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The scleractinian coral genus Cyphastrea is widely distributed in the Indo-Pacific region and is common from the subtropical to the warm-temperate regions in Japan. Three new species in this genus have recently been reported from south-eastern Australia or the Red Sea. However, taxonomic and species diversity have been little studied so far in Japan. In this study, we analyzed 112 specimens of Cyphastrea collected from the subtropical to the warm-temperate regions in Japan to clarify the species diversity in the country. This analysis was based on skeletal morphological and molecular analyses using three genetic markers of the nuclear 28S rDNA, histone H3 gene, and the mitochondrial noncoding intergenic region between COI and tRNAmet. The molecular phylogenetic trees showed that our specimens are separated mainly into four clades. Considering the morphological data with the molecular phylogenetic relationships, we confirmed a total of nine species, including two species, C. magna and C. salae, recorded for the first time in Japan. Although eight out of nine species were genetically included within Cyphastrea, one species, C. agassizi, was genetically distant from all other species and was closely related to the genus Leptastrea, suggesting the return of this species to the genus to which it was originally ascribed. Two newly recorded species were reciprocally monophyletic, while the other six species (excluding C. agassizi) clustered in two clades without forming species-specific lineages, including three polyphyletic species. Thus, the species boundary between species in Cyphastrea remains unclear in most species using these three sequenced loci.
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Antozoos , Animales , Filogenia , Antozoos/genética , Japón , ADN Ribosómico/genética , Mitocondrias/genética , Análisis de Secuencia de ADNRESUMEN
An unique laparoscopic ureteroneocystostomy technique was performed to treat an iatrogenic ureterovaginal fistula that was formed in a 69-year-old woman following open modified radical hysterectomy for endometrial cancer. Severe adhesions between the distal ureter and the surrounding tissues, including the iliac artery, were observed. Owing to difficulties in identifying the distal ureter, the proximal ureter was identified and dissected downward to free the ureter, thereby allowing anastomosis. This report shows that laparoscopic ureteroneocystostomy for ureterovaginal fistula repair may prove useful owing to its minimally invasive and broad approach.
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In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.
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Introduction: The treatment of thrombi in the renal vein (RV) and inferior vena cava (IVC) requires advanced laparoscopic experience. We present three cases of hand-assisted laparoscopic nephrectomy (HALN) using a novel technique for treating advanced renal cell carcinoma (RCC) with thrombi in the RV and IVC. Case Presentation. Three patients with RCC with RV or IVC thrombus below level I underwent HALN. Two patients had right RCC with RV and IVC thrombi. One patient had left RCC with an RV thrombus. We hooked a vessel loop to the end of the thrombus and pulled it up manually to make space for vascular processing. The RV was narrowed and dissected using Hem-o-lok clips or an Endo GIA stapler. Conclusion: In carefully selected cases, renal vascular processing could be easily and safely performed using a vessel loop in HALN with thrombectomy.
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In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.
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Criopreservación/métodos , Crioprotectores/química , Manejo de Especímenes/métodos , Espermatozoides/citología , Glicerol/química , Humanos , Masculino , Polilisina/química , Rafinosa/químicaRESUMEN
The first-in-human trial of induced pluripotent stem cell (iPSC)-based autologous transplantation was successfully performed on a female patient with age-related macular degeneration. Here we delineated the base-resolution methylome of the iPSC-derived retinal pigment epithelium (iRPE) used in this trial. The methylome of iRPE closely resembled that of native RPE (nRPE), although partially methylated domains (PMDs) emerged in iRPE but not nRPE. Most differentially methylated regions between iRPE and nRPE appeared to originate from (de)methylation errors during differentiation, whereas errors at reprogramming resulted in aberrant genomic imprinting and X chromosome reactivation. Moreover, non-CpG methylation was prominent in nRPE but not iRPE. Intriguingly, xenotransplantation to mouse remodeled the iRPE methylome to demethylate a subset of suppressed genes and accumulate non-CpG methylation, but failed to resolve PMDs and hypermethylated CpG islands. Although the impacts of these alterations remain elusive, our findings should provide a useful guide for methylome analyses of other iPSC-derived cells.
