Switching defective/sucrose non-fermenting chromatin remodeling complex coordinates meiotic gene activation via promoter remodeling and Meiosin activation in female germline.

In mammals, primordial germ cells (PGCs) enter meiosis and differentiate into primary oocytes in embryonic ovaries. Previously, we demonstrated that meiotic gene induction and meiotic initiation were impaired in female germline cells of conditional knockout (CKO) mice lacking the Smarcb1 (Snf5) gene, which encodes a core subunit of the switching defective/sucrose non-fermenting (SWI/SNF) complex. In this study, we classified meiotic genes expressed at lower levels in Snf5 CKO females into two groups based on promoter accessibility. The promoters of 74% of these genes showed lower accessibility in mutant mice, whereas those of the remaining genes were opened without the SWI/SNF complex. Notably, the former genes included Meiosin, which encodes a transcriptional regulator essential for meiotic gene activation. The promoters of the former and the latter genes were mainly modified with H3K27me3/bivalent and H3K4me3 histone marks, respectively. A subset of the former genes was precociously activated in female PGCs deficient in polycomb repressive complexes (PRCs). Our results point to a mechanism through which the SWI/SNF complex coordinates meiotic gene activation via the remodeling of PRC-repressed genes, including Meiosin, in female germline cells.

Mid-term outcomes of surgical aortic valve replacement using a mosaic porcine bioprosthesis with concomitant mitral valve repair.

This study retrospectively evaluated the mid-term outcomes of surgical aortic valve replacement (SAVR) using a stented porcine aortic valve bioprosthesis (Mosaic; Medtronic Inc., Minneapolis, MN, USA) with concomitant mitral valve (MV) repair. From 1999 to 2014, 157 patients (median [interquartile range] age, 75 [70-79] years; 47% women) underwent SAVR with concomitant MV repair (SAVR + MV repair), and 1045 patients (median [interquartile range] age, 76 [70-80] years; 54% women) underwent SAVR only at 10 centers in Japan as part of the long-term multicenter Japan Mosaic valve (J-MOVE) study. The 5-year overall survival rate was 81.5% ± 4.1% in the SAVR + MV repair group and 85.1% ± 1.4% in the SAVR only group, and the 8-year overall survival rates were 75.2% ± 5.7% and 78.1% ± 2.1%, respectively. Cox proportional hazards analysis showed no significant difference in the survival rates between the two groups (hazard ratio, 0.87; 95% confidence interval, 0.54-1.40; P = 0.576). Among women with mild or moderate mitral regurgitation who were not receiving dialysis, those who underwent SAVR + MV repair, were aged > 75 years, and had a preoperative left ventricular ejection fraction of 30-75% tended to have a lower mortality risk. In conclusion, this subgroup analysis of the J-MOVE cohort showed relevant mid-term outcomes after SAVR + MV repair.

Epigenome editing reveals core DNA methylation for imprinting control in the Dlk1-Dio3 imprinted domain.

The Dlk1-Dio3 imprinted domain is controlled by an imprinting control region (ICR) called IG-DMR that is hypomethylated on the maternal allele and hypermethylated on the paternal allele. Although several genetic mutation experiments have shown that IG-DMR is essential for imprinting control of the domain, how DNA methylation itself functions has not been elucidated. Here, we performed both gain and loss of DNA methylation experiments targeting IG-DMR by transiently introducing CRISPR/Cas9 based-targeted DNA methylation editing tools along with one guide RNA into mouse ES cells. Altered DNA methylation, particularly at IG-DMR-Rep, which is a tandem repeat containing ZFP57 methylated DNA-binding protein binding motifs, affected the imprinting state of the whole domain, including DNA methylation, imprinted gene expression, and histone modifications. Moreover, the altered imprinting states were persistent through neuronal differentiation. Our results suggest that the DNA methylation state at IG-DMR-Rep, but not other sites in IG-DMR, is a master element to determine whether the allele behaves as the intrinsic maternal or paternal allele. Meanwhile, this study provides a robust strategy and methodology to study core DNA methylation in cis-regulatory elements, such as ICRs and enhancers.

1-year outcomes of patients implanted with the Perceval sutureless valve: the Japanese post-marketing surveillance study.

