ED95 of remimazolam in nasal administration for attenuating preoperative anxiety in children.

Background: Preoperative anxiety often prevails in children at higher levels than adults, which is a common impediment for surgeons and anesthesiologists. It is of great necessity to explore an appropriate medication to improve this situation. Remimazolam, a type of benzodiazepine drug, has been indicated for the induction and maintenance of procedural sedation in adults since 2020. To date, rare studies were reported to investigate the effect of remimazolam on children. In this study, we investigated the safety and efficacy of intranasal drops of remimazolam and tried to determine the 95% effective dose (ED95) of remimazolam in single intranasal administration in attenuating preoperative anxiety in children. Methods: In this study, 114 children were enrolled who underwent laparoscopic high-level inguinal hernia ligation between January 2021 and December 2022 and were divided into an early childhood children group and a pre-school children group. The biased coin design (BCD) was used to determine the target doses. A positive response was defined as the effective relief of preoperative anxiety (modified Yale Preoperative Anxiety Scale, mYPAS < 30). The initial nasal dose of remimazolam was 0.5 mg·kg-1 in the two groups. An increment or decrement of 0.1 mg·kg-1 was applied depending on the sedative responses. Isotonic regression and bootstrapping methods were used to calculate the ED95 and 95% confidence intervals (CIs), respectively. Results: A total of 80 children completed the study, including 40 in the early childhood group and 40 in the pre-school children group. As statistical analysis indicated, the ED95 of a single intranasal infusion of remimazolam for the relief of preoperative anxiety is 1.57 mg·kg-1 (95% CI: 1.45-1.59 mg·kg-1) in early childhood children and 1.09 mg·kg-1 (95% CI: 0.99-1.11 mg·kg-1) in pre-school children, and the CIs did not overlap each other. Conclusion: Remimazolam is an effective medication to relieve preoperative anxiety in children. Moreover, the ED95 of single nasal administration of remimazolam for effective relief of preoperative anxiety was 1.57 and 1.09 mg·kg-1 in early childhood children and pre-school children, respectively.

[Cloning of ACE-2 gene encoding the functional receptor for the SARS coronavirus and its expression in eukaryotic cells].

OBJECTIVE: Angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), so its gene was cloned and eukaryotic expressed for further insight into mechanisms in SARS-CoV entry and pathogenesis, as well as development of a safe and reliable neutralization assay for SARS-CoV. METHODS: Total RNA was extracted from right atrial tissue of a patient with right heart failure resected during a valvular replacement surgery by Trizol one-step method, and the full-length ACE-2 encoding gene was acquired by RT-nested-PCR. The ACE-2 encoding gene was then cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to construct the recombinant plasmid pcDNA4/ ACE-2, which was then transfected into 293 T cell and ACE-2 eukaryotic transient expression was detected by Western Blot. Syncytia inhibition assay was established to detect SARS-CoV neutralizing antibody, and compared parallelly with SARS pseudovirus neutralization assay. RESULTS: The recombinant plasmid pcDNA4/ ACE-2 could express ACE-2 protein in eukaryotic cells and induce cell-cell fusion between S protein- and ACE2-expressing cells. This cell-cell fusion assay could be used to detect SARS-CoV neutralizing antibody. CONCLUSION: SARS-CoV receptor ACE-2 gene was successfully cloned and eukaryotic expressed, and used to establish syncytia inhibition assay for SARS-CoV neutralizing antibody assay.

[Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application].

OBJECTIVE: To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS. METHODS: SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly. RESULTS: The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates. CONCLUSION: The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.