Impact of changes in recommendation of optimal gestational weight gain in Japan: A retrospective study in a single tertiary center.

AIM: In March 2021, the Japanese Ministry of Health, Labour and Welfare revised the optimal gestational weight gain standards. In this study, we examined whether this revision affected gestational weight gain and low birth weight rates. METHODS: We analyzed the records of singleton pregnant women who underwent checkups from their 1st trimester and delivered at our institute after 37 weeks between 2020 and 2021 (before the revision) and between 2022 and 2023 (after the revision). Pregnancy outcomes were assessed in the following four groups stratified by pre-pregnancy body mass index (BMI): underweight (BMI: <18.5 kg/m2), normal-weight (BMI: 18.5-24.9 kg/m2), overweight (BMI: 25-29.9 kg/m2), and obese (BMI: ≥30 kg/m2). Leaflets on the optimal gestational weight gain standards for each group were distributed to all pregnant women at the first prenatal checkup. RESULTS: In each group, gestational weight gain did not change before and after the revision, with the corresponding values of 10.8 kg and 11.1 kg in the underweight (p = 0.94), 10.7 kg and 10.4 kg in the normal weight (p = 0.14), 9.7 kg and 9.2 kg in the overweight (p = 0.32), and 7.4 kg and 6.7 kg in the obese (p = 0.44) groups. Furthermore, the prevalence of low birth weight did not decrease in all groups. CONCLUSIONS: No significant differences in gestational weight gain or low birth weight were observed after the revision of the 2021 gestational weight gain recommendations. Merely distributing leaflets to pregnant women may not be sufficient to improve gestational weight gain or reduce low birth weight rates.

Pierisin, Cytotoxic and Apoptosis-Inducing DNA ADP-Ribosylating Protein in Cabbage Butterfly.

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology.

Impact of interpregnancy weight changes and perinatal outcomes: A retrospective study in Japan.

Previous studies have shown that interpregnancy weight fluctuations impact perinatal outcomes. In order to examine this in Japanese women, we analyzed the data of 2,861 women in their first and second pregnancies who delivered singletons between 2000 and 2022. We compared the second pregnancy perinatal outcomes of women whose interpregnancy body mass index (BMI) change was -1 to 1 unit with those of women whose BMI change was < -1 or ≥ 1 unit. An interpregnancy BMI change ≥ 1 unit was associated with an increased risk of developing gestational diabetes mellitus (adjusted odds ratio [aOR], 1.51; 95% confidence interval [CI], 1.18-1.95) and delivering a large for gestational age neonate (aOR, 1.67; 95% CI, 1.15-2.42) but a decreased risk of preterm birth (aOR, 0.66; 95% CI, 0.46-0.95). An interpregnancy BMI change < -1 unit was associated with a decreased risk of developing gestational diabetes mellitus (aOR, 0.51; 95% CI, 0.31-0.85). In a subgroup analysis of three groups divided according to prepregnancy BMI, interpregnancy BMI changes ≥ 1 unit in women with a BMI of < 18.5 kg/m2 before their first pregnancy were associated with a remarkable risk reduction of developing preterm birth (aOR, 0.30; 95% CI, 0.11-0.81). Interpregnancy BMI changes < -1 unit in women with a BMI of ≥ 25 kg/m2 before their first pregnancy were associated with a remarkable risk reduction of developing gestational diabetes mellitus (aOR, 0.33; 95% CI, 0.12-0.88). Weight gain during interpregnancy period was related to an increased risk of gestational diabetes mellitus and delivery of a large-for-gestational-age neonate, whereas weight loss was related to a decreased risk of developing gestational diabetes mellitus. These results indicate the importance of interpregnancy weight control as part of preconception care; therefore, women considering additional pregnancies should be educated on the importance of maintaining a healthy weight.

Cytotoxin-mediated silk gland organ dysfunction diverts resources to enhance silkworm fecundity by potentiating nutrient-sensing IIS/TOR pathways.

Energy reserves, primarily stored in the insect's fat body, are essential for physiological processes such as reproduction and cocoon formation. However, whether these processes are mutually constraining is unknown. Here, we showed that cocoon-free silkworms accumulate amino acid constituents of silk proteins in the hemolymph and maintain lipid and sugar reserves in the pupal fat body by repressing the expression of sericin and fibroin genes in the middle and posterior silk glands, respectively, via butterfly pierisin-1A catalytic domain expression. This, in turn, upregulates insulin/insulin-like signaling and target of rapamycin (IIS/TOR) signaling, which enhances vitellogenesis and accelerates ovarian development, thus contributing to increased fecundity. The impacts of semi-starvation on fecundity and egg hatchability were also less pronounced in cocoon-free silkworms compared with wildtype silkworms. These data uncover the resource allocation trade-off between cocoon formation and fecundity and demonstrate that nutritional signaling plays a role in regulating silkworm reproduction.

