tbx2 plays an important role in heart development. To further investigate the gene function in the atrioventricular canal (AVC) development, tbx2b-/- mutation zebrafish was successfully established using the CRISPR/ Cas9 mediated gene knockout technique. The knockout efficiency of F0 was detected by T7E1 assay, and the result showed that the average knockout efficiency was about 57.5%. Sanger sequencing confirmed that tbx2b F1 mutant had a-11bp-base deletion and caused frameshift mutation. Homozygous mutations were lethal in 5 dpf and early embryos suffered from cardiac looping abnormalities. In situ hybridization in 3 dpf tbx2b-/- showed ectopic expression of nppa and nppb, which are chamber-specific marker genes, and significantly decreased expression of has2, which is AVC marker gene. tbx2b -/- mutant was efficiently constructed and effects on downstream genes were discussed, which laid a foundation for further research on the effect of cardiac AVC development and understanding of early cardiac regulatory networks.
Objective: Keratin was used as the drug carrier material, and the hydrophilic drug rhodamine and hydrophobic drug curcumin were loaded to prepare the intelligent responsive drug delivery system, and the pH and redox responsiveness of drug release were studied. Methods: The exopolysaccharide of Lactobacillus plantarum was introduced into the reaction system of sodium selenite and ascorbic acid, and KNP was synthesized at room temperature. The size, morphology, structure and stability of KNP were studied by tungsten filament scanning electron microscopy (SEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and drug release experiments in vitro. Results:KNP with uniform particle size of about 300 nm was successfully prepared, which can load hydrophilic and hydrophobic drugs. Keratin based nanoparticles exhibited pH and glutathione (GSH) dual responsive release characters. In addition, these drug-loaded particles showed potent prolongation duration, good stability, sustainable and controllable release than the original drug. Conclusion: KNP with high stability, good water dispersion and responsiveness can be prepared simply and safely, and the drug delivery system of keratin is an appropriate candidate drug carrier.
In order to obtain a more ideal skin wound repair dressing, human hair keratin (KTN) is compounded on the basis of sodium alginate (SA) and polyacrylamide (PAM) hydrogel to prepare KTN/SA/PAM hydrogel glue skin dressing.It was characterized by an electronic universal testing machine, scanning electron microscope, etc. The results showed that the tensile strength of the KTN/SA/PAM hydrogel skin dressing was 42.41 kPa and the elastic modulus was 11.19 kPa, which were close to human skin tissue; The adhesion of the skin can be as high as 5.1 kPa, and the water absorption rate in 2 hours is 144.3%, which satisfy the basic requirements of skin wound repair dressing. Further rat skin wound repair experiments show that KTN/SA/PAM hydrogel skin dressings have better repairing ability than commercially available wound tapes and SA/PAM hydrogels. Histological analysis of wound skin tissue sections shows that after KTN/SA/PAM skin dressing treatment, the immature tissue areas of the wound were the smallest and the collagen fibers were arranged most neatly. After 14 days, the degree of wound healing was almost similar to that of normal skin. The results indicated that KTN/SA/PAM skin dressing may be a more promising skin wound repair dressing.
H2O2 is an important signaling molecule that participates in a variety of physiological metabolic activities in plants. However, excessive H2O2 destroys biological macromolecules, thus poisoning cells. Thioredoxin peroxidase (Tpx) plays an important role in protecting plants from oxidative damage by scavenging H2O2. The prokaryotic expression vector of tomato Tpx gene (SlTpx) was established, and SlTpx protein was induced and purified, and we found that the size of SlTpx protein was about 21 kDa. In vitro mixed functional oxidase (MFO) assay showed that SlTpx could protect DNA from harmful reactive oxygen species. In vitro experiments on SlTpx protein showed that SlTpx could improve the anti-heavy metal toxicity and antioxidant capacity of Escherichia coli. It laid a foundation for further study on the function and mechanism of SlTpx in plants.
