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BACKGROUND/AIM: We report an in vitro three-dimensional (3D) culture system optimized for the growth of HepG2 hepatocarcinoma cells. MATERIALS AND METHODS: The 3D culture system was fabricated based on polyethylene glycol (PEG)-based hydrogels; their mechanical strength was controlled by differences in the arm number and concentration of PEG-vinylsulfone. Moreover, cellular growth was evaluated after culturing HepG2 cells in PEG-based hydrogels with various mechanical strengths. RESULTS: HepG2 cell culture in the 3D PEG-based hydrogels induced the formation of spherical colonies. Moreover, the highest number of spherical colonies formed from HepG2 cells at the single-cell level, and the formation of spherical colonies with a uniform size was observed in HepG2 cells cultured in 5% (w/v) 8-arm PEG-based hydrogels. CONCLUSION: 5% (w/v) 8-arm PEG-based hydrogels may be developed as a 3D culture system optimized for stimulating the in vitro growth of HepG2 cells.
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Hidrogéis , Polietilenoglicóis , Humanos , Polietilenoglicóis/farmacologia , Células Hep G2 , Hidrogéis/farmacologia , Linhagem Celular , Técnicas de Cultura de Células/métodos , Materiais BiocompatíveisRESUMO
BACKGROUND: A piglet model for peritoneal metastasis (PM) of ovarian cancer was developed. It will contribute to establishing innovative chemotherapeutical and surgical strategies without any limitation on rodent models. METHODS: A total of 12 four- to five-week-old piglets of 7 to 8 kg were used. Two phases of ovarian cancer cell injections were performed with laparoscopic surgery. In phase I trial, 5.0 × 106 SK-OV-3 cells in 0.1 ml suspension were inoculated into the omentum, peritoneum, and uterine horns of two piglets twice with a one-week interval. In the phase II trial, 5.0 × 106 SNU-008 cells in 0.1 ml suspension were injected only into uterine horns within the same time frame because tumor implantation after inoculation of SK-OV-3 cells was not observed at the omentum or peritoneum in the phase I trial. Modified peritoneal cancer index (PCI) score was used to monitor tumorigenesis up to 4 weeks after inoculation. Tumor tissues disseminated in the peritoneum 4 weeks after injection were used for histological examination with hematoxylin and eosin (H&E) and paired-box gene 8 (PAX-8) staining. RESULTS: In the phase I trial, two piglets showed PM with modified PCI scores of 5 and 4 at 3 weeks after the first inoculation, which increased to 14 and 15 after 4 weeks, respectively. In the phase II trial, PM was detected in eight of ten piglets, which showed modified PCI scores of 6 to 12 at 4 weeks after the first inoculation. The overall incidence of PM from the total of 12 piglets after inoculation was 75%. Immunohistochemical H&E and PAX-8 staining confirmed metastatic tumors. CONCLUSIONS: This study provides strong evidence that piglets can be employed as a model for PM by inoculating ovarian cancer cell lines from humans. Using two cell lines, the PM rate is 75%.
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Neoplasias Ovarianas , Neoplasias Peritoneais , Animais , Carcinoma Epitelial do Ovário/patologia , Feminino , Humanos , Omento/patologia , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/patologia , Peritônio/patologia , SuínosRESUMO
To assess whether the follicle-stimulating hormone (FSH) subunits observed in patients with gonadotroph adenomas (GA) can cause infertility, the effects of subunits and heterodimeric FSH on the in vitro follicle development were evaluated in mice. The partial forms of FSH in follicle culture did not induce development into pseudoantral follicles, whereas follicles cultured with native FSH developed into pseudoantral follicles and produced mature metaphase II oocyte. Therefore, intact FSH is needed for folliculogenesis, implying that production of FSH with a partial structure in GA may result in infertility.
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Hormônio Foliculoestimulante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Feminino , Camundongos , Subunidades Proteicas/farmacologia , Técnicas de Cultura de TecidosRESUMO
The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.
