Discussion on the need for correction during isotopic analysis of nitrogen by the denitrifier method.

The nitrogen and oxygen isotopes of NO3 - are effectively used to trace the main nitrogen sources and migration processes in the atmosphere, water and soil. NO3 - can be converted into N2O by the bacterial denitrification method, which is an advanced method with high sensitivity. However, due to the existence of a small but inevitable blank during the whole experimental process, the N isotopic signal of N2O produced by denitrification superimposes on that of the N blank. Currently, the standard curve correction method is used to correct measured nitrogen isotope results to mitigate blank interference. It has been reported that high variability of the nitrogen isotope results have been produced by the denitrifier method by conducting an interlaboratory comparison of denitrifier methods and other methods on standards and environmental samples, and to reduce this problem, the nitrogen isotope calibration process with a standard curve is examined in depth in this paper, which uses PreCon-GC-IRMS to determine the nitrogen isotopes in N2O. We demonstrate for the first time that reliable results can be obtained without correction for samples with nitrogen isotope composition ranging from -9.9 to 19.5‰, which covers the natural sample range. This study establishes the double test approach for the bacterial denitrification method, ensuring the accuracy and long-term stability of different batches of nitrogen isotope results.

Development of a risk prediction nomogram for sarcopenia in hemodialysis patients.

BACKGROUND: Sarcopenia is associated with various adverse outcomes in hemodialysis patients. However, current tools for assessing and diagnosing sarcopenia have limited applicability. In this study, we aimed to develop a simple and reliable nomogram to predict the risk of sarcopenia in hemodialysis patients that could assist physicians identify high-risk patients early. METHODS: A total of 615 patients undergoing hemodialysis at the First Affiliated Hospital College of Medicine Zhejiang University between March to June 2021 were included. They were randomly divided into either the development cohort (n = 369) or the validation cohort (n = 246). Multivariable logistic regression analysis was used to screen statistically significant variables for constructing the risk prediction nomogram for Sarcopenia. The line plots were drawn to evaluate the effectiveness of the nomogram in three aspects, namely differentiation, calibration, and clinical net benefit, and were further validated by the Bootstrap method. RESULTS: The study finally included five clinical factors to construct the nomogram, including age, C-reactive protein, serum phosphorus, body mass index, and mid-upper arm muscle circumference, and constructed a nomogram. The area under the ROC curve of the line chart model was 0.869, with a sensitivity and specificity of 77% sensitivity and 83%, the Youden index was 0.60, and the internal verification C-statistic was 0.783. CONCLUSIONS: This study developed and validated a nomogram model to predict the risk of sarcopenia in hemodialysis patients, which can be used for early identification and timely intervention in high-risk groups.

Identification of nitrate sources in the Jing River using dual stable isotopes, Northwest China.

Nitrate (NO3-) contamination has become a dominant international problem in the aquatic environment, so identifying the sources and transformations of NO3- is the basis for improving water quality. Since the Jing River is the largest tributary of the Wei River, to understand its water quality, this study collected surface water samples from the Shaanxi section of the Jing River during the dry season. The potential sources of NO3- were analyzed by hydrochemical and bi-isotopic methods, and the SIAR model was used to estimate the proportional contribution of each source. Results indicated that NO3--N was the main form of inorganic nitrogen in this area, and the average total nitrogen content was 10.23 mg·L-1, which showed that nitrogen pollution was highly serious; the transformation process of nitrogen in this study area was mainly nitrification; The results of Bayesian model showed that manure and sewage contributed to the most NO3- (64.39%) in the dry season, followed by soil nitrogen, which was 26.35%. These results help to adopt better nitrogen management measures to meet the national environmental quality standards for surface water.

[Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides].

Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.

Molecular engineering of chitinase from Bacillus sp. DAU101 for enzymatic production of chitooligosaccharides.

Enzymatic production of chitooligosaccharides has high value in medicine and other fields. However, low chitinase activity and yield of chitooligosaccharides limit the production and application. Herein, we used a series of molecular biology strategies to increase the expression of chitinase in Bacillus subtilis WB600. Upon addition of the signal peptide NprB, Chisb was successfully secreted to the outside of the cell and extracellular expression level reached 35.54 U/mL. Furthermore, optimizing Ribosome Binding Sites (RBSs) with spacer sequences, and combining molecular docking technology with site-directed mutagenesis, the expression level and the specific activity of Chisb was further increased to 51.67 U/mL and 249.62 U/mg, respectively. When colloidal chitin was used as the substrate, the chitooligosaccharides detected by ion chromatography were (GlcNAc)1-5, and the total yield of chitooligosaccharides was 14.4%. Our results indicate that strategies for increasing Chisb expression contribute to the study and application of chitinase and the production of chitooligosaccharides.