Expanding the pH range of glutamate decarboxylase from L. pltarum LC84 by site-directed mutagenesis

Introduction: Glutamate decarboxylase is a class Ⅱ amino acid decarboxylase dependent onpyridoxal-5′-phosphate (PLP), which catalyzes the decarboxylation of substrateL-glutamate (L-Glu) to synthesize γ-aminobutyric acid (GABA). The low activity ofglutamic acid decarboxylase (GAD) and its ability to catalyze only under acidicconditions limit its application in biosynthesis of GABA.Methods: Taking glutamic acid decarboxylase from Lactobacillus plantarum, which produces GABA, as the research object, the mutation site was determined by amino acid sequence analysis of GAD, the mutation was introduced by primers, and the mutant was constructed by whole plasmid PCR and expressed in Escherichia coli. Then, the enzymatic properties of the mutant were analyzed. Finally, the three-dimensional structure of the mutant was simulated to support the experimental results.Results and Discussion: In this case, mutants E313S and Q347H of glutamate decarboxylase from L. pltarum LC84 (LpGAD) were constructed by targeted mutagenesis. Compared with the wild-type, their enzyme activity increased by 62.4% and 12.0% at the optimum pH 4.8, respectively. In the range of pH 4.0–7.0, their enzyme activity was higher than that of the wild-type, and enzyme activity of mutant E313S was 5 times that of the wild-type at pH 6.2. Visualization software PyMOL analyzed the 3D structure of the mutant predicted by homologous modeling, and the results showed that mutant E313S may broadened the reaction pH of LpGAD through the influence of surface charge, while mutant Q347H may broadened the reaction pH of LpGAD through the stacking effect of aromatic rings. In a word, mutants E313S and Q347H were improved the enzyme activity and were broadened the reaction pH of the enzyme, which made it possible for it to be applied in food industry and laid the foundation for the industrial production of GABA

Magnetic-field-induced splitting of Rydberg Electromagnetically Induced Transparency (EIT) and Autler-Townes (AT) spectra in 87^{87}Rb vapor cell

We theoretically and experimentally investigate the Rydberg electromagnetically induced transparency (EIT) and Autler-Townes (AT) splitting of $^{87}$Rb vapor under the combined influence of a magnetic field and a microwave field. In the presence of static magnetic field, the effect of the microwave field leads to the dressing and splitting of each $m_F$ state, resulting in multiple spectral peaks in the EIT-AT spectrum. A simplified analytical formula was developed to explain the EIT-AT spectrum in a static magnetic field, and the calculations are in excellent agreement with experimental results.We further studied the enhancement of the Rydberg atom microwave electric field sensor performance by making use of the splitting interval between the two maximum absolute $m_F$ states under static magnetic field. The traceable measurement limit of weak electric field by EIT-AT splitting method was extended by an order of magnitude, which is promising for precise microwave electric field measurement.Comment: 12 pages, 4 figure

Microwave electrometry with Rydberg atoms in a vapor cell using microwave amplitude modulation

We have theoretically and experimentally studied the dispersive signal of the Rydberg atomic electromagnetically induced transparency (EIT) - Autler-Townes (AT) splitting spectra obtained using amplitude modulation of the microwave (MW) field. In addition to the two zero-crossing points, the dispersion signal has two positive maxima with an interval defined as the shoulder interval of the dispersion signal $\Delta f_{\text{sho}}$. The relationship of MW field strength $E_{\text{MW}}$ and $\Delta f_{\text{sho}}$ are studied at the MW frequencies of 31.6 GHz, 22.1 GHz, and 9.2 GHz respectively. The results show that $\Delta f_{\text{sho}}$ can be used to character the much weaker $E_{\text{MW}}$ than the interval of two zero-crossing points $\Delta f_{\text{zeros}}$ and the traditional EIT-AT splitting interval $\Delta f_{\text{m}}$, the minimum $E_{\text{MW}}$ measured by $\Delta f_{\text{sho}}$ is about 30 times smaller than that by $\Delta f_{\text{m}}$. As an example, the minimum $E_{\text{MW}}$ at 9.2 GHz that can be characterized by $\Delta f_{\text{sho}}$ is 0.056 mV/cm, which is the minimum value characterized by frequency interval using vapour cell without adding any auxiliary fields. The proposed method can improve the weak limit and sensitivity of $E_{\text{MW}}$ measured by spectral frequency interval, which is important in the direct measurement of weak $E_{\text{MW}}$

Experimental Study of the Effects of Marrow Mesenchymal Stem Cells Transfected with Hypoxia-Inducible Factor-1α Gene

