[Clinical Application of Screening Cell Combination Method in the Prediction of Red Blood Cell Alloantibody].

OBJECTIVE: To explore the clinical application of screening cell combination method in the prediction of red blood cell alloantibody, so as to provide basis for clinical diagnosis. METHODS: From October 2018 to April 2020, 9 680 samples were screened with automatic blood group instrument, 79 patients with positive alloantibodies were identified by 4 sets of screening cells from different manufacturers (referred to as combined method). At the same time, cell panel Panocell-16 was used for comparative analysis. Meanwhile, the combined method was also used to identify the antibodies of 20 samples from National Center for Clinical Laboratories external quality assessment (EQA) in China and 12 samples from WHO EQA. RESULTS: The 79 alloantibodies included anti-Mia antibody (7 cases), anti-M antibody (13 cases), anti-Lea antibody (9 cases), anti-P1 antibody (2 cases), anti-E antibody (22 cases), anti-c + E antibody (9 cases), anti-D antibody (4 cases), anti-e antibody (2 cases), anti-C + e antibody (3 cases), anti-C antibody (3 cases), anti-H antibody (1 case), anti-Fyb antibody (1 case), anti-E + M antibody (1 case), autoantibody + anti-E (2 cases). However, 7 cases of anti-Mia antibody and 1 case of anti- Lea antibody were missed in Panocell-16 identification results. The results of antibody identification in 20 samples from National Center for Clinical Laboratories EQA in China and 12 samples from WHO EQA were 100% accurate by combination method. The identification results between combined method identification and Panocell-16 identification showed no significant difference. CONCLUSION: The combined method can identify the alloantibodies of red blood cells in Chinese population. The screening cells can be used for screening of irregular antibodies without wasting reagents at the same time.

Hydroximoyl fluorides as the precursors of nitrile oxides: synthesis, stability and [3 + 2]-cycloaddition with alkynes.

The transformation of hydroximoyl fluorides to nitrile oxides for [3 + 2]-cycloaddition with alkynes has been achieved for the first time. The hydroximoyl fluorides used in this work appeared to be not stable, which was proved by a series of experiments. A DFT calculation was performed to better understand the properties of hydroximoyl fluorides. Although not stable, the hydroximoyl fluorides could be successfully converted to the corresponding nitrile oxides for in situ [3 + 2]-cycloaddition with alkynes to yield the isoxazoles. Furthermore, it was feasible to conduct [3 + 2]-cycloaddition reaction without purification after the synthesis of hydroximoyl fluorides from gem-difluoroalkenes. By investigating a class of interesting yet previously rarely explored fluorinated compounds, this work sheds new light on the stability and reactivity of a C-F bond on a C[double bond, length as m-dash]N double bond.

Identification of a novel fumarase C from Streptomyces lividans TK54 as a good candidate for L-malate production.

Fumarase is a key enzyme that catalyzes the reversible hydration of fumarate to L-malate in the tricarboxylic acid cycle. This reaction has been extensively utilized for industrial applications in producing L-malate. In this study, a fumarase C gene from Streptomyces lividans TK54 (slFumC) was cloned and expressed as a fused protein (SlFumC) in Escherichia coli. The molecular mass of SlFumC was about 49 kDa determined by SDS-PAGE. Kinetic studies showed that the K m value of SlFumC for L-malate increased by approximately 8.5-fold at pH 6.5 (6.7 ± 0.81 mM) to 8.0 (57.0 ± 1.12 mM), which was higher than some known fumarases. The catalytic efficiency (k cat) and the specific activity increased by about 9.5-fold at pH 6.5 (65 s(-1)) to 8.0 (620 s(-1)) and from 79 U/mg at pH 6.5 to 752 U/mg at pH 8.0, respectively. Therefore, SlFumC may acquire strong catalytic ability by increasing pH to partially compensate for the loss of substrate affinity. The enzyme also showed substrate inhibition phenomenon, which is pH-dependent. Specific activity of SlFumC was gradually enhanced with increasing phosphate concentrations. However, no inhibition was observed at high concentration of phosphate ion, which was distinctly different in case of other Class II fumarases. In industrial process, the reaction temperatures for L-malate production are usually set between 40 and 60 °C. The recombinant SlFumC displayed maximal activity at 45 °C and remained over 85 % of original activity after 48 h incubation at 40 °C, which was more thermostable than other fumarases from Streptomyces and make it an efficient enzyme for use in the industrial production of L-malate.