study, the role of PRDX4 in transformed cells was studied by establishing a malignant transformation model and transforming cell overexpression, the experiment proves that PRDX4 is responsible for growth and proliferation by reducing the content of reactive oxygen species (ROS) in transformed cells. In the microenvironment, it promotes the malignant transformation of cells, that is, PRDX4 promotes the development of gastric cancer via eliminating ROS.

Abstract:In order to further improve the hemostatic performance of wound dressings, human platelet-rich plasma (PRP) containing various growth factors was introduced into chitosan solution. The silk fibroin solutions with different volume ratios (1:1, 1:3, 3:1 and 1:0) were added to improve the porosity and hemostasis of materials. The hPRP-chitosan/silk wound dressings with different ratios was prepared by freeze-drying and pure chitosan dressing was considered as the control group to study the effects of PRP and silk fibroin in different proportions on the hemostasis properties and the growth factors burst release. The hemostasis of chitosan dressing was improved by introducing PRP, but the porous structure and water absorption were not significantly improved. If silk fibroin solution was added in the ratio of 1:1, the more uniform porous structure and better hemostatic performance could be obtained. The porosity and water absorption could reach 86.83%±3.84% and 1 474%±114% respectively. In addition, the dressings with ratio of 1:1 had positive effects on reducing the burst release of growth factors on initial stage. Therefore, PRP-chitosan/silk fibroin composite dressing can become a kind of wound dressing that can achieve rapid hemostasis and promote wound healing.

Abstract:Sonication is one of the essential strategies of chromatin fragmentation in Chromatin immunoprecipitation (ChIP) assay. The impact of proteins with different molecular weights generated under different duration of sonication on the results of Chromatin immunoprecipitation sequencing (ChIP-seq) experiments involving the Polycomb Repressive Complex 2 (PRC2) related protein EZH2, which is a histone methyltransferase, and its product H3K27me3 was investigated. The results indicate that in the promoter region or nonpromoter region, there were hardly any differences among the H3K27me3 peaks annotated genes from different duration of sonication, which suggesting that the duration of sonication had little effect on histone ChIP-seq results. In contrast, in the promoter region newly gained EZH2 peaks annonated genes at 20 min sonication time were significantly clustered in the gene ontology (GO) pathways related to actin filament bundle compared with 10 min. In the nonpromoter region, compared with 10 min, the GO pathways of newly gained EZH2 peaks annonated genes at 20 min sonication time is much more than that of lost EZH2 peaks annonated genes. And in the nonpromoter region, compared with 20 min, the GO pathways of lost EZH2 peaks annonated genes at 30 min sonication time is much more than that of newly gained EZH2 peaks annonated genes. Most of these pathways are associated with RNA polymerase II (RNAPII), organ development and cell morphogenesis. These suggest that the genomic information of EZH2 will be lost if the duration of sonication is not enough or too long. Different duration of sonication mainly affect the EZH2 peaks in PRC2 unoccupied region and the bivalent promoter in the promoter region, as well as the PRC2 occupied region, PRC2 unoccupied region and the activated enhancer in the nonpromoter region. Therefore, the sonication for the chromatin related proteins with high molecular weights need to be optimized to make chromatin fragments size vary from 100 bp to 500 bp, which will yield relatively comprehensive genomic information of the target protein. For histones, which are of small molecular weights, duration of sonication has little effect on them.

Abstract:It is important to find monomers of traditional Chinese medicine that can inhibit the activation of IL-6/STAT3 signaling pathway to suppress the growth and deterioration of tumors. A novel expression vector containing STAT3 enhancer sequence and NanoLuc (NLuc) reporter gene sequence was constructed by gene recombination technology, then a cell line expressing NLuc luciferase and regulated by STAT3 was further established. The cell line was used to quantitatively detect the regulatory effects of various traditional Chinese medicine monomers on the IL-6/STAT3 signal pathway. The effects of the traditional Chinese medicine monomers inhibiting the IL-6/STAT3 signal pathway were verified by Western blotting immunoassay and Real-time PCR analysis. By enzyme digestion and DNA sequencing analysis, it showed that the reporter gene expression vector pQCXIP-STAT3-Nluc was constructed successfully. The addition of Interleukin-6 (IL-6), a stimulator of STAT3 transcription factor, to the constructed cell line specifically enhanced the luciferase enzymatic reaction in a dose dependent manner. These results demonstrated that the cell line expressing NLuc luciferase and regulated by STAT3 has been constructed successfully. We used the the cell line screened out dendrobiine and tetrandrine which could significantly inhibit IL-6/STAT3 signal pathway and down-regulated the expression of Bcl-2 and Bcl-x in a dose-dependent manner. In conclusion, we have constructed an efficient reporter gene system to detect the transcriptional activity of STAT3, by which the Chinese medicine monomers inhibiting IL-6/STAT3 signaling pathway were successfully screened out, which has some potential theoretical and practical values.

Abstract:Solanum tuberosum Zinc transporter 11 (StZnT11) is very important for maintaining zinc homeostasis in cells. The study on the expression of StZnT11 under abiotic stress and biotic stress laid a foundation for verifying the role of potato StZnT11 in the process of biotic stress of Ralstonia solanacearum species complex. According to the designated EST sequence, the homology of the original sequence was analyzed by using the Blast tool in NCBI, and a homologous object sequence with the highest similarity, coverage and e expectation value was selected. StZnT11 gene is obtained by Silico Cloning. The sequence and coding amino acid composition, physicochemical properties, molecular evolution, phosphorylation site and advanced structure of Solanum tuberosum StZnT11 gene were analyzed by bioinformatics method. The results showed that the cDNA gene is 1 300 bp in length, encoding a protein containing 348 amino acid residues, including 23 phosphorylation sites, one signal peptide and nine transmembrane regions, and is a hydrophobic protein located on the plasma membrane. Through amino acid sequence alignment, StZnT11 protein has a high homology with zinc transporter from tobacco, tomato, pepper and other plants. The results of real-time fluorescence quantitative polymerase chain reaction showed that, StZnT11 is up-regulated by different concentrations of exogenous plant hormone abscisic acid (ABA). Tissue localization showed that StZnT11 was mainly expressed in specific tissues (phloem and leaf vascular bundles of stem vascular system). These results provide a theoretical basis for further experimental cloning and functional verification of the gene.

Abstract:High expression of zearalenone (ZEN) degrading enzyme gene (zlhy-6) in Pichia pastoris strain GS115 was achieved by codon optimization and multi-copy construction in vitro. The codon-optimized zlhy-6 gene sequence was synthesized with the alpha factor signal peptide coding sequence and inserted into the pAO815 plasmid. The expression plasmid containing 1–6 expression cassettes was constructed by enzyme digestion and transferred into P. pastoris GS115 strain to obtain the ZEN degrading enzyme recombinant strain. The molecular weight of the recombinant protein was 28.9 kDa, which was consistent with the theoretical value. After 3 days of induction fermentation, the protein concentration reached the highest level and then decreased; the expression level was the highest in the induction culture at pH 5.0 and 4.5, while the expression level at other pH was very low; the expression level was the highest when 0.8% methanol was added every day and 10% inoculation was added; the expression level of four-copy transformants was the highest, and the enzyme activity reached 10 U/mL after 3 days of flask fermentation, The degradation rate of ZEN in 1 g corn ballast was 44.08%–75.51% when 0.1–0.5 mL fermentation supernatant added and hydrolyzed for 24 hours. The results of this study laid a foundation for improving the industrial fermentation level of ZEN degrading enzyme and its application in eliminating ZEN in food and feed.