Transcription factor (TF)-based biosensors have been widely applied in metabolic engineering, synthetic biology, metabolites monitoring, etc. These biosensors are praised for the high orthogonality, modularity, and operability. However, most natural TFs with weak responses and low specificity still demand optimization for desired performance in applications. Herein, we comprehensively summarize the recent advances in the engineering and optimization of TF-based biosensors with the assistance of computational simulation and artificial intelligence. This review includes the regulatory protein engineering aided by protein structure prediction and ligand binding simulation and the regulatory protein responses predicted by a mathematical model obtained from machine learning of mutagenesis data. In comparison with conventional tools, computational simulation and artificial intelligence enable more accurate and rapid design and construction of biosensors. Thus, these technologies will greatly promote the development of novel biosensors for applications.
Biosensing Techniques/methods* ; Transcription Factors/metabolism* ; Artificial Intelligence ; Protein Engineering/methods* ; Computer Simulation ; Synthetic Biology ; Machine Learning

Objective:To establish the characteristic spectrum of Liushenqu standard decoction using ultra-high performance liquid chromatography (UPLC); To determine the contents of related index components; To evaluate the quality of Liushenqu standard decoction.Methods:UPLC method was used to establish characteristic spectrum of Liushenqu standard decoction. Chromatographic Fingerprint Similarity Evaluation System (2012 edition) was used for similarity analysis, the characteristic peak was assigned, and the content of its index components was determined.Results:The characteristic peaks of Liushenqu standard decoction were calibrated and 8 components were identified, namely uridine, adenosine, guanosine, 5-hydroxymethylfurfural, tryptophan, vanillic acid, ferulic acid and shaftaside. The contents of uridine, adenosine, tryptophan ferulic acid and shaftaside in 10 batches of Liushenqu standard decoction were simultaneously determined, and ranged from 0.036 1～0.383 9 mg/g, 0.030 7～0.170 2 mg/g, 0.007 0～0.060 2 mg/g, 0.001 0～0.005 0 mg/g, 0.000 8～0.013 8 mg/g, respectively. The transfer rates ranged from 44.2% to 50.8%, 60.1% to 67.7%, 60.4% to 76.4%, 62.7% to 77.4%, 50.7% to 61.4%, respectively.Conclusion:The established UPLC characteristic spectrum and content determination method are accurate and repeatable, which can provide references for quality control of Liushenqu standard granules.

Objective:To establish UPLC characteristic spectrum of Hujiao standard decoction and the determination method for the content of piperine.Methods:15 batches of freeze-dried powder of Hujiao standard decoction were prepared according to the traditional decocting method. The range of paste yield was determined, and the UPLC characteristic spectrum of the standard decoction was established. High-resolution mass spectrometry and control products were used to identify common peaks. Based on the common peak area, the weights of each peak were compared using entropy weight method, and correlation analysis and similarity evaluation were conducted using clustering analysis and grey correlation method; a method for determining the content of piperine in Hujiao decoction pieces and freeze-dried powder of standard decoction was simultaneously established, and the transfer rate was calculated.Results:The extraction rate of 15 batches of freeze-dried powder of Hujiao standard decoction ranged from 10.4% to 16.8%, with an average of 14.0%. The content of piperine ranged from 12.2 to 30.0 mg/g, with an average of 18.5 mg/g, and the transfer rate ranged from 4.0% to 7.8%, with an average of 5.8%. Six common peaks were identified in the established characteristic spectrum and identified by high-resolution mass spectrometry and control products respectively. Peak 1 was N-trans-feruloyltyramine, peak 3 was piperine and the similarity was 0.959-1.000. Clustering analysis and grey correlation analysis showed that there was little difference between quality of Piperis Fructus and origins.Conclusion:In this study, the characteristic spectrum and content determination method of freeze-dried powder of Hujiao standard decoction are established, which can provide references for quality detection and control of Hujiao standard decoction or its derivative products.

