Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod).

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/µL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

An extraction-free method for rapid detection of CYP2C19 * 2/3/17 polymorphisms in one tube using melting curve analysis.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction (PCR). This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies µL-1 ; that of genomic samples was 0.1 ng µL-1 . The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1 h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n = 93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing. Due to its accuracy, rapidity, simple operation, and low cost, detection technology based on real-time polymerase amplification combined with melting curve analysis is expected to become a powerful tool for detecting and guiding clopidogrel use in countries with limited resources.

SOX4 promotes beige adipocyte-mediated adaptive thermogenesis by facilitating PRDM16-PPARγ complex.

Brown and beige fat protect against cold environments and obesity by catabolizing stored energy to generate heat. This process is achieved by controlling thermogenesis-related gene expression and the development of brown/beige fat through the induction of transcription factors, most notably PPARγ. However, the cofactors that induce the expression of thermogenic genes with PPARγ are still not well understood. In this study, we explored the role of SOX4 in adaptive thermogenesis and its relationship with PPARγ. Methods: Whole transcriptome deep sequencing (RNA-seq) analysis of inguinal subcutaneous white adipose tissue (iWAT) after cold stimulation was performed to identify genes with differential expression in mice. Indirect calorimetry detected oxygen consumption rate and heat generation. mRNA levels were analyzed by qPCR assays. Proteins were detected by immunoblotting and immunofluorescence. Interaction of proteins was detected by endogenous and exogenous Co-IP. ChIP-qPCR, FAIRE assay and luciferase reporter assays were used to investigate transcriptional regulation. Results: SOX4 was identified as the main transcriptional effector of thermogenesis. Mice with either adipocyte-specific or UCP1+ cells deletion of SOX4 exhibited significant cold intolerance, decreased energy expenditure, and beige adipocyte formation, which was attributed to decreased thermogenic gene expression. In addition, these mice developed obesity on a high-fat diet, with severe hepatic steatosis, insulin resistance, and inflammation. At the cell level, loss of SOX4 from preadipocytes inhibited the development of beige adipocytes, and loss of SOX4 from mature beige adipocytes reduced the expression of thermogenesis-related genes and energy metabolism. Mechanistically, SOX4 stimulated the transcriptional activity of Ucp1 by binding to PPARγ and activating its transcriptional function. These actions of SOX4 were, at least partly, mediated by recruiting PRDM16 to PPARγ, thus forming a transcriptional complex to elevate the expression of thermogenic genes. Conclusion: SOX4, as a coactivator of PPARγ, drives the thermogenic gene expression program and thermogenesis of beige fat, promoting energy expenditure. It has important physiological significance in resisting cold and obesity.

Construction and Analysis of Gene Co-Expression Networks in Escherichia coli.

Network-based systems biology has become an important method for analyzing high-throughput gene expression data and gene function mining. Escherichia coli (E. coli) has long been a popular model organism for basic biological research. In this paper, weighted gene co-expression network analysis (WGCNA) algorithm was applied to construct gene co-expression networks in E. coli. Thirty-one gene co-expression modules were detected from 1391 microarrays of E. coli data. Further characterization of these modules with the database for annotation, visualization, and integrated discovery (DAVID) tool showed that these modules are associated with several kinds of biological processes, such as carbohydrate catabolism, fatty acid metabolism, amino acid metabolism, transportation, translation, and ncRNA metabolism. Hub genes were also screened by intra-modular connectivity. Genes with unknown functions were annotated by guilt-by-association. Comparison with a previous prediction tool, EcoliNet, suggests that our dataset can expand gene predictions. In summary, 31 functional modules were identified in E. coli, 24 of which were functionally annotated. The analysis provides a resource for future gene discovery.