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Ácido Aspártico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Influenza A/enzimologia , Mutação , Proteínas Nucleares/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico/química , Ácido Aspártico/genética , Galinhas , DNA Polimerase Dirigida por DNA/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Infecções por Orthomyxoviridae/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Homologia de Sequência , Proteínas Virais/genéticaRESUMO
Speech emotion recognition (SER) is a natural method of recognizing individual emotions in everyday life. To distribute SER models to real-world applications, some key challenges must be overcome, such as the lack of datasets tagged with emotion labels and the weak generalization of the SER model for an unseen target domain. This study proposes a multi-path and group-loss-based network (MPGLN) for SER to support multi-domain adaptation. The proposed model includes a bidirectional long short-term memory-based temporal feature generator and a transferred feature extractor from the pre-trained VGG-like audio classification model (VGGish), and it learns simultaneously based on multiple losses according to the association of emotion labels in the discrete and dimensional models. For the evaluation of the MPGLN SER as applied to multi-cultural domain datasets, the Korean Emotional Speech Database (KESD), including KESDy18 and KESDy19, is constructed, and the English-speaking Interactive Emotional Dyadic Motion Capture database (IEMOCAP) is used. The evaluation of multi-domain adaptation and domain generalization showed 3.7% and 3.5% improvements, respectively, of the F1 score when comparing the performance of MPGLN SER with a baseline SER model that uses a temporal feature generator. We show that the MPGLN SER efficiently supports multi-domain adaptation and reinforces model generalization.
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Bases de Dados Factuais , Emoções/classificação , Aprendizado de Máquina , Reconhecimento Automatizado de Padrão , Fala , HumanosRESUMO
Adenomyosis is defined as the presence of ectopic nests of endometrial glands and stroma within the myometrium. Adenomyosis is a common cause of dysmenorrhea, menorrhagia, and chronic pelvic pain but is often underdiagnosed. Despite its prevalence and severity of symptoms, its pathogenesis and etiology are poorly understood. Our previous study showed that aberrant activation of ß-catenin results in adenomyosis through epithelial-mesenchymal transition. Using transcriptomic and ChIP-seq analysis, we identified activation of TGF-ß signaling in the uteri of mutant mice that expressed dominant stabilized ß-catenin in the uterus. There was a strong positive correlation between ß-catenin and TGF-ß2 proteins in women with adenomyosis. Furthermore, treatment with pirfenidone, a TGF-ß inhibitor, increased E-cadherin expression and reduced cell invasiveness in Ishikawa cells with nuclear ß-catenin. Our results suggest that ß-catenin activates TGF-ß-induced epithelial-mesenchymal transition in adenomyosis. This finding describes the molecular pathogenesis of adenomyosis and the use of TGF-ß as a potential therapeutic target for adenomyosis.
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Adenomiose/metabolismo , Suscetibilidade a Doenças , Transição Epitelial-Mesenquimal , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismo , Adenomiose/etiologia , Adenomiose/patologia , Animais , Sítios de Ligação , Caderinas/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fator de Crescimento Transformador beta/farmacologiaRESUMO
This study examined whether neonatal chicken bone marrow cells (cBMCs) could support the osteogenesis of human stromal cells in a three-dimensional (3D) extracellular bioprinting niche. The majority (>95%) of 4-day-old cBMCs subcultured 5 times were positive for osteochondrogenesis-related genes (Col I, Col II, Col X, aggrecan, Sox9, osterix, Bmp2, osteocalcin, Runx2, and osteopontin) and their related proteins (Sox9, collagen type I, and collagen type II). LC-MS/MS analysis demonstrated that cBMC-conditioned medium (c-medium) contained proteins related to bone regeneration, such as periostin and members of the TGF-ß family. Next, a significant increase in osteogenesis was detected in three human adipose tissue-derived stromal cell (hASC) lines, after exposure to c-medium concentrates in 2D culture (p < 0.05). To evaluate biological function in a 3D environment, we employed the cBMC-derived bioactive components as a cell-supporting biomaterial in collagen bioink, which was printed to construct a 3D hASC-laden scaffold for observing osteogenesis. Complete osteogenesis was detected in vitro. Moreover, after transplantation of the hASC-laden structure into rats, prominent bone formation was observed compared with that in control rats receiving scaffold-free hASC transplantation. These results demonstrated that substance(s) secreted by chick bone marrow cells clearly activated the osteogenesis of hASCs in 2D- or 3D-niches.
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Bioimpressão , Células da Medula Óssea/citologia , Tinta , Impressão Tridimensional , Células Estromais/citologia , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Humanos , Estrutura Molecular , Osteogênese , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Propriedades de SuperfícieRESUMO
BACKGROUND: Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. The ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. METHODS: In this study, we targeted chicken ANP32A using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. RESULTS: We showed that chicken ANP32A only, not ANP32B and ANP32E, plays a pivotal role in supporting vPol activity of AIVs. Furthermore, we found that the human ANP32C, ANP32D, and ANP32E have suppressive effects on vPol activity in contrast to human ANP32A and ANP32B. CONCLUSIONS: Chicken and human ANP32 family members had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.