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Epigenoma , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Madre Pluripotentes Inducidas/citología , Epitelio Pigmentado de la Retina/citología , Trasplante de Células Madre , Reprogramación Celular , Biología Computacional/métodos , Islas de CpG , Metilación de ADN , Humanos , Degeneración Macular/etiología , Degeneración Macular/metabolismo , Degeneración Macular/terapia , Trasplante Autólogo , Secuenciación Completa del GenomaRESUMEN
As corals in tropical regions are threatened by increasing water temperatures, poleward range expansion of reef-building corals has been observed, and temperate regions are expected to serve as refugia in the face of climate change. To elucidate the important indicators of the sustainability of coral populations, we examined the genetic diversity and connectivity of the common reef-building coral Acropora hyacinthus along the Kuroshio Current, including recently expanded (<50 years) populations. Among the three cryptic lineages found, only one was distributed in temperate regions, which could indicate the presence of Kuroshio-associated larval dispersal barriers between temperate and subtropical regions, as shown by oceanographic simulations as well as differences in environmental factors. The level of genetic diversity gradually decreased towards the edge of the species distribution. This study provides an example of the reduced genetic diversity in recently expanded marginal populations, thus indicating the possible vulnerability of these populations to environmental changes. This finding underpins the importance of assessing the genetic diversity of newly colonized populations associated with climate change for conservation purposes. In addition, this study highlights the importance of pre-existing temperate regions as coral refugia, which has been rather underappreciated in local coastal management.
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Antozoos/genética , Cambio Climático , Animales , Antozoos/crecimiento & desarrollo , Variación Genética , Genotipo , Japón , Refugio de Fauna , TemperaturaRESUMEN
Age-dependent decline of mitochondrial function has been proposed to be a main cause of decline of embryo quality. Then, l-carnitine plays important roles in reducing the membranous toxicity of free-fatty acids by forming acyl-carnitine and promoting ß-oxidation, preventing cell damage. Recent research revealed that l-carnitine played important roles in vitro in oocyte growth, oocyte maturation and embryo development. However, such beneficial effects of l-carnitine in vivo have yet to be verified. The effect of oral l-carnitine supplementation on embryo quality and implantation potential was examined. A total of 214 patients were included in this study. They all previously received in vitro fertilization-embryo transfer (IVF-ET) and failed to conceive. Then they were administered l-carnitine for 82 days on average and underwent IVF-ET again. There were no significant differences in the total number of retrieved oocytes, and their maturation and fertilization rates between before and after l-carnitine administration. The quality of embryos on Days 3 and 5 after insemination was improved following l-carnitine administration (p < .05) in cycles after l-carnitine administration compared with previous cycles. Healthy neonates were born after IVF-ET following l-carnitine administration. Our data suggested that oral administration of l-carnitine to fertility patients improved the developmental competence of their oocytes after insemination.
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Carnitina/uso terapéutico , Desarrollo Embrionario/efectos de los fármacos , Fertilización In Vitro/estadística & datos numéricos , Infertilidad Femenina/tratamiento farmacológico , Administración Oral , Adulto , Carnitina/farmacología , Femenino , Humanos , Insuficiencia del TratamientoRESUMEN
Current induction methods of hepatocytes from human induced pluripotent stem cells (hiPSCs) are neither low cost nor stable. By screening a chemical library of 1,120 bioactive compounds and known drugs, we identified the α1-adrenergic receptor agonist methoxamine hydrochloride as a small molecule that promotes the differentiation of hiPSC-derived hepatoblasts into ALBUMIN+ hepatocyte-like cells. Other α1-adrenergic receptor agonists also induced the differentiation of hepatocyte-like cells, and an α1-receptor antagonist blocked the hepatic-inducing activity of methoxamine hydrochloride and that of the combination of hepatocyte growth factor (HGF) and Oncostatin M (OsM), two growth factors often used for the induction of hepatoblasts into hepatocyte-like cells. We also confirmed that treatment with methoxamine hydrochloride activates the signal transducer and activator of transcription 3 (STAT3) pathway downstream of IL-6 family cytokines including OsM. These findings allowed us to establish hepatic differentiation protocols for both mouse embryonic stem cells (mESCs) and hiPSCs using small molecules at the step from hepatoblasts into hepatocyte-like cells. The results of the present study suggest that α1-adrenergic agonists induce hepatocyte-like cells by working downstream of HGF and OsM to activate STAT3.
