Tetrodotoxin (TTX) is an alkaloid neurotoxin, and its poisoning cases occurr worldwide and therefore seriously threat human health. However, there is no specific antidote yet for TTX, so the detection of TTX is of great importance in the field of food safety. To obtain a more efficient TTX recognition element, guided by molecular simulations, a DNA aptamer (TTX-27) previously discovered by SELEX screening was continuously optimized. First, the stem-loop structure, which hinders the TTX binding, was replaced with a mini-hairpin structure to make the TTX bind more easily to the truncated aptamer; next, T39 and C40 bases were mutated to C and T bases, respectively, and C39 was also modified with 2'-OH to enhance the hydrogen bonding and van der Waals interactions of the bases with TTX. Microscale thermophoresis (MST) experiments confirmed that the affinities of the aptamer variants were increased by the truncation, base mutation and chemical modification. The dissociation equilibrium constant Kd of the binding of the chemically modified variant TTX-D2-X-R to TTX was 1.08 nmol/L, which increased 75.5 times compared to that of TTX-27. Thus, this study demonstrates that the molecular simulation-based truncation-mutation-chemical modification is an effective approach to the post-SELEX optimization of nucleic-acid aptamers, and the resulting aptamer variant TTX-D2-X-R has potential applications in the field of TTX detection.
Objective: To investigate the osteogenic properties of dental-derived stem cell composite with grooved porous hydroxyapatite (HAG) scaffolds to provide a new approach for bone defect repair. Methods: Periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs) were separated from healthy adults’ third molars and seeded on HAG scaffolds for multi-directional differentiation identification and alkaline phosphatase (ALP) activity detection; the cell proliferation ability was detected by CCK-8; reverse transcription polymerase chain reaction (qRT-PCR) was used to detect bone morphogenetic protein 2 (BMP-2), osteocalcin (OCN) and osteopontin (OPN).In vivo study, the HAG scaffolds loaded with two kinds of cells were transplanted into the back subcutaneous tissue of nude mice, and the tissue engineered bones were taken after 8 weeks. After tissue sections, hematoxylin and eosin (HE) staining was used to observe the formation of new bones. Western blot was used to observe expressions of osteogenesis-related proteins such as ALP and OCN. Results: In the in vitro study, the cell proliferation ability, ALP activity, and the expressions of osteogenesis-related genes ALP, BMP2, and OCN in the DPSCs combined with HAG scaffold group were higher than those in the PDLSCs combined with HAG scaffold group. In vivo study, HE staining showed that the PDLSCs combined with HAG scaffold group and the DPSCs combined with HAG scaffold group had more cell growth areas, fibrocyte proliferation and bone matrix formation than the blank HAG scaffold group, and the DPSCs combined with HAG scaffold group had more bone matrix area. The expression of bone-related proteins in the PDLSCs combined with HAG scaffold group and the DPSCs combined with HAG scaffold group was higher than that in the blank HAG group, and the expression of ALP protein in the DPSCs combined with HAG scaffold group was significantly higher than that in the PDLSCs combined with HAG scaffold group. Conclusion: PDLSC and DPSC composites with HAG scaffolds showed good osteogenic properties in vitro and in vivo, and the osteogenic properties of DPSC composite with HAG scaffolds were even better.
Larix gmelinii is a very important coniferous tree species for afforestation. It has the characteristics of rapid growth in early stage, strong stress resistance and good ecological benefits. Trehalose participates in the regulation of drought, cold, salt damage and other stresses. Trehalose-6-phosphate phosphatase (TPP) is an important enzyme in the trehalose synthesis pathway. In this study, the full-length sequence of LgTPPI.1 was screened from the transcriptome of L. gmelinii under stress and its coding sequence(CDS) was cloned. The recombinant vector was constructed and homozygous lines of transgenic Arabidopsis thaliana overexpressing LgTPPI.1 were obtained. The results showed that the full-length CDS of LgTPPI.1 was 1 236 bp, encoding 411 amino acids; the expression level of LgTPPI.1 was lower in roots and stems, but higher in needles. Under salt treatment, the LgTPPI.1 overexpression lines conferred stronger tolerance than the wild type A. thaliana, with elevated trehalose and proline content; increased superoxide dismutase(SOD) and catalase(CAT) activity and up-regulated expresssion of the stress-responsive marker genes. These results indicated that gymnosperm utilized similar trehalose pathway as angiosperm to telerate abiotic stress. This study provided a theoretical basis for further analysis of the function of trehalose synthetic genes and the response mechanism of conifers under stress.