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RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Influenza Aviária/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Replicação Viral/genética , Animais , Galinhas , Cães , Técnicas de Silenciamento de Genes , Influenza Aviária/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular , Células Madin Darby de Rim Canino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNARESUMO
Here, as a basic study in revealing the correlation between extracellular matrix components and spontaneous abortion, we defined the types of integrins expressed on the surface of endometrial stromal (ES) cells retrieved from the uterus of a patient experiencing spontaneous abortion. For these, the types of integrin subunits in the ES cells retrieved from a woman with spontaneous abortion were identified at the transcriptional and translational levels, and functional assay was conducted for confirming the combinations of integrin α and ß subunits. Among the genes encoding 25 integrin subunits, significantly high transcription was seen in integrins α1, α2, α3, α4, α5, αV, ß1, ß3, and ß5. Translation of integrins α1, α3, α5, αV, and ß1 on the cell surface was detected in almost all ES cells, whereas integrins α2, α4, ß3, and ß4 were expressed translationally only in some ES cells. Subsequently, ES cells showed significantly increased adhesion to collagen I, laminin, fibronectin, and vitronectin, and functional blocking of integrin α1, α3, α5, and αV significantly inhibited adhesion to these molecules. These results demonstrated that active heterodimers composed of integrins α1ß1, α3ß1, α5ß1, and αVß1 were co-localized on the surface of ES cells derived from a patient experiencing spontaneous abortion.
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Aborto Espontâneo/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Integrinas/metabolismo , Células Estromais/metabolismo , Feminino , HumanosRESUMO
BACKGROUND: Endometrial cancer is the most common gynecological cancer. G-protein coupled receptor 64 (GPR64) belongs to a family of adhesion GPCRs and plays an important role in male fertility. However, the function of GPR64 has not been studied in endometrial cancer. Our objective is to investigate the role of GPR64 in endometrial cancer. METHODS: We examined the levels of GPR64 in human endometrioid endometrial carcinoma by immunohistochemistry analysis. To determine a tumor suppressor role of GPR64 in endometrial cancer, we used a siRNA loss of function approach in human endometrial adenocarcinoma cell lines. RESULTS: GPR64 levels were remarkably lower in 10 of 21 (47.62%) of endometrial carcinoma samples compared to control. Depletion of GPR64 by siRNA transfection revealed an increase of colony formation ability, cell proliferation, cell migration, and invasion activity in Ishikawa and HEC1A cells. The expression of Connexin 43 (Cx43), a member of the large family of gap junction proteins, was reduced through activation of AMP-activated protein kinase (AMPK) in Ishikawa cells with GPR64-deficicy. CONCLUSIONS: These results suggest that GPR64 plays an important tumor suppressor role in endometrial cancer.
Assuntos
Carcinoma Endometrioide/patologia , Neoplasias do Endométrio/patologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Conexina 43/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Fosforilação , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genéticaRESUMO
Calcium (Ca2+) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1⯵M medroxyprogesterone acetate) and cyclic AMP (0.5â¯mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release and IP3R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca2+ release. Finally, intracellular Ca2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs.
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Cálcio/metabolismo , Decídua/citologia , Decídua/metabolismo , Endométrio/citologia , Endométrio/metabolismo , Células Estromais/metabolismo , Adulto , Antígenos CD/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Retículo Endoplasmático/metabolismo , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Moléculas de Interação Estromal/metabolismoRESUMO
Embryonic stem cells (ESCs) maintain pluripotency through unique epigenetic states. When ESCs commit to a specific lineage, epigenetic changes in histones and DNA accompany the transition to specialized cell types. Investigating how epigenetic regulation controls lineage specification is critical in order to generate the required cell types for clinical applications. Uhrf1 is a widely known hemi-methylated DNA-binding protein, playing a role in DNA methylation through the recruitment of Dnmt1 and in heterochromatin formation alongside G9a, Trim28, and HDACs. Although Uhrf1 is not essential in ESC self-renewal, it remains elusive how Uhrf1 regulates cell specification. Here we report that Uhrf1 forms a complex with the active trithorax group, the Setd1a/COMPASS complex, to maintain bivalent histone marks, particularly those associated with neuroectoderm and mesoderm specification. Overall, our data demonstrate that Uhrf1 safeguards proper differentiation via bivalent histone modifications.