Objective:To establish HPLC chromatogram for Aspongopus; To determine 8 nucleoside components of uracil, adenine, uridine, uric acid, hypoxanthine, adenosine, xanthine and canine quinolinic acid; To provide reference for quality control and evaluation.Methods:The Agilent ZORBAX SB-Aq chromatographic column (4.6 mm×250 mm, 5 μm) was used for gradient elution with mobile phases consisting of a methanol (A) and 0.05% phosphoric acid (B). The column temperature was 25 ℃, the flow rate was 0.8 ml/min, and the detection wavelength was 254 nm. HPLC chromatograms for Aspongopus were established and the contents of 8 components were determined.Results:The characteristic chromatogram of 15 batches aspongopus herbs was established. A total of 10 common characteristic peaks were identified and 8 were identified. The similarity between the characteristic chromatogram of samples and the control chromatogram was 0.969-0.997. The content determination showed that the linear range of uracil, adenine, urin, uric acid, hypoxanthine, adenosine, xanthine and xanuric acid was among 0.002 0-0.644 0, 0.001 4-0.448 0, 0.001 0-1.257 0, 0.005 4-6.221 0, 0.001 0-0.724 0, 0.001 0-0.644 0, 0.002 0-1.113 0, 0.003 8-2.059 0 μg, respectively, with a good linear relationship ( r≥0.999); the repeatability and stability of RSD were <2.0%, and the average sampling recovery rate was between 99.36% and 103.40%. Conclusion:The characteristic chromatogram and content determination method established in this study are simple, reliable, reproducible and accurate, and can be used for the qualitative and quantitative analysis of Aspongopus and can provide a reference for the quality evaluation method of the Aspongopus.

Objective:To conduct a multidimensional quantification and analysis of the development trajectory of both "quality" and "quantity" in China's higher education from 2001 to 2021, and lay a solid foundation for the construction of a high-quality education system.Methods:Based on macro statistical data on education in China from 2001 to 2021, a comprehensive indicator system for the development level of higher education was established. This system included six dimensions: educational conditions, financial support, institutional safeguards, scientific research, social services, and international collaboration. Methods such as entropy-weighted TOPSIS, general difference index, spatial autocorrelation, and hot spot analysis were used to evaluate the level of high-quality development of higher education and its spatial differentiation. Additionally, an obstacle degree analysis model was used to evaluate the factors hindering the high-quality development of higher education.Results:The high-quality development level of China's higher education has steadily improved, with a growth rate of 2.85%. The spatial distribution exhibited a clustering pattern, with stable hotspot regions established in the eastern region characterized by significant "spillover" effects. Concurrently, the western regions narrowed the development gap compared with other areas. Scientific research represents the primary challenge to achieving high-quality development in higher education, with an average obstacle degree of 18.46%. Books per student, fixed assets per student, foreign technology imported per institution, research & development project funds, research & development projects per institution, and personnel investment in research & development projects were important constraints on the development of higher education in China and the provinces.Conclusions:Resource sharing and linkage need to be guaranteed by policy and institutional support, and each subject of higher education should give full play to its own advantages to contribute to the high-quality development of higher education through interregional linkage development, mutual synergy, and common progress.

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.
Glycoside Hydrolases/metabolism* ; Hydrogen-Ion Concentration ; Rhamnose ; Substrate Specificity ; Temperature