Intrauterine fetal death due to fetal intracranial hemorrhage associated with maternal vitamin K deficiency: A case report.

Vitamin K deficiency can cause coagulopathy; therefore, supplementation is recommended to prevent intracranial hemorrhage in newborns. Some reports have shown that maternal vitamin K deficiency is associated with intracranial hemorrhage in the fetus. However, no clear guidelines exist for the diagnosis and treatment of maternal vitamin K deficiency to prevent fetal intracranial hemorrhage. We report a case of intrauterine fetal death due to intracranial hemorrhage associated with maternal vitamin K deficiency resulting from hyperemesis gravidarum. In this case, maternal protein induced by vitamin K absence II (PIVKA-II) was high at the time of intrauterine fetal death. Therefore, measuring maternal PIVKA-II levels in high-risk cases may help determine the timing of therapeutic interventions for vitamin K deficiency during pregnancy.

Multimodal analyses of a non-human primate model harboring mutant amyloid precursor protein transgenes driven by the human EF1α promoter.

Alzheimer's disease (AD) is the leading cause of dementia which afflicts tens of millions of people worldwide. Despite many scientific progresses to dissect the AD's molecular basis from studies on various mouse models, it has been suffered from evolutionary species differences. Here, we report generation of a non-human primate (NHP), common marmoset model ubiquitously expressing Amyloid-beta precursor protein (APP) transgenes with the Swedish (KM670/671NL) and Indiana (V717F) mutations. The transgene integration of generated two transgenic marmosets (TG1&TG2) was thoroughly investigated by genomic PCR, whole-genome sequencing, and fluorescence in situ hybridization. By reprogramming, we confirmed the validity of transgene expression in induced neurons in vitro. Moreover, we discovered structural changes in specific brain regions of transgenic marmosets by magnetic resonance imaging analysis, including in the entorhinal cortex and hippocampus. In immunohistochemistry, we detected increased Aß plaque-like structures in TG1 brain at 7 years old, although evident neuronal loss or glial inflammation was not observed. Thus, this study summarizes our attempt to establish an NHP AD model. Although the transgenesis approach alone seemed not sufficient to fully recapitulate AD in NHPs, it may be beneficial for drug development and further disease modeling by combination with other genetically engineered models and disease-inducing approaches.

Humanized liver TK-NOG mice with functional deletion of hepatic murine cytochrome P450s as a model for studying human drug metabolism.

Chimeric TK-NOG mice with a humanized liver (normal Hu-liver) are a unique animal model for predicting drug metabolism in humans. However, residual mouse hepatocytes occasionally prevent the precise evaluation of human drug metabolism. Herein, we developed a novel humanized liver TK-NOG mouse with a conditional knockout of liver-specific cytochrome P450 oxidoreductase (POR cKO Hu-liver). Immunohistochemical analysis revealed only a few POR-expressing cells around the portal vein in POR cKO mouse livers. NADPH-cytochrome c reductase and cytochrome P450 (P450)-mediated drug oxidation activity in liver microsomes from POR cKO mice was negligible. After the intravenous administration of S-warfarin, high circulating and urinary levels of S-7-hydroxywarfarin (a major human metabolite) were observed in POR cKO Hu-liver mice. Notably, the circulating and urinary levels of S-4'-hydroxywarfarin (a major warfarin metabolite in mice) were much lower in POR cKO Hu-liver mice than in normal Hu-liver mice. POR cKO Hu-liver mice with minimal interference from mouse hepatic P450 oxidation activity are a valuable model for predicting human drug metabolism.

Effects of breastfeeding on postpartum weight change in Japanese women: The Japan Environment and Children's Study (JECS).

