Apai enzyme

The strains genetic relatedness was observed by DNA macrorestriction with 30 U of ApaI enzyme (New England BioLabs Inc., United States) followed by pulsed-field gel electrophoresis (PFGE), as previously described (Durmaz et al., 2009 (link)). The electrophoresis was run with 0.5X TBE buffer at 14°C, with 6 V/cm², for 20 h and linear ramping, with initial and final switch times of 5 and 30 s, respectively, using a CHEF Mapper system (CHEF Mapper XA Pulsed Field Electrophoresis System—Bio-Rad Laboratories Inc., United States). After electrophoresis, the gel was stained with 0.01% SYBRSafe (Invitrogen, Life Technologies, United States) in 0.5x TBE buffer for 1 h, followed by visualization using the ChemiDoc XRS (Bio-Rad Laboratories Inc., United States).
We compared the DNA band profiles using the Bionumerics software (version 7.6, Applied-Maths, Belgium) with 0.5% optimization and 1.25% tolerance parameters, with cluster analysis done by the unweighted pair group method using the arithmetic average (UPGMA). Isolates with 100% similarity by PFGE were considered indistinguishable and clustered in the same pulsotype and subtype; isolates with similarity 80% were considered closely related and clustered in the same pulsotype, but different subtypes; isolates with similarity <80% were considered possibly related and are classified as belonging to different pulsotypes.

PFGE was carried out for all isolates in a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules and CA) by ApaI enzyme (Abbo et al., 2005 (link)) (New England Biolab, Massachusetts).
When indistinguishable isolates of Acinetobacter spp. were cultured from two or more neonates treated in the NICU during an interval of up to 15 days, they were considered as a single episode of cross-transmission (Roy et al., 2010 (link)).

Wild-type human CCNB3 were constructed and then recombined with the eukaryotic expression vector pcDNA3.1. A FLAG-tag was fused at the C-terminus of CCNB3. The vectors were constructed by GenScript (Nanjing). The variants were generated using Fast Mutagenesis Kit (Vazyme, Nanjing, C214). The plasmids were linearized with ApaI enzyme (New England Biolabs, # R0114S) and then be transcribed to cRNAs using HiScribe T7 ARCA mRNA Kit (New England Biolabs, E2060) according to the manufacturer’s standard mRNA synthesis protocols.