Sutureless offers an alternative to standard valves in surgical aortic valve replacement (SAVR). We sought to confirm the efficacy and safety of the Perceval sutureless valve in Japanese patients. Prospective observational study of 204 patients who underwent SAVR with Perceval at 19 sites in Japan between March and December 2019. The primary outcomes were 30-day mortality and postoperative complications; the secondary outcome was all-cause mortality at 1 year. Efficacy outcomes were changed in New York Heart Association (NYHA) class, pressure gradients, effective orifice area (EOA), EOA indexed to body surface area (EOAi) and severity of aortic regurgitation. Mean age was 77.7 years, 62.7% were female. Procedural success rate was 99.0%. The median cross-clamp and cardiopulmonary bypass times were 68.0 and 108 min. Perceval size S and M were implanted in 95 (46.6%) and in 76 (37.3%) of patients, respectively. The 30-day and late mortality rate were 0.5% and 4.4%, while the new permanent pacemaker implantation rate was 4.4%. Mean pressure gradient was 13.0 mmHg at discharge, reaching 11.0 mmHg at 1 year; while the mean EOA was 1.5 cm2 at discharge remaining stable up to 1 year. No moderate or severe leakages were present at discharge or at 1 year. NYHA class improved by ≥ 1 level in 55.1% of the patients at discharge and in 69.4% of the patients at 1 year. 1-year outcomes of SAVR with the Perceval sutureless valve in Japanese patients were favorable. This valve offers a promising alternative to conventional biological AVR in this Japanese population.

Long-Term Outcomes of the Mosaic Mitral Porcine Bioprosthesis in Japan　- Results From the Japan Mosaic Valve Long-Term Multicenter Study.

BACKGROUND: This study retrospectively evaluated the long-term patient outcomes and durability of the Mosaic mitral porcine bioprosthesis in the Japan Mosaic valve long-term multicenter study.Methods and Results:The medical records of 390 patients who underwent mitral valve replacement with the Mosaic bioprosthesis at 10 centers in Japan (1999-2014) were reviewed. Patient data were collected using the Research Electronic Data Capture software. Patient survival was determined using the Kaplan-Meier method. Freedom from structural valve deterioration (SVD) and valve-related reoperation and death were determined using actuarial methods. The median (interquartile range [IQR]) age of the cohort was 73 (69-77) years. The median (IQR) follow-up period was 4.83 (1.84-8.26) years. The longest follow-up period was 15.8 years. The 30-day mortality rate was 5.4%. The 12-year actuarial survival rate was 54.1±4.5%, and the freedom from valve-related death was 85.3±3.4%. The freedom from reoperation at 12 years was 74.3±5.7%. The freedom from SVD at 12 years was 81.4±6.6% for patients aged ≥65 years and 71.6±11.1% for those aged <65 years. The median (IQR) mean pressure gradient was 4.1 (3.0-6.0) and 5.6 (4.0-6.7) mmHg at 1 and 10 years, respectively. The median (IQR) effective orifice area was 1.7 (1.4-2.0) and 1.4 (1.2-1.6) cm2at 1 and 10 years, respectively. CONCLUSIONS: The Mosaic porcine bioprosthesis offered satisfactory long-term outcomes for up to 12 years.

Management of Nuisance Macromoths in Expressways through Academic-Industrial Collaboration: Light Trap Designed on the Basis of Moths' Preferences for Light Attributes.

Aggregation of flying insects such as gypsy moths at commercial light sources in summer not only has an aesthetically negative impact on public facilities but also permits the establishment of new insect populations there from the next year. Although energy-efficient light traps equipped with light-emitting diodes (LEDs) have recently been used for controlling pest insects in agriculture, there are very few maintenance-free light traps that are available on the market. Based on the results of field surveys, we fabricated a prototype light trap in which the preferences of insects for light irradiation angle and wavelength are implemented. Field experiments revealed that flying moths were attracted more to light with a narrow irradiation angle than to light with a wide irradiation angle. Moreover, there was a tendency for fewer moths to be collected when fluorescent paint was applied to the surface of the flight-interception board, indicating that a high contrast made by illumination and the background is preferred by flying moths. Taken together with our previous results, we found that the moth catch was influenced more by modification of the light design than by change in visible light wavelengths. A semi-portable light trap, named the "Kurihara trap" after the primary contributor to its development, is made of light-weight plastic and is driven by solar power. This light trap is omnidirectional and maintenance-free and is therefore suitable for deployment in the backyards of rest areas as well as at houses for long-term macromoth sampling.

Seed management using NGS technology to rapidly eliminate a deleterious allele from rice breeder seeds.