Objective: To provide a method that can significantly improve the stable transfection of lentivirus into human pluripotent stem cells, and establish a simple and a non-invasive screening method for transfected cells. Methods: In the process of lentivirus transfection of human pluripotent stem cells, we compared the dynamic changes of cell morphology with and without Y-27632, and the differences of lentivirus particles uptake ability under different cell morphologies, so as to optimize and establish an efficient lentivirus transfection method. After that, a visualized and simple micromanipulation device was designed and developed to explore the technology of picking up the transfected positive monoclone cells to establish a line with the aid of a fluorescence microscope, and establish a relatively simple new method for purification of transfected cells. Results: The morphology of normal cultured human pluripotent stem cells colony changed significantly 6 hours after Y-27632 was added. The cells were in loose colony showing a long spindle shape, and an increased cell surface area; The colonies returned to normal 6 hours after removal; In conventionally cultured pluripotent stem cells, lentivirus tended to enter the colony periphery or partial cells; after Y-27632 was treated for 6 hours in advance, the pluripotent stem cells showed a loose colony and a significant increased surface, making the lentivirus infect more evenly into the periphery and internal of the colony. It significantly improved the efficiency of lentiviral transfection. Using the capillary glass tube, we designed and manufactured independently a single colony selection device that was visualized under a microscope. With the aid of a microscope, the selection and establishment of successfully transfected colony can be easily performed in the laboratory. It can replace puromycin screening with certain cell damage and the flow cytometry which requires professional equipment. Conclusions: In the process of lentiviral transfection, the hESC / IPSC colonies cultured in conventional condition were relatively dense and resistant to lentivirus. Y-27632, a small molecule compound, made the hESC / IPSC colonies relatively loose in structure and increased the surface area, which significantly improved the susceptibility of cells to lentivirus and improved the infection efficiency; a simple and non-toxic micromanipulation device was successfully designed under conventional laboratory conditions, and it can effectively replace flow cytometry and drug screening, and realize the selection of cell clones and finally the establishment of cell lines.
Brevibacillin, a broad-spectrum antimicrobial peptide, has shown strong antibacterial effect on bacteria and fungi, which can be regard as a kind of potential substitute for antibiotics. The wild strain Brevibacillus laterosporus was carried out the conventional mutagenesis techniques including UV mutagenesis, atmospheric and room temperature plasma mutagenesis and nitrosoguanidine mutagenesis to obtain four mutant strains. After two rounds of genome shuffling, strain F2-24 was obtained, and the yield of brevibacillin reached (340.5±16.35) μg/mL, which was 1.92-fold than that of wild strain fmb70. Furthermore, the fusion strain F2-24 possessed great stability after five generations. Finally, the fermentation process of fusion strain F2-24 was optimized. The results of the best carbon source, nitrogen source and inorganic salt ion was sucrose, beef extract and Mg 2+, respectively and the addition dose was 4%, 2% and 0.5%, respectively. The yield of brevibacillin was significantly enhanced via fermentation at 30℃ pH6.0 for 24 h, and meanwhile, the results suggested that the yield of brevibacillin was remarkably improved by a series of optimization and the yield reached (442.45±9.58) μg/mL, which was 2.50-fold than that produced by wild strain fmb70.
Lentiviral vectors (LVVs) are used for various gene therapeutic applications in vitro, and have shown very promising results in several clinical trials. This possibility of a durable cure for monogenic diseases affecting the hematopoietic system has made gene therapy very attractive. Yet, efficiency, safety, and cost of LVVs gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LVVs used on the cells. LVVs are pseudotyped with different viral envelope glycoproteins to alter and improve their tropism for different target cells. Another strategy to optimize the entry and post-entry steps of LVVs is the addition of transduction enhancers (TEs) during the transduction procedure, which improves the transduction efficiency and keeps stable expression of transferred genes in vivo. The combination of pseudotyping with heterologous viral envelopes and adding TEs increased the transduction efficiency of LVVs, and has the potential to improve clinical protocols.
Long non-coding RNAs (lncRNAs) are generally defined as RNA transcripts of more than 200 nucleotides in length with limited protein-coding capacity; however their functions are diverse and complex. Previous studies have demonstrated that lncRNAs are closely related to cancer development and are potential cancer regulators. lncRNAs can participate in the regulation of intracellular biological processes in different ways. It is a potential cancer regulator. Among them, lncRNAs can affect cancer progression mainly through regulating the level of epigenetic modification. Epigenetic modifications exist in the cells during the pathogenesis of cancer such as DNA modification, RNA modification, and post-translational modification of proteins including methylation, acetylation, phosphorylation, glycosylation, and ubiquitination, and the degree of abnormal modifications is different at different stages of cancer. This phenomenon will affect the biological processes of tumorigenesis. Studies have shown that lncRNAs can participate in the occurrence and development of cancer through self-modification or taking part in the epigenetic modification of other biomolecules. Therefore, the epigenetic modification lncRNAs participate in and the role of lncRNAs in epigenetic modification were reviewed. This paper explores how lncRNAs affect cancer progression through regulating the level of epigenetic modification in order to summarize and analyze the research progress and provide potential targets and biomarkers for cancer diagnosis and treatment.
