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Error-corrected sequences (ECSs) that utilize double-stranded DNA sequences are useful in detecting mutagen-induced mutations. However, relatively higher frequencies of G:C > T:A (1 × 10 -7  bp) and G:C > C:G (2 × 10 -7  bp) errors decrease the accuracy of detection of rare G:C mutations (approximately 10 -7  bp). Oxidized guanines in single-strand (SS) overhangs generated after shearing could serve as the source of these errors. To remove these errors, we first computationally discarded up to 20 read bases corresponding to the ends of the DNA fragments. Error frequencies decreased proportionately with trimming length; however, the results indicated that they were not sufficiently removed. To efficiently remove SS overhangs, we evaluated three mechanistically distinct SS-specific nucleases (S1 Nuclease, mung bean nuclease, and RecJf exonuclease) and found that they were more efficient than computational trimming. Consequently, we established Jade-Seq™, an ECS protocol with S1 Nuclease treatment, which reduced G:C > T:A and G:C > C:G errors to 0.50 × 10 -7  bp and 0.12 × 10 -7  bp, respectively. This was probably because S1 Nuclease removed SS regions, such as gaps and nicks, depending on its wide substrate specificity. Subsequently, we evaluated the mutation-detection sensitivity of Jade-Seq™ using DNA samples from TA100 cells exposed to 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene, which contained the rare G:C > T:A mutation (i.e., 2 × 10 -7  bp). Fold changes of G:C > T:A compared to the vehicle control were 1.2- and 1.3-times higher than those of samples without S1 Nuclease treatment, respectively. These findings indicate the potential of Jade-Seq™ for detecting rare mutations and determining the mutagenicity of environmental mutagens., (© 2021. The Author(s).)

It is difficult to identify mutagen-induced genome-wide somatic mutations using next generation sequencing; hence, mutagenic features of each mutagen and their roles in cancer development require further elucidation. We described Hawk-Seq™, a highly accurate genome sequencing method and the optimal conditions, for using it to construct libraries that would enable the accurate (c.a. 1 error/10 7 -10 8 bp) and efficient survey of genome-wide mutations. Genomic mutations in gpt delta mice or Salmonella typhimurium TA100 exposed to methylnitrosourea (MNU), ethylnitrosourea (ENU), diethylnitrosamine (DEN), benzo[a]pyrene (BP), and aristolochic acid (AA) were profiled using Hawk-Seq™ to analyse positions, substitution patterns, or frequencies. The resultant vast mutation data provided high-resolution mutational signatures, including for minor mutational fractions (e.g. G:C>A:T by AA), which enabled the clarification of the mutagenic features of all mutagens. The 96-type mutational signatures of MNU, AA, and BP indicate their partial similarity to signature 11, 22, and 4 or 29, respectively. Meanwhile, signatures attributable to ENU and DEN were highly similar to each other, but not to signature 11, suggesting that the mechanisms of these agents differed from those of typical alkylating agents. Thus, Hawk-Seq™ can clarify genome-wide chemical mutagenicity profiles at extraordinary resolutions, thereby providing insight into mutagen mechanisms and their roles in cancer development.

Purpose: To reconsider the pathogenesis of traumatic myopia by describing two cases and literature review., Methods: Refraction was measured, A-scan ultrasonography was performed, and ultrasound biomicroscopy (UBM) was used to observe the ciliary body in the acute and recovery stages., Results: The first patient had a myopic shift of -6.12 diopters (D) on the initial examination (day 2) compared with the recovered stage. UBM showed ciliochoroidal effusion in one half of the circumference and severe edema in three eighths of the ciliary body, which led to an anterior rotation of the ciliary body. Immediately after the cycloplegia, the myopic shift decreased by -3.9 D. On day 11, the refraction was -0.63 D, and the UBM findings were normalized. The second patient had a myopic shift of -4.38 D on the first examination compared with the recovered stage. UBM showed an annular ciliochoroidal effusion leading to anterior rotation of the ciliary body. Immediately after the cycloplegia, there was a decrease of -2.75 D. Biometric measurements showed an increased anterior chamber depth and a decreased thickness of the lens. On day 16, the refraction was -1.0 D, and the UBM findings were normalized. In both cases, biometric measurements in the acute phase suggested a anterior chamber shallowing, a thickening of the lens, and an anterior shift of the lens-iris diaphragm, Conclusions: Cycloplegia decreased the myopic shift by more than 60 %. These findings indicate a significant contribution of ciliary spasm combined with ciliochoroidal effusion, anterior shift of the lens-iris diaphragm, and thickening of the lens.