Microalgal biotechnology for biofuels is at a crossroads and its development is still in flux. The development of microalga-derived multi-product technology will greatly improve the economic viability, particularly with a combination of the production of high value-added compounds. The results showed that the mutants with DWARF1 (DWF1) gene knocked out had higher pigment content and photosynthetic efficiency than the wild type, and could also significantly reduce the accumulation of cholesterol (which might act as a leading risk factor for human cardiovascular disease) from over 70% of total sterols (TSs) to null. In contrast, the production of its precursor 24-methylenecholesterol (a critical micronutrient of royal jelly that is beneficial to human health) was increased from null to more than 60% of TSs. Combined with the high content of omega-3 fatty acids of N. oceanica, we anticipate an appreciable profit by exploiting this strain on an industrial scale.
Oral vaccines have attracted much attention due to their advantages of high patient compliance, reduced generation of harmful waste, convenient vaccination, and their ability to cause mucosal immunity. However, unfavorable conditions such as the acidic environment of the stomach, proteases, intestinal mucus and tight junctions between intestinal epithelial cells make the gap between oral vaccines and injectable vaccines too large, which restricts the immune effect of oral vaccines. In order to improve the immune effect of oral vaccines, we combined morphology control and coating modification strategies to prepare a new type of oral vaccine carrier. Specifically, polylactic acid-glycolic acid copolymer (PLGA) rod-shaped particles were prepared by combining emulsion solvent evaporation method with fast membrane emulsification method, then β-glucan that enhances immune response and thiolated hydroxypropyl methyl cellulose phthalate (T-HPMCP) with higher degradation pH and stronger adhesion with intestinal epithelium were used for coating modification of PLGA rod-shaped particles. In the preparation of PLGA rod-shaped particles, the effects of PBS concentration and polyvinyl alcohol (PVA) concentration in the outer aqueous phase on the preparation of PLGA rod-shaped particles were explored. It is found that the deformation degree of PLGA particles first increased and then decreased with the increase of PBS concentration, but the deformation degree of PLGA particles always increased with the increase of PVA concentration. This is because PBS forms an electric double layer with -COO- of PLGA, which makes the emulsion more stable and makes deformation more difficult to occur. Finally, the optimal formula was determined to prepare PLGA rod-shaped particles with a length of 2-4 μm and a width of 1-2 μm suitable for the uptake of small intestinal epithelial cells. It is found that the protein entrapment rate of PLGA rod-shaped particles is higher than that of PLGA spherical particles because the electric double layer makes the emulsion more stable. The results of in vitro experiments show that the vaccine carrier modified by T-HPMCP is stable in an acidic environment and the amount of released protein is negligible, which can prevent the antigen from being corroded by the acidic environment and is conducive to the protection of antigen activity. It can be decomposed at pH ≥ 7.4 and then release antigen. The results of cell experiments show that its special rod-shaped morphology can be taken up by Caco-2 cells faster and can be rapidly transported by the Caco-2 cell monolayer model constructed in transwell chambers, and the modification of β-glucan can also promote dendritic cells (DCs) secretion of surface molecules MHC-I, MHC-II and CD80 to activate DCs. The results of animal experiments showed that PLGA rod-shaped particles modified by β-glucan and T-HPCMP could increase the levels of OVA-specific IgA and IgG antibodies, which reached their maximum on the 28th day, and they promoted immune central memory T cells and CD8+ effects generation of memory T cells. In conclusion, the prepared coated PLGA rod-shaped particles can improve the immune response of the body as an oral vaccine carrier, thereby producing a better immune effect and providing new materials and ideas for the research of oral vaccines.
Objective: To investigate and verify the candidate interactors of S100A7. Methods: The candidate interactors of S100A7 are identified using BioID and mass spectrum and verified by Co-IP and immunofluorescence. Results: 94 candidate proteins are identified in two groups by mass spectrum. Interaction between Annexin A2(AnxA2) and S100A7 was then verified using Co-IP in HEK293WT cells. It is demonstrated that AnxA2 interacts with S100A7 directly, and the immunofluorescence results show that S100A7 and AnxA2 are collocating in the cytoplasm. Conclusion: BioID is a new technology for screening S100A7 interactors and the interaction between S100A7 and AnxA2 is discovered using BioID.
The global pandemic of the COVID-19 has had a major impact on the entire human society, and human beings are facing challenges such as fiscal stimulus, financial stress, and debt restructuring. Before the emergence of specific therapeutic drugs and methods, large-scale population screening and isolation has become the most effective method for epidemic management. However, the new strain of coronavirus this time has shown a very high genetic variability, with a statistical mutation rate of more than 2.3‰ as of March 31st, 2022. So far, new highly infectious virus strains have been emerging, and the number of mutant strains officially warned by the World Health Organization has reached 7. Therefore, in the next virus prevention and control and research, we not only need to detect SARS-CoV-2, but also need to explore accurate and practical single nucleotide variation (SNV) genotyping techniques, especially for large-scale population screening. It is not only necessary to obtain information on the SRAS-CoV-2, but also to accurately and quickly distinguish variant strains with higher infectivity and virulence. This paper briefly introduces the infection and mutation mechanism of the virus, and focuses on the classification and review of the existing main SARS-CoV-2 SNV genotyping techniques, hoping to provide insight into the development of new detection technologies.