Rapid and accurate detection of pathogens is essential to achieve efficient epidemic prevention and control, accurate treatment of diseases, and timely disposal of polluted environment. However, the existing on-site rapid detection methods of pathogenic bacteria mainly focuses on qualitative analysis. False positive/negative results exist and their detection accuracy still needs to be improved. It is urgent to develop rapid detection technologies of pathogenic bacteria by taking use of new principles and methods. The CRISPR (clustered regularly interspaced short palindromic repeats) based biosensors have several unique advantages, such as high flexibility (only needing to change the crRNA sequence for different target genes), high specificity (single base resolution), high sensitivity (better than 10-18 mol/L concentration), programmability, modularity, low cost, and high efficiency and stability in various in vitro media. It has become the leader of the next generation of pathogen detection technologies without the limitations of traditional molecular diagnosis and detection technologies. In this technology, Cas effector proteins are used as highly specific sequence recognition elements. Through combined with various biosensor mechanisms, they can be used for rapid and sensitive detection of pathogens with high specificity. After summarizing the principle of the CRISPR/Cas biosensor technology, the research progress of the CRISPR/Cas12 and CRISPR/Cas13 biosensor technologies for pathogen detection was reviewed. The challenges of the CRISPR/Cas biosensor technology in practical application are discussed, and its future developments are prospected.
With the continuous update of high-throughput sequencing technologies, the third-generation sequencing technology that can read nucleotide sequences at the single-molecule level has developed rapidly. Nanopore sequencing technology is its representative single-molecule sequencing technology, which realizes base calling by detecting the characteristic changes of electrical current when the DNA single-stranded molecule is passing through a nanopore channel. Compared with the traditional first-generation and the next-generation sequencing (NGS) technologies, the nanopore sequencing of DNA has great advantages in device portability, base acquisition speed and read length, which has attracted much attention. With the continuous development of nanopore sequencing technologies, various signal processing schemes and biological information processing tools for nanopore sequencing have been developed, and base calling and model simulation are two of the key research directions. The fundamental principle and signal processing flow of nanopore sequencing are surveyed, the current challenges are discussed, then the development trend of base calling and nanopore model simulation in recent years are summarized, and the performance of different base calling methods are compared by using real sequencing reads. Then, an integrated simulation platform for the evaluation of signal processing algorithms of nanopore sequencing is developed. Furthermore, with the explosive growth of global data volume, DNA data storage is becoming a promising medium for future massive data storage, and the use of nanopore for sequencing and reading is a very effective method. The application progress of the nanopore sequencing technology for DNA data storage is summarized, and its feasibility is analyzed. The rapid readout method of artificial chromosome data storage based on nanopore sequencing is analyzed, and the application of the simulation of nanopore sequencing reads combined with actual sequencing data in DNA data storage is discussed, which provides a reference for the development of a suitable DNA data storage program.
Synthetic biology is an emerging filed that aims to de novo design and synthesis of biological functional modules, gene circuits or artificial life with the standardized bioparts based on engineering principles. Since its inception in 1998, phage recombinase-mediated site-specific recombination, also known as recombineering, has revolutionized synthetic biology. Due to high efficiency, high precision, and broad-spectrum applicability, phage recombinases have been developed into powerful synthetic biology tools such as gene editing, DNA assembly, and gene-directed insertion. Yet, some applications, such as gene circuits, biocomputing, and biotherapy, need controlled excision, and this requires the number of well-characterized integrases and RDFs. Here, the classification and applicability of phage recombinases and focused on the application of phage recombinase systems in gene circuits design and construction, in vivo genetic modification and in vitro recombination were briefly introduced. Besides, the challenges of phage recombinase as tools for genetic engineering and sophisticated gene expression regulation as well as prospects for phage recombinases were analyzed.
Pseudomonas contamination incidents are not seldom encountered in clinical treatment and daily diet, which may result in illness, death and other undesirable consequences. To this end,recent studies focus on the inhibiting of pathogenic Pseudomonas and the reducing of their drug resistance, in which it has been proved that the excruciating characters of Pseudomonas can be improved by natural active ingredients (for example, probiotics). As a kind of widely existing probiotics, Lactobacillus is utilized for the survey. By considering the studies all over the world up to date, effects of Lactobacillus on the biofilm structure, growth activity, ability to adhere to cell surfaces and biological toxicity of Pseudomonas are introduced. Also, the influence of mice infected by Pseudomonas are is demonstrated. Based on the surveys aforementioned, it is significant to find out biological principles of influence on Pseudomonas of Lactobacillus and other probiotics (including their metabolites), which are the key to prevent and control microbial contamination and infection.
Gamma aminobutyric acid (GABA) is a kind of non-protein amino acid which is very soluble in water. It is widely used in food and pharmaceutical industry, and the market demand is very large. It can be produced by chemical synthesis, plant enrichment, microbial direct fermentation and biotransformation. In recent years, the synthesis of GABA by biological methods has its relative advantages, and has been paid attention by researchers. In this paper, the production methods of GABA, the microorganisms producing GABA, the key metabolic pathway of GABA synthesis and the directed transformation strategy of GAD enzyme were discussed.