Spontaneous mutations are stochastic phenomena that occur in every population. However, deleterious mutated allele present in seeds distributed to farmers must be detected and removed. Here, we eliminated undesirable mutations from the parent population in one generation through a strategy based on next-generation sequencing (NGS). This study dealt with a spontaneous albino mutant in the 'Hinohikari' rice variety grown at the Miyazaki Comprehensive Agricultural Experiment Station, Japan. The incidence of albinism in the population was 1.36%. NGS analysis revealed the genomic basis for differences between green and albino phenotypes. Every albino plant had a C insertion in the Snow-White Leaf1 (SWL1) gene on chromosome 4 causing a frameshift mutation. Selfing plants heterozygous for the mutant allele, swl1-R332P, resulted in a 3:1 green/albino ratio, confirming that a single recessive gene controls albinism. Ultrastructural leaf features in the swl1-R332P mutants displayed deformed chlorophyll-associated organelles in albino plants that were similar to those of previously described swl1 mutants. Detection of the causative gene and its confirmation using heterozygous progenies were completed within a year. The NGS technique outlined here facilitates rapid identification of spontaneous mutations that can occur in breeder seeds.

Five multicopy gene family genes expressed during the maternal-to-zygotic transition are not essential for mouse development.

Upon fertilization, oocytes transform into totipotent and pluripotent cleavage stage cells through the maternal-to-zygotic transition (MZT), which is regulated by maternal factors and zygotic genome activation (ZGA). Here, we investigated the in vivo function of 16 genes expressed with strong biases in oocytes and cleavage stage embryos by generating knockout (KO) mice. These MZT-associated genes are conserved across many mammalian species and include five multicopy gene family genes: the Nlrp9, Khdc1, Rfpl4, Trim43, and Zscan5 genes. Intercrosses between female KO and male KO mice, including Nlrp9a/b/c triple KO (TKO), Khdc1a/b/c TKO, Rfpl4a/b double KO (DKO), Trim43a/b/c TKO, and Zscan5b KO mice led to the birth to healthy offspring that in turn produced healthy offspring. Our study not only demonstrated that these MZT-associated genes are not essential for mouse development, but also provides valuable resources for analyzing the functions of these genes in other genetic backgrounds, in the presence of stressors, and under pathogenic conditions.

Use of a cutting stapler to excise a left atrial appendage in minimally invasive cardiac surgery.

PURPOSE: A cutting stapler is a well-established instrument in many surgical fields. However, its efficacy and safety have not been proven yet in resecting a left atrial appendage (LAA) in minimally invasive cardiac surgery (MICS). METHODS: A cutting stapler was used to resect the LAA in 98 consecutive patients who underwent MICS. Of these, 26 patients underwent aortic valve replacement, 72 mitral valve repair/replacement, 25 tricuspid annuloplasty, 7 closure of atrial septal defect, and 26 the Maze procedure (contains overlapping). The ascending aorta was elevated using a retractor, and a 12-mm shaft motor-driven cutting stapler was inserted through the transverse sinus. As a control group, 150 patients who underwent suture resection/closure of the LAA either from inside or outside were compared. RESULTS: There was one mortality in each group. They were not related to the LAA resection/closure. In the suture group, the LAA suture was taken down in one patient because of a suspected obstruction of the circumflex artery, and two patients had re-exploration for bleeding from the LAA. In the stapler group, there was no complication related with the LAA. The rate of complication did not reach a statistical difference. CONCLUSION: A cutting stapler is considered to be a useful instrument to resect the LAA in MICS.

Mutations of histone demethylase genes encoded by X and Y chromosomes, Kdm5c and Kdm5d, lead to noncompaction cardiomyopathy in mice.

Mammalian X and Y chromosomes evolved from a pair of autosomes. Although most ancestral genes have been lost from the Y chromosome, a small number of ancestral X-Y gene pairs are still present on the sex chromosomes. The KDM5C and KDM5D genes, which encode H3K4 histone demethylases, are a surviving ancestral gene pair located on the X and Y chromosomes, respectively. Mutations in KDM5C cause X-linked intellectual disability in human males, suggesting functional divergence between KDM5C and KDM5D in the nervous system. In this study, to explore the functional conservation and divergence between these two genes in other organs, we generated female mice lacking Kdm5c (homozygous X5c- X5c- females) and male mice lacking both Kdm5c and Kdm5d (compound hemizygous X5c- Y5d- males). Both X5c- X5c- females and X5c- Y5d- males showed lower body weights and postnatal lethality. Histological examination of the hearts showed prominent trabecular extension and a thin layer of compacted myocardium in the left and right ventricles, indicating noncompaction cardiomyopathy. However, hemizygous males lacking either Kdm5c or Kdm5d showed no signs of noncompaction cardiomyopathy. These results clearly demonstrate that the function of Kdm5c and Kdm5d in heart development is conserved.

Involvement of Nlrp9a/b/c in mouse preimplantation development.