AIM: The aim of this study was to examine the relationship between breastfeeding and postpartum maternal weight change. METHOD: This study used data from the Japan Environment and Children's Study (JECS), an ongoing nationwide birth cohort study. Participants were categorized into two groups: full breastfeeding (FB) and non-full breastfeeding (NFB) groups. Postpartum weight changes between the FB (n = 26,340) and NFB (n = 38,129) groups were compared. RESULTS: At 6 months postpartum, mean weight retention was significantly lower in the FB group than in the NFB group (0.2 vs 0.8 kg, respectively, p<0.001). Weight retention differed by pre-pregnancy body mass index (BMI), with postpartum weights of overweight (pre-pregnancy BMI 25.0-29.9) and obese (pre-pregnancy BMI ≥30.0) participants being lower than pre-pregnancy weight; this trend was more pronounced in the FB group than in the NFB group (overweight: -2.2 vs -0.7 kg, respectively; obese: -4.8 vs -3.4 kg, respectively). Factors affecting weight retention at 6 months postpartum were weight gain during pregnancy (ß = 0.43; p<0.001), pre-pregnancy BMI (ß = -0.147; p<0.001) and feeding method. FB resulted in lower weight retention than NFB (ß = -0.107; p<0.001). CONCLUSION: Breastfeeding reduced maternal weight retention, which was greater in mothers who were obese before pregnancy. For obese women, active breastfeeding may improve their health.

Neurosurgical Treatment of Patients with Posterior Fossa Acute Subdural Hematoma Right after Cardiac Surgery.

As posterior fossa acute subdural hematoma (ASDH) right after cardiac surgery is extremely rare, the clinical course and optimal treatment strategy remain undetermined. We performed a retrospective analysis of patients with posterior fossa ASDH right after cardiac surgery requiring neurosurgical treatment at our institution over a 7-year period and, in this study, discussed the neurosurgical strategy and clinical course. Collected data included clinical history, laboratory results, time course, symptoms, neurosurgical treatment, outcome at discharge, and imaging studies. All six patients were women who had no history of head trauma and had received antithrombotic therapy during the perioperative period of cardiac surgery. All patients showed lower platelets count and were diagnosed with ASDH within 3 days (longest time 64 h) right after cardiac surgery. After discontinuation of anticoagulation therapy and administration of reversal agents, they underwent emergency hematoma evacuation craniotomy (n = 5) or burr hole drainage surgery (n = 1), which were performed in the prone (n = 4) or lateral (n = 2) positions. Four of these patients showed favorable outcomes, and two showed poor outcomes. One of the poor-outcome patients received three antithrombotic therapies, and another developed rapidly progressive ASDH. Posterior fossa ASDH associated with antithrombotic therapy right after cardiac surgery is frequently found in women, and emergent neurosurgical treatment with anticoagulation discontinuation and reversal agent administration can be performed safely. Burr hole drainage surgery might be acceptable in nonsevere cases. By contrast, we must pay attention to cases receiving both anticoagulant and antiplatelet drugs and rapid progression cases.

An improved TK-NOG mouse as a novel platform for humanized liver that overcomes limitations in both male and female animals.

We developed a novel immunodeficient NOG mouse expressing HSVtk mutant clone 30 cDNA under the control of mouse transthyretin gene enhancer/promoter (NOG-TKm30) to acquire fertility in males and high inducibility of liver injury in females. Maximum human albumin levels (approx. 15 mg/mL plasma) in both male and female NOG-TKm30 mice engrafted with human hepatocytes (humanized liver mice) were observed 8-12 weeks after transplantation. Immunohistochemical analyses revealed abundant expression of major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4) in reconstituted liver with original zonal distribution. In vivo drug-drug interactions were observed in humanized liver mice as decreased area under the curve of midazolam (CYP3A4/5 substrate) and omeprazole (CYP3A4/5 and CYP2C19 substrate) after oral administration of rifampicin. Furthermore, we developed a pregnant model for evaluating prenatal exposure to drugs. The detection of thalidomide metabolites in the fetuses of pregnant humanized liver mice indicates that the novel TK model can be used for developmental toxicity studies requiring the assessment of human drug metabolism. These results suggest that the limitations of traditional TK-NOG mice can be addressed using NOG-TKm30 mice, which constitute a novel platform for humanized liver for both in vivo and in vitro studies.

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii.

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott's methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.

Establishing an induced pluripotent stem cell line from neonatal common marmoset fibroblasts by an all-in-one episomal vector approach.

Epstein-Barr virus (EBV)-based episomal vector system enables persistent transgene expression, which is advantageous for efficient derivation of transgene-free induced pluripotent stem cells (iPSCs) without viral transduction. Here, we report establishment of an iPSC line from somatic fibroblasts of a neonatal common marmoset monkey (marmoset; Callithrix jacchus) using an all-in-one episomal vector that we newly developed. The established iPSC line, named NM-iPS, showed standard characteristics of pluripotency such as pluripotency-related marker expression, three germ layer differentiation, and normal karyotype (2n = 46). The novel iPSC line would be a useful resource for stem cell research using non-human primates.

Pathogenesis of murine astrovirus in experimentally infected mice.