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Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Hepatocitos/citología , Células Madre Embrionarias Humanas/citología , Células Madre Pluripotentes Inducidas/citología , Metoxamina/farmacología , Antagonistas de Receptores Adrenérgicos alfa 1/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Evaluación Preclínica de Medicamentos , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Oncostatina M/farmacología , Factor de Transcripción STAT3/metabolismo , Albúmina Sérica Humana/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).
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Células Madre Pluripotentes Inducidas/citología , Degeneración Macular/terapia , Epitelio Pigmentado de la Retina/citología , Anciano , Técnicas de Cultivo de Célula , Diferenciación Celular , Estudios de Factibilidad , Femenino , Fibroblastos , Humanos , Masculino , Epitelio Pigmentado de la Retina/trasplante , Trasplante AutólogoRESUMEN
The family Poritidae formerly included 6 genera: Alveopora, Goniopora, Machadoporites, Porites, Poritipora, and Stylaraea. Morphologically, the genera can be differentiated based on the number of tentacles, the number of septa and their arrangement, the length of the polyp column, and the diameter of the corallites. However, the phylogenetic relationships within and between the genera are unknown or contentious. On the one hand, Alveopora has been transferred to the Acroporidae recently because it was shown to be more closely related to this family than to the Poritidae by previous molecular studies. On the other hand, Goniopora is morphologically similar to 2 recently described genera, Machadoporites and Poritipora, particularly with regard to the number of septa (approximately 24), but they have not yet been investigated at the molecular level. In this study, we analyzed 93 samples from all 5 poritid genera and Alveopora using 2 genetic markers (the barcoding region of the mitochondrial COI and the ITS region of the nuclear rDNA) to investigate their phylogenetic relationships and to revise their taxonomy. The reconstructed molecular trees confirmed that Alveopora is genetically distant from all poritid genera but closely related to the family Acroporidae, whereas the other genera are genetically closely related. The molecular trees also revealed that Machadoporites and Poritipora were indistinguishable from Goniopora. However, Goniopora stutchburyi was genetically isolated from the other congeneric species and formed a sister group to Goniopora together with Porites and Stylaraea, thus suggesting that 24 septa could be an ancestral feature in the Poritidae. Based on these data, we move G. stutchburyi into a new genus, Bernardpora gen. nov., whereas Machadoporites and Poritipora are merged with Goniopora.
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Antozoos/anatomía & histología , Antozoos/clasificación , Antozoos/genética , Filogenia , Animales , Secuencia de Bases , Pesos y Medidas Corporales , Análisis por Conglomerados , ADN Espaciador Ribosómico/genética , Complejo IV de Transporte de Electrones/genética , Extremidades/anatomía & histología , Océano Índico , Japón , Malasia , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
The migration of Caenorhabditis elegans gonadal distal tip cells (DTCs) offers an excellent model to study the migration of epithelial tubes in organogenesis. mig-18 mutants cause meandering or wandering migration of DTCs during gonad formation, which is very similar to that observed in animals with mutations in mig-17, which encodes a secreted metalloprotease of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family. MIG-18 is a novel secreted protein that is conserved only among nematode species. The mig-17(null) and mig-18 double mutants exhibited phenotypes similar to those in mig-17(null) single mutants. In addition, the mutations in fbl-1/fibulin-1 and let-2/collagen IV that suppress mig-17 mutations also suppressed the mig-18 mutation, suggesting that mig-18 and mig-17 function in a common genetic pathway. The Venus-MIG-18 fusion protein was secreted from muscle cells and localized to the gonadal basement membrane, a tissue distribution reminiscent of that observed for MIG-17. Overexpression of MIG-18 in mig-17 mutants and vice versa partially rescued the relevant DTC migration defects, suggesting that MIG-18 and MIG-17 act cooperatively rather than sequentially. We propose that MIG-18 may be a cofactor of MIG-17/ADAMTS that functions in the regulation of the gonadal basement membrane to achieve proper direction of DTC migration during gonadogenesis.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Movimiento Celular , Desintegrinas/metabolismo , Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Basal/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Movimiento Celular/genética , Desintegrinas/genética , Expresión Génica , Gónadas/metabolismo , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Mutación , Unión Proteica , Transporte de Proteínas , Alineación de Secuencia , Transducción de SeñalRESUMEN
To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 µg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 µg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 µg per liter).