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To better understand the evolution of the SARS-CoV-2 Omicron subvariants, we performed molecular evolutionary analyses of the spike ( S ) protein gene/S protein using advanced bioinformatics technologies. First, time-scaled phylogenetic analysis estimated that a common ancestor of the Wuhan, Alpha, Beta, Delta variants, and Omicron variants/subvariants diverged in May 2020. After that, a common ancestor of the Omicron variant generated various Omicron subvariants over one year. Furthermore, a chimeric virus between the BM.1.1.1 and BJ.1 subvariants, known as XBB, diverged in July 2021, leading to the emergence of the prevalent subvariants XBB.1.5 and XBB.1.16. Next, similarity plot (SimPlot) data estimated that the recombination point (breakpoint) corresponded to nucleotide position 1373. As a result, XBB.1.5 subvariants had the 5' nucleotide side from the breakpoint as a strain with a BJ.1 sequence and the 3' nucleotide side as a strain with a BM.1.1.1 sequence. Genome network data showed that Omicron subvariants were genetically linked with the common ancestors of the Wuhan and Delta variants, resulting in many amino acid mutations. Selective pressure analysis estimated that the prevalent subvariants, XBB.1.5 and XBB.1.16, had specific amino acid mutations, such as V445P, G446S, N460K, and F486P, located in the RBD when compared with the BA.4 and BA.5 subvariants. Moreover, some representative immunogenicity-associated amino acid mutations, including L452R, F486V, R493Q, and V490S, were also found in these subvariants. These substitutions were involved in the conformational epitopes, implying that these mutations affect immunogenicity and vaccine evasion. Furthermore, these mutations were identified as positive selection sites. These results suggest that the S gene/S protein Omicron subvariants rapidly evolved, and mutations observed in the conformational epitopes may reduce the effectiveness of the current vaccine, including bivalent vaccines such as mRNA vaccines containing the BA.4/BA.5 subvariants.

Recently, there has been increasing interest in medial meniscal extrusion (MME), but few reports have evaluated MME via X-ray. In this study, the amount of MME and meniscal height at the medial border of the tibia were measured via X-ray with gradation processing. The extrusion length divided by the meniscal height yields the meniscal extrusion ratio, which was used as an index. In addition, the medial meniscal length of the part protruding from the medial border of the tibia on MRI was measured as an absolute value. Then, the correlation between the meniscal extrusion ratio and the amount of MME on MRI was examined, and there was a strong correlation between the meniscal extrusion ratio via X-ray and the amount of MME on MRI (correlation coefficient 0.860, p < 0.0001). The cut-off value of the meniscal extrusion ratio via X-ray for positive meniscal extrusion on MRI was 0.50, with an AUC of 0.9825, sensitivity of 0.9063, and specificity of 0.8663. From the present study, it was possible to measure the extrusion length and meniscal height via gradation processing, with X-ray and without MRI, and to calculate the meniscal extrusion ratio, which strongly correlates with the amount of MME on MRI.

Despite the increasing evidence of the clinical impact of Pseudomonas -derived cephalosporinase (PDC) sequence polymorphisms, the molecular evolution of its encoding gene, bla PDC , remains elusive. To elucidate this, we performed a comprehensive evolutionary analysis of bla PDC . A Bayesian Markov Chain Monte Carlo phylogenetic tree revealed that a common ancestor of bla PDC diverged approximately 4660 years ago, leading to the formation of eight clonal variants (clusters A-H). The phylogenetic distances within clusters A to G were short, whereas those within cluster H were relatively long. Two positive selection sites and many negative selection sites were estimated. Two PDC active sites overlapped with negative selection sites. In docking simulation models based on samples selected from clusters A and H, piperacillin was bound to the serine and the threonine residues of the PDC active sites, with the same binding mode for both models. These results suggest that, in P. aeruginosa , bla PDC is highly conserved, and PDC exhibits similar antibiotic resistance functionality regardless of its genotype.

DNA gyrase plays important roles in genome replication in various bacteria, including Pseudomonas aeruginosa . The gyrA gene encodes the gyrase subunit A protein (GyrA). Mutations in GyrA are associated with resistance to quinolone-based antibiotics. We performed a detailed molecular evolutionary analyses of the gyrA gene and associated resistance to the quinolone drug, ciprofloxacin, using bioinformatics techniques. We produced an evolutionary phylogenetic tree using the Bayesian Markov Chain Monte Carlo (MCMC) method. This tree indicated that a common ancestor of the gene was present over 760 years ago, and the offspring formed multiple clusters. Quinolone drug-resistance-associated amino-acid substitutions in GyrA, including T83I and D87N, emerged after the drug was used clinically. These substitutions appeared to be positive selection sites. The molecular affinity between ciprofloxacin and the GyrA protein containing T83I and/or D87N decreased significantly compared to that between the drug and GyrA protein, with no substitutions. The rate of evolution of the gene before quinolone drugs were first used in the clinic, in 1962, was significantly lower than that after the drug was used. These results suggest that the gyrA gene evolved to permit the bacterium to overcome quinolone treatment.