CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) ...mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as
EGFP
,
mCherry
,
Cre
, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.
The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause a ...systemic developmental syndrome including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic diseases have remained unclear. We performed whole-exome sequencing analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis, and we identified what we believe to be a novel probable pathogenic variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 laminin globular 4-5 (LG4-5) domain. Heterozygous and homozygous knockin mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels, which suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Reexaminations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.
Background
To clarify the clinical as well as pathological outcomes in Japanese women with germline pathogenic
BRCA1/2
variants who underwent risk-reducing salpingo-oophorectomy (RRSO).
Methods
This ...prospective study examined the rate of occult cancer and primary peritoneal cancer after RRSO at our institution in the period from 2011 to 2020. Clinical records of genetically confirmed patients with germline pathogenic
BRCA1/2
variants who desired to undergo RRSO were reviewed. Specimens obtained during RRSO were pathologically diagnosed as per SEE-FIM protocol. All the participants underwent magnetic resonance imaging (MRI) about 1 month preoperatively.
Results
One hundred and seventeen women underwent RRSO during this period. Of these, the numbers of women with germline pathogenic
BRCA1
and
BRCA2
variants were 72 and 45, respectively. The mean observational time after RRSO was 35.8 months. Despite negative preoperative screening results, three (2.6%) serous tubal intraepithelial carcinoma and three (2.6%) invasive carcinomas were identified. Of the three invasive carcinomas cases, two were International Federation of Gynecology and Obstetrics (FIGO) stage I primary fallopian tube cancer, and the third case was double cancer (ovarian cancer and fallopian tube cancer) with FIGO stage IC3.
Conclusions
The rate of occult neoplasms was similar to those reported by studies performed in other countries. Although women with occult cancer were diagnosed with FIGO stage I, the MRI performed 1 month preoperatively did not show any such malignant findings. Thus, RRSO is the only promising method that can improve the prognosis in women with germline pathogenic
BRCA1/2
variants.
Cinnamon, clove, and fennel are commonly used as spices and herbal medicines, and one of their medicinal uses is as aromatic stomachics. We investigated the effect on appetite in mice of inhaling ...volatile compounds contained in essential oils extracted from herbal medicines used as aromatic stomachics. The appetite-enhancing effects of cinnamon and fennel essential oils were similar to those of their main components
trans
-cinnamaldehyde and
trans
-anethole, respectively. The appetite-enhancing effects of clove essential oil were observed over a wide range of doses (4.5 × 10
−4
to 4.5 × 10
−3
mg/cage), even though the active compounds showed effects within a narrow range of doses (eugenol: 4.5 × 10
−4
to 2.5 × 10
−3
mg/cage; eugenol acetate: 1.1 × 10
−3
to 4.5 × 10
−3
mg/cage). The increase in appetite at doses that differed by tenfold in mice administered clove oil was due to synergistic effects between eugenol and eugenol acetate in clove oil. Thus, loss of appetite could be treated more effectively using essential oil containing both eugenol and eugenol acetate compared with the active compounds administered separately. Administering essential oils, such as cinnamon and clove, could improve loss of appetite without strict dosage adjustment.
To determine the impact of the coverage of risk-reducing salpingo-oophorectomy (RRSO) and mastectomy (RRM) as well as genetic testing for BRCA pathogenic variants by the national insurance system in ...Japan. We compared the clinical background of women who underwent RRSO at our institution before and after its coverage by the national insurance system. Those who underwent RRSO between January 2017 and December 2019 and between April 2020 and March 2022 were classified as Period. A and B, respectively. Overall, 134 women underwent RRSO during the study period. In Period A and B, 45 and 89 women underwent RRSO for the study period was 36 and 24 months, respectively. Compared with Period A, the number of women who underwent RRSO per month increased by threefold in Period B (p < 0.01). In addition, the number of women who underwent surgery for breast cancer along with RRSO increased in Period B (p < 0.01). Although the number of women who underwent concurrent RRM with RRSO in Period B increased, the difference was not statistically significant. Compared with Period A, the number of women diagnosed with BRCA pathogenic variant increased by 3.9-fold, and the proportion of women who underwent concurrent hysterectomy at the time of RRSO decreased from 66 to 7.9% in Period B (p < 0.01). Owing to the introduction of the national insurance system, the number of women who underwent RRSO and concurrent surgery for breast cancer at the time of RRSO increased in Japan.
Ovarian surveillance in women with hereditary breast and ovarian cancer who do not undergo risk-reducing salpingo-oophorectomy has been controversial. Therefore, this study aimed to demonstrate the ...clinical features of ovarian surveillance at our institution using a technique that combines serum cancer antigen 125 measurements, transvaginal ultrasonography, and uterine endometrial cytology.
We retrospectively examined 65 women, who had not undergone risk-reducing salpingo-oophorectomy diagnosed with hereditary breast and ovarian cancer between 2000 and 2021 at our hospital. Clinical information was obtained and analyzed through a chart review. The details of the treatment course were reviewed for patients who had developed ovarian cancer.
Overall, 5 of the 65 women were diagnosed with ovarian cancer based on abnormal findings during periodic surveillance. All patients who developed ovarian cancer were asymptomatic, even if the cancer was at an advanced stage. Two of the 65 patients had endometrial cytology abnormalities, both of whom had ovarian cancer. All patients who developed ovarian cancer underwent primary debulking surgery, and complete gross resection was achieved. None of the patients experienced ovarian cancer recurrence.
The ovarian surveillance strategy at our institution for women with hereditary breast and ovarian cancer who do not undergo risk-reducing salpingo-oophorectomy can identify asymptomatic ovarian cancer and contribute to achieving complete gross resection during primary surgery. Ovarian surveillance may contribute to a reduction in ovarian cancer mortality.
The present study was conducted to investigate the strength and power characteristics of the lower limb using the rebound jump (RJ) for collegiate female soccer players, focusing on the relationships ...between running performance, muscle strength and differences in competition level. The performance variables of 19 collegiate female soccer players in the 20 m sprint, pro agility test, squat, isokinetic knee strength, countermovement jump, and RJ were calculated. The kinetics data (ground reaction force, joint torque, power, joint work) in the RJ were then calculated. The results revealed no relationship between performance variables in the RJ and various parameters related to running performance and muscle strength. However, in the starter group, the 20 m sprint time and pro agility time were significantly faster, the RJ-index was significantly higher, and contact time in the RJ was significantly shorter. In addition, in the starter group, the maximum ground reaction force, ankle joint torque, power, and knee joint torque in the first half of the take-off phase were large. Therefore, it is suggested that female soccer players with a high competition level have excellent strength and power abilities, particularly in terms of ankle joint and knee joint muscle strength and power exertion ability to achieve the jumping height in a short contact time.
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