Objective. To construct the eukaryotic expression vector hypoxia-inducible factor 1α-pcDNA3.1 and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF-1α gene in MSCs. Methods. mRNA of Wistar Rats' myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF-1α was amplified by polymerase chain reaction (PCR), and constructed into pcDNA3.1. Transfected HIF-1α-pcDNA3.1 into MSCs by liposome mediated method. The expression of HIF-1α in the cells was detected by Western Blot Analysis and ELISA. Results. Eukaryotic expression vector HIF-1α-pcDNA3.1 was constructed successfully. Analyzed by flow cytometer, The MSCs' surfaces mark were CD44+, SH3(CD73)+, CD34−, CD45− and the CD44+ cells and SH3(CD73)+ cells were 94.7% and 97.3%, respectively, showing the high purity of the cultured MSCs. After inducing, the cultured MSCs can differentiate into osteoblasts and adipocytes successfully. In HIF-1α gene transfected MSCs, the expression of HIF-1α mRNA and HIF-1α protein were both increased obviously. Conclusion. HIF-1α was cloned successfully. HIF-1α-pcDNA3.1 can be transfected into MSCs by liposome-mediated method effectively and which resulting stable expression of HIF-1α in transfected MSCs

Population pharmacokinetics and initial dose optimization of tacrolimus in children with severe combined immunodeficiency undergoing hematopoietic stem cell transplantation

The present study aimed to explore the population pharmacokinetics and initial dose optimization of tacrolimus in children with severe combined immunodeficiency (SCID) undergoing hematopoietic stem cell transplantation (HSCT). Children with SCID undergoing HSCT treated with tacrolimus were enrolled for analysis. Population pharmacokinetics of tacrolimus was built up by a nonlinear mixed-effects model (NONMEM), and initial dose optimization of tacrolimus was simulated with the Monte Carlo method in children weighing <20 kg at different doses. A total of 18 children with SCID undergoing HSCT were included for analysis, with 130 tacrolimus concentrations. Body weight was included as a covariable in the final model. Tacrolimus CL/F was 0.36–0.26 L/h/kg from body weights of 5–20 kg. Meanwhile, we simulated the tacrolimus concentrations using different body weights (5–20 kg) and different dose regimens (0.1–0.8 mg/kg/day). Finally, the initial dose regimen of 0.6 mg/kg/day tacrolimus was recommended for children with SCID undergoing HSCT whose body weights were 5–20 kg. It was the first time to establish tacrolimus population pharmacokinetics in children with SCID undergoing HSCT; in addition, the initial dose optimization of tacrolimus was recommended

Nucleated red blood cells as a novel biomarker in the diagnosis and prediction of sepsis severity in children

IntroductionSepsis is a vitally serious disease leading to high mortality. Nucleated red blood cells (NRBCs) are present in some noninfectious diseases, but the relationship between NRBCs and sepsis in children remains unknown. The purpose of this study was to compare the clinical characteristics and outcomes of sepsis with positive NRBCs and negative NRBCs in children, and to further explore whether the count of NRBCs has a relationship with the severity of sepsis.MethodsWe enrolled children with sepsis who were admitted to the Children’s Hospital of Chongqing Medical University between January 2020 and December 2022. The children’s clinical data, laboratory data and outcomes were recorded and analyzed.ResultsOne hundred and fifteen children met the inclusion criteria in our study. Compared to negative NRBCs patients, the C-reactive protein, alanine transaminase, urea nitrogen values, mortality rate and length of hospitalization were found to be significantly increased, while platelet counts, and hemoglobin were significantly decreased in sepsis patients with positive NRBC (P < 0.05). Receiver operating characteristic (ROC) curves analysis showed that the optimal cutoff value of the NRBC count in the diagnosis of severe sepsis was 3, with a sensitivity of 87.5% and specificity of 94.9%. The area under the ROC curve was 0.877 (95% CI: 0.798-0.957).DiscussionThese findings demonstrated that NRBC count has the potential to be a biomarker for the diagnosis of sepsis in children, especially an NRBC count greater than 3, which may predict the severity and poor prognosis in children suffering from sepsis

Poly(lactic acid)/cellulose nanocrystal composites via the Pickering emulsion approach: Rheological, thermal and mechanical properties

The use of nanocellulose is an attractive method to improve the characteristics of biodegradable polymers, but its effects are often affected by uneven dispersion. In this work, cellulose nanocrystals (CNCs) were evenly dispersed into poly(lactic acid) (PLA) via the Pickering emulsion approach. The PLA/CNC composites prepared were studied by rheological, thermal as well as mechanical measurements. Changes in the rheological characteristics of the composites showed that CNC promoted the transition of the composites from fluid to solid-like behavior at high temperatures. The introduction of 5 wt% CNC improved the crystallinity of PLA considerably and increased the onset temperature of crystallization by about 10 °C. The storage modulus of the composites increased throughout the entire temperature range of testing. Flexural modulus was improved considerably. All the results indicated that the Pickering emulsion approach improved the dispersion of CNC in the PLA matrix and CNC improved efficiently most properties of PLA