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

Long-Term Outcomes of the Mosaic Aortic Porcine Bioprosthesis in Japan　- Results From the Japan Mosaic Valve Long-Term Multicenter Study.

BACKGROUND: This study retrospectively evaluated the long-term patient outcomes and durability of the Mosaic aortic porcine bioprosthesis in the Japan Mosaic valve long-term multicenter study.Methods and Results:We reviewed the records of 1,202 patients who underwent aortic valve replacement with the Mosaic bioprosthesis at 10 centers in Japan (1999-2014). Patient data were collected using Research Electronic Data Capture. Patient survival was determined by Kaplan-Meier methodology. Freedom from structural valve deterioration (SVD) and valve-related reoperation and death were determined by actuarial methods. The median (interquartile range [IQR]) age of the cohort was 76 (70-80) years. The median (IQR) follow-up period was 3.52 (1.71-5.35) years. The longest follow-up was 15.8 years. The 30-day mortality rate was 2.3%. The 12-year actuarial survival rate was 59.9±7.5%, and the freedom from valve-related death was 81.1±7.9%. The freedom from reoperation was 86.4±2.6% at 12 years. The freedom from SVD at 12 years was 93.5±2.9% for patients aged ≥65 years and 98.2±1.8% for those aged <65 years. The median (IQR) systolic pressure gradient was 17 (12-23) and 19 (12-25) mmHg at 1 and 10 years, respectively. The median (IQR) effective orifice area was 1.2 (1.1-1.5) and 1.1 (1-1.5) cm2at 1 and 10 years, respectively. CONCLUSIONS: The Mosaic porcine bioprosthesis showed satisfactory long-term outcomes over 12 years.

A novel 'two-window' technique to facilitate totally 3D-endoscopic mitral valve repair.

We describe our experience of a novel 'two-window' technique designed to assist in totally 3D-endoscopic mitral valve repair. A 10-mm trocar for a 3D-endoscope was inserted through the 4th intercostal space in the mid-axillary line. A main small incision (3 cm, 4th intercostal space) without rib spreading, and a second 1 cm incision (1-2 intercostal spaces above,) were made in the anterior axillary line. Soft tissue retractors were applied on both ports. A left atrial retractor, a left ventricular vent, and right-hand instruments were inserted through the main working port. A flexible aortic cross-clamp, an antegrade cardioplegia line, and left-hand instruments were inserted through the second port. Our two-window technique is safe, effective, and reproducible for totally endoscopic mitral valve repair.

[Totally Endoscopic Aortic Valve Replacement Using the Three-dimensional Endoscope].

Totally endoscopic aortic valve replacement (TE-AVR) is still challenging, and few series report exist even today. In 2015, we started to use three-dimensional (3D) endoscope and we also introduced TE-AVR. Patient is placed in the partial left lateral position. The main wound is created in right antero-lateral 4th intercostal space through 4 cm skin incision. No rib spreader is used. 3D endoscope is inserted on the mid-axillary line. A 5 mm trocar was inserted in the 3rd intercostal space, thus creating 3-port setting similarly to that for endoscopic mitral valve surgery. All sutures are tied using a knot-pusher. We have performed 106 cases of TE-AVR. Compared with transaxillary AVR, there were no significant differences between the 2 groups in the hospital deaths or MACCE. Postoperative hospital stays became shorter in totally endoscopic group. In conclusion, TE-AVR was possible through 3 ports created in the right antero-lateral chest similarly to the endoscopic mitral valve surgery. Transaxillary approach seemed to be suitable for the TE-AVR. By adopting common approach for both mitral valve surgery and aortic valve surgery, endoscopic double valve surgery could be performed seamlessly.

Three-port totally endoscopic repair vs conventional median sternotomy for atrial septal defect.

PURPOSES: We assessed the validity of three-port totally endoscopic repair (3PTER) for atrial septal defect (ASD). METHODS: Between February, 2000 and November, 2017, 151 patients underwent surgery for ASD. Forty-seven patients underwent 3PTER as minimally invasive cardiac surgery (MICS) and 104 patients underwent conventional median sternotomy (CMS). Propensity matching yielded 94 matched patients (47 vs 47). We compared the early results between the groups. The 3PTER technique was performed with the patient in the partial left lateral position, under cardio-pulmonary bypass (CPB) established through a groin incision. The three ports consisted of a main incision (3 cm), a trocar for the left-handed instrument, and a camera port in right antero-lateral chest. RESULTS: MICS needed longer cross clamp and CPB times (57, 48-86 vs 24, 16-30 min, p < 0.01 and 115, 106-131 vs 53, 43-80 min, p < 0.01, respectively)*, although the operation time and hospital stay were significantly shorter (180, 159-203 vs 190, 161-225 min, p = 0.024 and 6.0, 6-8 vs 15, 13-19 days, p < 0.01, respectively)*. The intra-operative and postoperative bleeding were significantly less in MICS than CMS (20, 5-40 vs 225, 130-287.5 p < 0.01 and 200, 145-290 vs 340, 250-535 ml, p < 0.01, respectively)*. *: median, 25th-75th percentile. CONCLUSION: Irrespective of the longer CPB and cross-clamp time than for CMS, MICS had a shorter operation time, less bleeding, and resulted in quicker recovery. The 3PTER was safe and cosmetically excellent.

