Research experimental design for the construction and identification of the pGEX-BCKD-E4A recombinant point-mutant plasmid.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that eventually develops into cirrhosis and even liver cancer. In recent years, the incidence rate has been increasing, and the early diagnosis and treatment of PBC are crucial. In the early diagnosis method of PBC, anti-mitochondrial antibodies (AMAs) are an important diagnostic basis, especially the M2 subtype (AMA-M2) with almost 100% specificity. We selected the BCOADC-E2 protein, a mitochondrial autoantigen that reacts specifically with AMA-M2 antibodies, and carried out DNA recombination and protein mutation experiments by cloning in vitro the homologous target gene sequence BCKD that expresses the antigenic epitope of BCOADC-E2 protein, to provide experience for later exploring the effect of mutations of amino acids around the lysine in the active center of BCOADC-E2 protein on its specific binding to AMA-M2, and to lay the foundation for determining the key amino acids of BCOADC-E2 for the diagnosis and treatment of PBC. In addition, we apply this scientific research content to graduate course teaching. Experimental technology of microbial molecular ecology is a course with the cross-integration of multidisciplinary knowledge and experimental skills offered at our college since 2018. This article derives from the part of this course on the construction of recombinant plasmids. The students first constructed the recombinant plasmid pGEX-BCKD using the vector plasmid pGEX-4T1 and the target gene fragment BCKD provided by the laboratory and used this as a template to construct the pGEX-BCKD-E4A point mutation plasmid by the overlap extension PCR (SOE PCR) technique to achieve the effect of mutating the fifth amino acid glutamate in front of lysine, the active centre of the BCOADC-E2 lipid acyl binding domain, to alanine for subsequent studies. Through the research experiment, combining theoretical knowledge and experimental operation, we aim to deepen the student's understanding of DNA recombination technology, let them feel the practical application prospect of experimental technology, stimulate students' interest in professional knowledge learning, and cultivate students' scientific thinking and innovation consciousness. We examined the quality of the teaching through the process and summative evaluation of the students. In this study, the students successfully completed the construction of pGEX-BCKD-E4A point mutant plasmid, and the average test score increased from 40.4% before teaching to 91.1%. The teaching effect was remarkable. This kind of research experimental teaching mode has good application prospects, and other education and teachers can refer to and reference it.

Are medical record front page data suitable for risk adjustment in hospital performance measurement? Development and validation of a risk model of in-hospital mortality after acute myocardial infarction.

OBJECTIVES: To develop a model of in-hospital mortality using medical record front page (MRFP) data and assess its validity in case-mix standardisation by comparison with a model developed using the complete medical record data. DESIGN: A nationally representative retrospective study. SETTING: Representative hospitals in China, covering 161 hospitals in modelling cohort and 156 hospitals in validation cohort. PARTICIPANTS: Representative patients admitted for acute myocardial infarction. 8370 patients in modelling cohort and 9704 patients in validation cohort. PRIMARY OUTCOME MEASURES: In-hospital mortality, which was defined explicitly as death that occurred during hospitalisation, and the hospital-level risk standardised mortality rate (RSMR). RESULTS: A total of 14 variables were included in the model predicting in-hospital mortality based on MRFP data, with the area under receiver operating characteristic curve of 0.78 among modelling cohort and 0.79 among validation cohort. The median of absolute difference between the hospital RSMR predicted by hierarchical generalised linear models established based on MRFP data and complete medical record data, which was built as 'reference model', was 0.08% (10th and 90th percentiles: -1.8% and 1.6%). In the regression model comparing the RSMR between two models, the slope and intercept of the regression equation is 0.90 and 0.007 in modelling cohort, while 0.85 and 0.010 in validation cohort, which indicated that the evaluation capability from two models were very similar. CONCLUSIONS: The models based on MRFP data showed good discrimination and calibration capability, as well as similar risk prediction effect in comparison with the model based on complete medical record data, which proved that MRFP data could be suitable for risk adjustment in hospital performance measurement.

An environmentally friendly and fast approach to prepare reduced graphite oxide with water and organic solvents solubility.

In this paper, reduced graphite oxide (RGO) was prepared using thiourea dioxide as reductant and polyvinylpyrrolidone (PVP) as stabilizer. Thiourea dioxide, a cheap and nontoxic industrialized material, was demonstrated to be an efficient reducing agent for graphite oxide (GO) in this paper. Ultraviolet and visible (UV-vis) spectroscopy results revealed that the reduction of GO could be readily achieved in 10 min, a reaction time which is much shorter than those required in common reduction reactions. The procedures of reduction including the by-products are all nontoxic, thus it is absolutely environmentally friendly. To the best of our knowledge, this is the first time that thiourea dioxide was successfully used to prepare RGO. Moreover, the stabilizer, PVP, which could be easily absorbed onto the surface of RGO, provided RGO with good water and organic solvents solubility, with low conductivity though. However, by controlling the content of PVP, RGO with balanced solubility and conductivity can be obtained. The resultant RGO could be used as nanofillers to prepare conductive materials and biomaterials with potential applications as electrical devices or biosensors.

Wet-grinding assisted ultrasonic dispersion of pristine multi-walled carbon nanotubes (MWCNTs) in chitosan solution.

Ultrasonication is often used to disperse nano-particles in aqueous solution. However, a good dispersion of nano-particles in aqueous solution is not always achieved, due to the fact that incoming ultrasonicwaves in liquid are usually reflected and damped at the gas/liquid interface. In this work, we report a so-called wet-grinding assisted ultrasonication (GU) method, in which wet-grinding of multi-walled carbon nanotubes (MWCNTs) in chitosan solution is carried out before ultrasonication. The dispersions of MWCNTs were characterized by visual comparison, UV/vis spectroscopy, and scanning electron microscopy (SEM). The results demonstrate that the dispersion quality of chitosan/MWCNT suspension prepared by wet-grinding assisted ultrasonication is much better than that by ultrasonication or wet-grinding alone. It was found that wet-grinding could improve the water wettability of MWCNTs and eliminate the barrier of air layer around MWCNTs to ultrasonicwaves. Meanwhile, the composite from the chitosan/MWCNTs suspension prepared by GU method has an obvious improvement in mechanical property compared to pure chitosan. This simple method for integrating MWCNTs and biocompatible chitosan into a homogeneous dispersion may have great potential application in biotechnology, such as preparing composite materials for medicine, bio-fiber, biosensor, antibacterial coating, and cell cultivation.

A simple and efficient method to prepare graphene by reduction of graphite oxide with sodium hydrosulfite.

Inspired by an ancient reducing method used in textile production, sodium hydrosulfite was used to reduce graphite oxide as an efficient reducing agent in our work. The reduced materials were characterized by x-ray photoelectron spectroscopy, thermogravimetric analysis, wide-angle x-ray scattering, Raman spectroscopy, solid state (13)C NMR spectroscopy and electrical conductivity measurements, respectively. The results showed that graphite oxide can be reduced with sodium hydrosulfite in a few minutes, with a degree of reduction comparable to those achieved with hydrazine. It provides an efficient method to reduce graphite oxide and could be used as a method to prepare novel composites.