Objective:To screen out and explore the core gene (Hub gene) involvement and the potential role of cyclin B2 (CCNB2) in the development and prognosis of hepatocellular carcinoma (HCC) through bioinformatics methods.Methods:Four HCC-related datasets were screened, and downloaded from the GEO database. GEO2R tool was used to analyze data and identify the differentially expressed genes (DEGs). Gene Ontology (GO) and KEGG signal pathway enrichment analysis were completed using DAVID database and Cytoscape (ClueGO) plug-in, respectively. Protein-protein interaction network (PPI) of DEGs was established using the STRING database. Cytoscape software was used to visualize PPI network, key modules (cluster) construction and core genes identification. UCSC and UALCAN database were used to analyze the differential expression and survival of TCGA hepatocellular carcinoma core genes. Firebrowse, Oncomine and UALCAN databases were used to analyze the expression of core genes in multiple tumors including HCC. Real-time quantitative reverse transcription PCR (RT-qPCR) was used to detect the expression levels of candidate genes in HCC tissues and liver cancer cell lines.Results:A total of 73 DEGs were identified from the four datasets, including 15 up-regulated genes and 58 down-regulated genes. KEGG pathway enrichment analysis signal showed that DEGs were mainly enriched in tumor-related pathways. PPI network based on DEGs had screened the key modules and 10 core genes. CCNB2 and NCAPG were highly expressed in liver cancer tissues in multiple databases. CCNB2 was positively correlated with NCAPG and was considered as a key gene related to prognosis ( P < 0.01). RT-qPCR results showed that CCNB2 was highly expressed in human HCC tissues and cell lines ( P < 0.01). Conclusion:Successfully screened DEGs and core genes related to HCC. Among them, CCNB2 is highly expressed in HCC and is related to the survival and prognosis of patients, so it is expected to become a biomarker for the diagnosis and prognosis of HCC.

Objective To explore the effectiveness of self-care intervention for elderly patients with diabetes mellitus in rural community. Methods We randomly selected 100 elderly patients with diabetes from January 2013 to December 2013 in rural community health service station in Yizhou, Guangxi Zhuang Autonomous Region. Individualized intervention of diabetes-related knowledge was delivered to patients. Fasting blood glucose and glycated hemoglobin were compared before and after the intervention. The effectiveness of intervention was investigated by diabetes self-care scale. Results Levels of fasting blood glucose [(5. 91 ± 0. 10)mmol/L] and glycated hemoglobin [(4. 62 ± 0. 14)%] from 100 patients were lower than those before the intervention after 1 year intervention (t=11. 840,12. 379;P<0. 01). After intervention, the total score of diabetes self-care scale was improved to (89. 66 ± 1. 56) from (75. 32 ± 1. 50) (t = -7. 597,P <0. 01). Conclusions Blood glucose can be effectively controlled by helping the rural elderly diabetic patients to learn about self-care.

OBJECTIVE: To investigate the infection in spotted fever group Rickettsiae (SFGR) in wild rodents from Heilongjiang, China. METHODS: Polymerase chain reaction (PCR) was used to detect the OmpA gene of SFGR in rodents collected in Heilongjiang. The PCR products amplified from rodent specimens were sequenced and compared with the corresponding part of the sequences deposited in the GenBank. Phylogenetic trees were constructed with Mega 5.0 software. RESULTS: A total of 514 rodents were collected from Heilongjiang during 2009-2011 and 11 species were included. The infection rate of SFGR in the rodents was 9.3% (95% CI: 7.1%-12.2%). Statistical analysis showed a significant difference in different areas of Heilongjiang (P=0.023). The highest prevalence was observed in Mudanjing area (12.42%). There were significant differences in different species of rodents (P=0.002). The infection rate of SFGR determined in Clethrionomys rufocanus was the highest (22.1%). Sequence analysis revealed SFGR belonged to R.heilongjiangensis and a new unknown rickettsia genotype. CONCLUSION R.heilongjiangensis has been presented in rodents in Heilongjiang, and a new SFGR genotype different from other rickettsiae genotypes may exist in this area.
Animals ; China ; DNA, Bacterial ; genetics ; isolation & purification ; Forests ; Phylogeny ; Polymerase Chain Reaction ; Rats ; Rickettsia ; classification ; genetics ; isolation & purification ; Rickettsia Infections ; microbiology ; veterinary ; Rodentia ; microbiology ; Sequence Analysis

As an efficient and promising protein engineering strategy, directed evolution includes the construction of mutant libraries and screening of desirable mutants. A rapid and high-throughput screening method has played a critical role in the successful application of directed evolution strategy. We reviewed several high-throughput screening tools which have great potential to be applied in directed evolution. The development of powerful high-throughput screening tools will make great contributions to the advancement of protein engineering.
Directed Molecular Evolution ; methods ; High-Throughput Screening Assays ; methods ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; genetics ; Protein Engineering ; methods