Reshaping Phosphatase Substrate Preference for Controlled Biosynthesis Using a "Design-Build-Test-Learn" Framework.

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time-consuming trial-and-error process. This study presents a quantum mechanics (QM)-incorporated design-build-test-learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N-acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field-based methods, resulting in a 1.4-fold increase in substrate preference (kcat/Km) toward GlcNAc-6-phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol-1 reduction in activation energy. Furthermore, an iterative computer-aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5-fold increase in kcat-GlcNAc6P/Km-GlcNAc6P and a 59% decrease in kcat-Glc6P/Km-Glc6P is highly desirable compared to the wild type in the GlcNAc-producing chassis. The GlcNAc titer increases to 217.3 g L-1 with a yield of 0.597 g (g glucose)-1 in a 50-L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.

Determination and Correlation of Solubility of N-Acetylglucosamine in Four Aqueous Binary Solvents from 283.15 to 323.15 K.

N-Acetylglucosamine (NAG) is a significant novel functional monosaccharide with a wide range of potential applications, such as in the medical and cosmetic fields. A gravimetric technique was used to assess the solubility of NAG in water, methanol, N,N-dimethylformamide, water + methanol, water + n-propanol, water + N,N-dimethylformamide, and water + acetonitrile at temperatures ranging from 283.15 to 323.15 K. The outcomes of the experiment demonstrated that the temperature and water content were positively correlated with the solubility of NAG in four experimental binary solvents. The order of solubility in the four aqueous solvent mixtures is water + DMF > water + methanol > water + n-propanol > water + acetonitrile. The solubility data was well correlated using the modified Apelblat model, the CNIBS/R-K model, and the Apelblat-Jouyban-Acree model. Experimental data from NAG will help guide the design of cooling and dissolution in crystallization processes.

Elevated NKT cell levels in adults with severe chronic immune thrombocytopenia.

The aim of this study was to investigate the frequency of circulating natural killer T (NKT) cells and regulatory T cells (Tregs), as well as serum cytokine profiles, in adult chronic primary immune thrombocytopenia (ITP). The frequency of circulating T cell receptor (TCR) Vα24+Vß11+CD3+ NKT cells and CD4+CD25+CD127-/low Tregs was measured using multi-color flow cytometry. The serum concentrations of 11 cytokines were determined with a cytometric bead assay. The frequency of circulating NKT cells in patients with ITP was 0.13±0.03%, whereas the frequency in healthy controls was 0.07±0.01% of CD3+ (P>0.05). However, the frequency of NKT cells in patients with ITP with platelet counts ≤20×109/l (0.22±0.05%) was significantly higher than that in patients with platelet counts >20×109/l (0.05±0.01%; P<0.05) and that in healthy controls (0.07±0.01%; P<0.05). The frequency of peripheral Tregs was comparable between patients with ITP (3.97±0.44% of CD4+) and healthy controls (3.69±0.31%; P>0.05). No significant differences were observed in the serum concentrations of 11 cytokines between patients with ITP and healthy controls, despite the fact that the serum levels of interleukin (IL)-12p70, IL-8, IL-4, interferon (IFN)-γ and tumor necrosis factor (TNF)-α in patients with ITP were higher than those in the healthy controls. The platelet count was negatively correlated with the frequency of circulating NKT cells in chronic ITP. These results indicate that NKT cells may be involved in ITP with severe thrombocytopenia, and NKT and Tregs may be important in cytokine deregulation in chronic ITP.

[Expression and relationship of p27(kip1) and its related molecules Jab1 and CRM1 during proliferation of lymphoma cells U937].

OBJECTIVE: To investigate the expression and relationship of p27(kip1) and its related molecules Jab1 and CRM1 during proliferation of lymphoma cells U937. METHODS: U937 cells were treated with serum starvation and release, and the effects of these treatments on the cell growth was tested with cell number counting. The expression and localization of p27(kip1), Jab1 and CRM1 in U937 cells were detected by Western blot, double immunolabelling and laser scanning confocal microscopy. RESULTS: The growth of U937 cells was blocked by serum starvation. The total protein of p27(kip1) was increased while Ser10-phosphorylated p27(kip1) -related molecules Jab1 and CRM1 were decreased. Meanwhile, the location of p27(kip1) was changed from cytoplasm into nuclei. After serum release, the location of p27(kip1) expression reappeared in the cytoplasm again. CONCLUSION: During the proliferation process of lymphoma U937 cells, Jab1 and CRM1 may influence the location and expression of p27kip1, and may participate in regulation of growth of NHL cells.