Intercellular translocation of molecules via plasmodesmata in the multiseriate filamentous brown alga, Halopteris congesta (Sphacelariales, Phaeophyceae).

Despite the high number of studies on the fine structure of brown algal cells, only limited information is available on the intercelluar transportation of molecules via plasmodesmata in brown algae. In this study, plasmodesmatal permeability of Halopteris congesta was examined by observing the translocation of microinjected fluorescent tracers of different molecular sizes. The tip region of H. congesta consists of a cylindrical apical cell, while the basal region is multiseriate. Fluorescein isothiocyanate-dextran (FD; 3, 10, and 20 kDa) and recombinant green fluorescent protein (27 kDa) were injected into the apical cell and were observed to diffuse into the neighboring cells. FD of 40 kDa was detected only in the injected apical cell. The plasmodesmatal size exclusion limit was considered to be more than 20 kDa and less than 40 kDa. The extent of translocation of 3 and 10 kDa FD from the apical to neighboring cells 2 h postinjection was estimated based on the fluorescence intensity. It was suggested that the diffusing capacity of plasmodesmata varied according to molecular size. In order to examine acropetal and/or basipetal direction of molecular movement, 3 and 10 kDa FD were injected into the third cell from the apical cell. Successive observations indicated that the diffusion of fluorescence in the acropetal direction took longer than that in the basipetal direction. No ultrastructural difference in plasmodesmata was noted among the cross walls.

Distribution of alginate and cellulose and regulatory role of calcium in the cell wall of the brown alga Ectocarpus siliculosus (Ectocarpales, Phaeophyceae).

MAIN CONCLUSION: This work investigated a correlation between the three-dimensional architecture and compound-components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity. Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.

Characterization of a novel negevirus isolated from Aedes larvae collected in a subarctic region of Japan.

We isolated and characterized a novel positive-sense, single-stranded RNA virus from Aedes larvae collected on Okushiri Island, Hokkaido, Japan. This virus, designated Okushiri virus (OKV), replicated in the Aedes albopictus cell line C6/36 with severe cytopathic effects and produced a large number of spherical viral particles that were 50-70 nm in diameter and released into the cell culture medium. The OKV genome consisted of 9,704 nucleotides, excluding the poly(A) tail at the 3'-terminus, and contained three major open reading frames (ORF1, ORF2, and ORF3). ORF1 encoded a putative protein of approximately 268 kDa that included a methyltransferase domain, FtsJ-like methyltransferase domain, helicase domain, and RNA-dependent RNA polymerase domain. The genome organization and results of a phylogenetic analysis based on the amino acid sequence predicted from the nucleotide sequence indicated that OKV is a member of a new insect virus group of negeviruses with a possible evolutionary relationship to some plant viruses. ORF2 and ORF3 were suggested to encode hypothetical membrane-associated proteins of approximately 45 kDa and 22 kDa, respectively. This is the first study on a novel negevirus isolated from mosquito larvae in Japan.

[Minimally Invasive Mitral Valve Plasty;My Technique].

We perform minimally invasive mitral valve plasty under totally endoscopic view. Skin incisions are composed of a 3 to 5 cm of main wound along the right 4th intercostal space, 1 trocar port in the 3rd intercostal space, and a camera port in the 5th intercostal space. Matal rib spreader is not used. A 3-dimensional endoscope was recently introduced. Forceps controlled by the left hand are inserted through the independent trocar port in the 3rd intercostal space. Left atrial retractor, aortic clamp, and all the cannulae are inserted through the main incision. Cardio-pulmonary bypass is established through the right femoral artery and vein cannulation. No additional venous cannula through the right jugular vein is used. Posterior leaflet lesions are repaired by resection and suture technique. Anterior leaflet prolapse is repaired mainly using the loop technique. Bleeding from the chest wall is meticulously checked before closing the chest.