ABSTRACT
Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.
ABSTRACT
Hepatocyte nuclear factor (HNF)-1alpha is one of the liver-enriched transcription factors involved in many tissue-specific expressions of hepatic genes. The molecular mechanisms for determining HNF1alpha-mediated transactivation have not been explained fully. To identify unknown proteins that interact with HNF1alpha, we developed a co-IP-MS strategy to search HNF1alpha interactions, and high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel HNF1alpha-interacting protein. In vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation studies confirmed an interaction between HMGB1 and HNF1alpha. The protein-protein interaction was mediated through the HMG box domains of HMGB1 and the homeodomain of HNF1alpha. Furthermore, electrophoretic mobility shift assay and chromatin-immunoprecipitation assay demonstrated that HMGB1 was recruited to endogenous HNF1alpha-responsive promoters and enhanced HNF1alpha binding to its cognate DNA sequences. Moreover, luciferase reporter analyses showed that HMGB1 potentiated the transcriptional activities of HNF1alpha in cultured cells, and downregulation of HMGB1 by RNA interference specifically affected the HNF1alpha-dependent gene expression in HepG2 cell. Taken together, these findings raise the intriguing possibility that HMGB1 is a new cofactor of HNF1alpha and participates in HNF1alpha-mediated transcription regulation through protein-protein interaction.