Esophageal cancer is the tenth most common cancer in the world, with high morbidity and mortality. The main subtypes of esophageal cancer include esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), the patients of which are usually diagnosed at a late stage. The standard treatment methods of esophageal cancer include radiotherapy and chemotherapy, endoscopic therapy and surgery, but the prognosis is still not good as expected. The patient-derived xenograft (PDX) model retains the cellular morphology, tissue structure and genetic characteristics of the original tumor to the greatest extent. The PDX model provides a new platform and guarantee for studying the reactivity of patients with esophageal cancer to radiotherapy and chemotherapy, seeking new therapeutic targets and improving prognosis, which makes personalized precision therapy research enter a new stage. This article first reviews the characteristics of esophageal cancer PDX model, the commonly used experimental animals, the ways and methods for optimizing the model establishment, and the application of PDX model in the research of esophageal cancer, and then discusses the limitations and future development prospects of esophageal cancer PDX model, in order to provide a new research direction for personalized precision therapy and improve the prognosis of patients with esophageal cancer.
The establishment of efficient genetic transformation systems can promote the study of plant functional genomics and the cultivation of new crop varieties. At present, low regeneration efficiency is one of the main technical barriers to the establishment of efficient genetic transformation systems in many plants. With the deepening of research on plant meristem and somatic embryo formation, some key regulatory genes have been identified, which are collectively called developmental regulators. The application of developmental regulatory factors in plant genetic transformation can effectively improve the establishment of plant meristem induction and regeneration ability, which provides an important opportunity to improve the efficiency of genetic transformation. In this paper, the research progress of seven types of developmental regulatory factors in improving the efficiency of plant genetic transformation was reviewed, with emphasis on the application of three types of developmental regulatory factors in promoting maize genetic transformation. Finally, the development direction of establishing efficient plant genetic transformation system was presented.
The cell wall is a complex and dynamic network structure, playing a vital regulatory role in plant growth and development, stress response, and immune resistance. As the main polysaccharide component in the primary cell wall structure, pectin is composed of homogalacturonic acid (HG), the most abundant linear polymer. The degree of methyl esterification of HG leads to its enzymatic hydrolysis to form a gel, thus affecting the stability of the pectin structure. Depending on post-translational mechanisms, pectin methylesterase inhibitors (PMEIs) regulate the PME activity by fine-tuning the balance of methyl esterification of pectin polysaccharides to maintain cell wall integrity and biomechanical properties. An increasing number of studies have shown that PMEI-PME interaction modulates the homeostasis of pectin methyl esterification, which is a crucial factor determining cell adhesion, cell wall porosity, elasticity, and organ morphogenesis. It is also an associated molecular pattern of cell wall-mediated immune response to environmental cues by releasing the anti-stress signals. This review presents the recent research progress on the physiological importance of PMEIs in regulating plant growth and development, especially regulatory mechanisms underlying the function of the stress tolerance in response to various stress stimuli. Since much remains unknown concerning the in vivo activity and regulatory mechanisms of PMEIs in woody plants, this review provides a theoretical basis and strategic reference for future research.
In recent years, with global warming and the increasing intensity of water eutrophication, cyanobacterial blooms occur frequently. Microcystins are the most harmful cyanobacterial toxins produced and released by harmful cyanobacteria, which have had a harmful impact on the ecological environment and public health. Therefore, seeking effective microcystin degradation methods has become a research hotspot in the global scientific fields. The biological treatment technologies of microcystins are reviewed, the production, physicochemical properties and biotoxicity of microcystins are described, and the degradation efficiency of microcystins by microorganisms, aquatic plants and zooplankton is summarized. On this basis, the removal effects of biological treatment technologies such as biological filter, constructed wetland, ecological floating bed, and membrane biofilm reactor on microcystins are overviewed, and the advantages and limitations of existing microcystins’ biological treatment methods are analyzed. The further research subjects are also proposed. All these provide ideas for solving the pollution problem of microcystins in the water environment.
2-Phenylethanol (2-PE) is an important aromatic with broad application prospects. Due to the complexity of chemical synthesis and the high cost of natural extraction, the synthesis of 2-PE by microbial fermentation has gained extensive attention recently. Various microorganisms have the ability to synthesize 2-PE naturally with low yield. In recent years, using genetic engineering and synthetic biology technology, the microbial yield of 2-PE has been greatly improved by up-regulating the expression level of rate-limiting enzyme gene, improving precursors transport and enhancing the tolerance of 2-PE. The research progress of microbial synthesis of 2-PE is reviewed, the mechanism of key metabolic regulation is analyzed, current problems are addressed, and suggestions for improvement are proposed.