Astroviruses are often associated with gastrointestinal diseases in mammals and birds. Murine astrovirus (MuAstV) is frequently detected in laboratory mice. Previous studies on MuAstV in mice did not report any symptoms or lesions. However, little information is available regarding its pathogenicity in immunodeficient mice. Therefore, in this study, we experimentally infected germ-free NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic (NOG) mice, which are severely immunodeficient, with MuAstV. Germ-free mice were used for experimental infection to eliminate the effects of intestinal bacteria. Mice in each group were then necropsied and subjected to PCR for MuAstV detection, MuAstV RNA quantification in each organ, and histopathological examination at 4 and 28 days post inoculation (DPI). Tissue samples from the small intestine were examined by transmission electron microscopy. No symptoms or abnormalities were detected in any mice during necropsy. The MuAstV concentration was highest in the lower small intestine, where it increased approximately 8-fold from 4 to 28 DPI. Transmission electron microscopy revealed circular virus particles of approximately 25 nm in diameter in the cytoplasm of the villous epithelial cells of the lower small intestine. Histopathological examination did not reveal any abnormalities, such as atrophy, in the intestinal villi. Our results suggest that MuAstV proliferates in the villous epithelial cells of the lower small intestine and has weak pathogenicity.

Generation of a common marmoset embryonic stem cell line CMES40-OC harboring a POU5F1 (OCT4)-2A-mCerulean3 knock-in reporter allele.

POU class 5 homeobox 1 (POU5F1, also known as OCT4) is critical for maintenance of pluripotency, germ cell fate, reprogramming into a pluripotent state, and early embryogenesis. We generated an embryonic stem cell (ESC) line of the common marmoset (Callithrix jacchus) harboring a heterozygous knock-in allele of OCT4-P2A-mCerulean-T2A-pac. The ESC line (CMES40-OC) will be valuable for investigation of primed/naïve pluripotency and germ cell fate. Homozygous OCT4 knock-in clones were generated but could not be sustained in an undifferentiated state in long-term culture. The OCT4 knock-in system facilitated simultaneous knock-in of a reporter construct at another locus, DDX4 (VASA).

Factors for the Variability of Three Acceptable Maximal Expiratory Flow-Volume Curves in Chronic Obstructive Pulmonary Disease.

BACKGROUND: Generally, the maximal expiratory flow-volume (MEFV) curve must be measured for the diagnosis and staging of chronic obstructive pulmonary disease (COPD). As this test is effort dependent, international guidelines recommend that three acceptable trials are required for each test. However, no study has examined the magnitude and factors for the variability in parameters among three acceptable trials. METHODS: We evaluated the intra-individual variations in several parameters among three acceptable MEFV curves obtained at one-time point in patients with COPD (n = 28, stage 1; n = 36, stage 2; n = 21, stages 3-4). Next, the factors for such variations were examined using forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC). RESULTS: The averages of coefficient of variation (CV) for FEV1 and FVC were 2.0% (range: 1.0-3.0%) and 1.6% (0.9-2.2%), respectively. Both parameters were significantly better than peak expiratory flow rate, forced expiratory flow at 50% of expired FVC, and forced expiratory flow at 75% of expired FVC (CVs: 5.0-6.9%). A higher spirometric stage was significantly associated with higher CVs for FVC and FEV1, and older age was significantly correlated with a higher variation in FEV1 alone. Furthermore, a significantly inverse association was observed between emphysema severity, and the CVs for FEV1, but not that for FVC, regardless of spirometric stage. CONCLUSION: Both FVC and FEV1 are highly reproducible; nevertheless, older age, lower FEV1 at baseline, and non-emphysema phenotype are factors for a higher variability in FEV1 in patients with COPD.

Prenatal ultrasonographic findings and fetal/neonatal outcomes of body stalk anomaly.

