Effect of paternal aging and vitrification on mitochondrial DNA copy number and telomere length of mouse blastocysts.

In this study, we examined the effects of paternal aging on the mitochondrial DNA copy number (mt-cn), telomere length (TL), and gene expression in mouse embryos. The effects of vitrification on the mt-cn and TL of the embryos derived from young and aged male parents (YF and AF, respectively) were examined. C57BL/6N male mice were used for embryo production at 13-23 and 50-55 weeks of age. Two-cell stage embryos were collected from the oviducts of superovulated female mice (8-15 weeks old) and cultured for 24 h until the 8-cell stage, followed by embryo vitrification. Fresh and vitrified-warmed embryos were incubated for 2 days until the blastocyst stage, and mt-cn and TL were investigated. The cell-free mitochondrial DNA copy number (cf-mt-cn) in the spent culture medium (SCM) of the embryos was then investigated. RNA sequencing of blastocysts revealed that metabolic pathways, including oxidative phosphorylation and mTOR pathways, were enriched in differentially expressed genes. The mt-cn and TL of AF-derived blastocysts were lower and shorter, respectively, than those of YF-derived blastocysts. Paternal aging did not affect the blastocyst rate after vitrification. Vitrification of the 8-cell stage embryos did not affect the mt-cn of the blastocysts. However, it increased the cf-mt-cn (cell-free mt-cn) in the SCM of both YF- and AF-derived embryos. Vitrification did not affect the TL of either YF- or AF-derived embryos. Thus, paternal aging affected the mt-cn and TL of the embryos, but vitrification did not affect these parameters in either age groups.

A safe sequential treatment approach for patients who have acute cholecystitis with severe inflammation: Transmural gallbladder drainage followed by laparoscopic cholecystectomy under the guidance of fluorescence imaging.

INTRODUCTION: For patients who have acute severe cholecystitis, urgent/early biliary drainage followed by delayed/elective laparoscopic cholecystectomy is recommended according to the Tokyo Guidelines 2018. Percutaneous transhepatic gallbladder drainage is an established technique. Recently, transmural gallbladder drainage under the guidance of endoscopic ultrasonography (EUS-GBD) was reported as a safe alternative. During surgery, fluorescence imaging using indocyanine green (ICG) has been increasingly used for visualizing the bile ducts. Herein, we report a sequential treatment approach which ensures safety without impairing normal activities before cholecystectomy: EUS-GBD followed by laparoscopic cholecystectomy using ICG fluorescence imaging. MATERIALS AND SURGICAL TECHNIQUE: A 66-year-old man with acute cholecystitis underwent urgent EUS-GBD and had the drainage tube placement through the duodenum into the gallbladder. During 2.5 months of the waiting period, he had no clinical troubles. After insertion of a laparoscope, we found a structure between the gallbladder and the duodenum. We injected 0.025 mg/mL of ICG into the nasobiliary drainage tube (placed in the gallbladder through the duodenum) and confirmed that the structure was a fistula. After removing the tube, the fistula was divided using a surgical stapler under the guidance of fluorescence imaging. The cystic and common bile ducts were also clearly visualized as fluorescence. DISCUSSION: We reported a safe sequential treatment approach for the patient who required biliary drainage: EUS-GBD followed by laparoscopic cholecystectomy under the guidance of ICG fluorescence imaging. This sequential approach may improve patients' satisfaction with respect to quality of life during the waiting period and may ensure the safety of laparoscopic cholecystectomy.

Characterization of tryptic hydrolysis of alpha-lactalbumin/saponin mixture and structural change of alpha-lactalbumin interacting with soybean saponin.

Bovine milk alpha-lactalbumin (alpha-La) was mixed with soybean saponin, and the resulting mixture was hydrolyzed by trypsin. Saponin increased the tryptic-hydrolysis level of alpha-La only at relatively high phosphate buffer concentrations (> or = 0.05 M). T(1) experiments with acetylated soybean saponin demonstrated that there were some interactions between alpha-La and saponin not only at high concentrations of phosphate buffers but even at low concentrations as well. Circular dichroism spectra of alpha-La showed that the tertiary structure of alpha-La was changed through interactions with saponin only at high buffer concentrations. Furthermore, by analyzing the tryptic peptides from an alpha-La/saponin mixture, hydrolyzing rates at all or some of K5, R10, and K16 of alpha-La were accelerated by saponin interactions. The increase in the tryptic hydrolysis of alpha-La by saponin addition was considered due to modification of the tertiary structure of alpha-La by saponin.