We studied 27 cases that were post or prenatally diagnosed with body stalk anomaly (BSA) using medical records of prenatal ultrasound findings, pregnancy outcomes, and fetal/neonatal prognosis during 1992 to 2018. Termination of pregnancy was chosen in 15 cases. Of the remaining 12 cases, seven were stillbirths and five were live births. Of seven stillbirths, intrauterine fetal demise occurred before onset of labor in four cases at 17 to 20th weeks of pregnancy. Pregnancy was continued in eight cases. Median gestational age of delivery was 33rd weeks of pregnancy. Median birth weight was 1198 g (range:482-1914 g). Vaginal delivery was chosen in six and caesarean delivery in two cases. Among six vaginally delivered cases, three (50%) fetuses were stillborn. All five live born neonates died within a few hours (16-133 minutes). Eighteen cases were confirmed as BSA postnatally by placental examination or autopsy at our hospital. Main prenatal ultrasonographic findings of them were abdominal wall defect (100%), absence of the umbilical cord (72%), abnormal spine (61%), and abnormal legs (50%). The most characteristic prenatal ultrasonography findings of BSA were the absence of free umbilical cord in the amniotic cavity and the presence of abdominal organs into the extraembryonic celom through abdominal wall defects. The autopsy showed severe pulmonary hypoplasia with the median lung/body weight ratio of 0.61% (range:0.34-0.85%). There were no cases of maternal morbidities. Our study provides important information about the pregnancy outcome and the fetal/neonatal outcome of BSA cases for the parents whose fetuses are diagnosed with BSA prenatally.

A novel step-down infusion method of barbiturate therapy: Its safety and effectiveness for intracranial pressure control.

Intracranial pressure (ICP) has to be maintained quite constant, because increased ICP caused by cerebrovascular disease and head trauma is fatal. Although controlling ICP is clinically critical, only few therapeutic methods are currently available. Barbiturates, a group of sedative-hypnotic drugs, are recognized as secondary treatment for controlling ICP. We proposed a novel "step-down infusion" method, administrating barbiturate (thiamylal) after different time point from the start of treatment under normothermia, at doses of 3.0 (0-24 h), 2.0 (24-48 h), 1.5 (48-72 h), and 1.0 mg/kg/h (72-96 h), and evaluated its safety and effectiveness in clinical. In 22 patients with severe traumatic brain injury or severe cerebrovascular disease (Glasgow coma scale ≤8), thiamylal concentrations and ICP were monitored. The step-down infusion method under normothermia maintained stable thiamylal concentrations (<26.1 µg/ml) without any abnormal accumulation/elevation, and could successfully keep ICP <20 mmHg (targeted management value: ICP <20 mmHg) in all patients. Moreover the mean value of cerebral perfusion pressure (CPP) was also maintained over 65 mmHg during all time course (targeted management value: CPP >65 mmHg), and no threatening changes in serum potassium or any hemodynamic instability were observed. Our novel "step-down infusion" method under normothermia enabled to maintain stable, safe thiamylal concentrations to ensure both ICP reduction and CPP maintenance without any serious side effects, may provide a novel and clinically effective treatment option for patients with increased ICP.

Generation and validation of a common marmoset embryonic stem cell line ActiCre-B1 that ubiquitously expresses a tamoxifen-inducible Cre-driver.

We previously reported the efficient targeted introduction of transgenes into the genomic DNA of the common marmoset (Callithrix jacchus) using CRISPR-Cas9. In this study, we generated a marmoset embryonic stem cell (ESC) line that ubiquitously expresses the tamoxifen-inducible Cre-driver ERT2CreERT2. We validated the pluripotency of the ESC line and also successfully demonstrated the temporal control of the Cre-driver in a tamoxifen-dependent manner in the ESCs. This ESC line, named ActiCre-B1, will be a valuable resource for in vitro investigation of phenotypes related to embryonic lethality by targeted knockout of functionally important genes.

Two cases of chronic obstructive pulmonary disease with undetectable diffusing capacity for carbon monoxide.

Pulmonary diffusing capacity for carbon monoxide (DLCO) is a valuable pulmonary function test to evaluate the gas exchange capacity of the lungs. Generally, DLCO values are significantly lower in patients with chronic obstructive pulmonary disease (COPD), particularly in those with a predominantly emphysema phenotype. However, it is extremely rare that DLCO values cannot be obtained for reasons other than technical errors. Herein, we report two patients with COPD in whom DLCO values were undetectable without prolonging the breath-holding time for the test. We discuss possible mechanisms for these peculiar findings.

Prevalence of murine astrovirus in laboratory animal facilities in Japan.

To investigate the prevalence of murine astrovirus (MuAstV) in mice in laboratory animal facilities in Japan, a polymerase chain reaction (PCR) test targeting the RNA-dependent RNA polymerase (RdRP) gene was performed on the cecum contents of 1,212 mice (1,183 immunocompetent mice and 29 immunodeficient mice) from 226 facilities. The results showed that 118 (52.2%) of the 226 facilities were positive for MuAstV. Out of the 1,212 mice, 424 (35.0%) were positive. No gross lesions were observed in any of the mice examined. A phylogenetic analysis for 15 selected strains revealed that 13 strains formed one cluster, while two were genetically distant from that cluster. These results suggest that multiple strains are prevalent in laboratory mice in Japan.