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Reprogramação Celular/genética , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT , Técnicas de Reprogramação Celular , Quimera , Metilação de DNA/fisiologia , Epigênese Genética , Feminino , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/isolamento & purificação , Histonas/metabolismo , Humanos , Masculino , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Células-Tronco Embrionárias Murinas , Placa Neural/citologia , Placa Neural/fisiologia , Proteínas Nucleares/genética , Cultura Primária de Células , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Acquisition of pluripotency by transcriptional regulatory factors is an initial developmental event that is required for regulation of cell fate and lineage specification during early embryonic development. The evolutionarily conserved core transcriptional factors regulating the pluripotency network in fishes, amphibians, and mammals have been elucidated. There are also species-specific maternally inherited transcriptional factors and their intricate transcriptional networks important in the acquisition of pluripotency. In avian species, however, the core transcriptional network that governs the acquisition of pluripotency during early embryonic development is not well understood. RESULTS: We found that chicken NANOG (cNANOG) was expressed in the stages between the pre-ovulatory follicle and oocyte and was continuously detected in Eyal-Giladi and Kochav stage I (EGK.I) to X. However, cPOUV was not expressed during folliculogenesis, but began to be detectable between EGK.V and VI. Unexpectedly, cSOX2 could not be detected during folliculogenesis and intrauterine embryonic development. Instead of cSOX2, cSOX3 was maternally inherited and continuously expressed during chicken intrauterine development. In addition, we found that the pluripotency-related genes such as cENS-1, cKIT, cLIN28A, cMYC, cPRDM14, and cSALL4 began to be dramatically upregulated between EGK.VI and VIII. CONCLUSION: These results suggest that chickens have a unique pluripotent circuitry since maternally inherited cNANOG and cSOX3 may play an important role in the initial acquisition of pluripotency. Moreover, the acquisition of pluripotency in chicken embryos occurs at around EGK.VI to VIII.
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This paper reports soft actuator based tactile stimulation interfaces applicable to wearable devices. The soft actuator is prepared by multi-layered accumulation of thin electro-active polymer (EAP) films. The multi-layered actuator is designed to produce electrically-induced convex protrusive deformation, which can be dynamically programmable for wide range of tactile stimuli. The maximum vertical protrusion is and the output force is up to 255 mN. The soft actuators are embedded into the fingertip part of a glove and front part of a forearm band, respectively. We have conducted two kinds of experiments with 15 subjects. Perceived magnitudes of actuator's protrusion and vibrotactile intensity were measured with frequency of 1 Hz and 191 Hz, respectively. Analysis of the user tests shows participants perceive variation of protrusion height at the finger pad and modulation of vibration intensity through the proposed soft actuator based tactile interface.
Assuntos
Sistemas Homem-Máquina , Tato , Interface Usuário-Computador , Dispositivos Eletrônicos Vestíveis , Desenho de Equipamento , Retroalimentação Sensorial , Dedos , Humanos , Estimulação Física , Polímeros , Percepção do Tato , VibraçãoRESUMO
Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( Pgrcre/+Rbpjf/f; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.
Assuntos
Aborto Habitual/metabolismo , Endométrio/fisiologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Miométrio/fisiologia , Regeneração , Aborto Habitual/genética , Aborto Habitual/patologia , Adulto , Animais , Endométrio/patologia , Feminino , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Interferon gama/genética , Interferon gama/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Miométrio/patologia , Período Pós-Parto/genética , Período Pós-Parto/metabolismo , GravidezRESUMO
OBJECTIVE: Remarkable difference in cellular activity was found between early and late subpassaged embryonic stem cell (ESCs) lines, which can be created by subtle changes in cell manipulation protocol. This study subsequently examined whether post-thaw subculture of early subpassaged ESC lines could further affect the activity of the ESCs. METHODS: Fresh (as a control treatment) or cryopreserved F1 hybrid (B6CBAF1) early ESC lines (C57BL/6xCBA) of the 4 (P4) or the 19 passage (P19) were subcultured once, twice or six times under the same condition. The post-thaw survival of the ESCs was monitored after the post-treatment subculture and the ability of cell proliferation, reactive oxygen species (ROS) generation, apoptosis and mitochondrial ATP synthesis was subsequently examined. RESULTS: Regardless of the subculture number, P19 ESCs showed better (p<0.05) doubling time and less ATP production than P4 ESCs and such difference was not influenced by fresh or cryopreservation. The difference between P4 and P19 ESC lines became decreased as the post-treatment subculture was increased and the six times subculture eliminated such difference. Similarly, transient but prominent difference in ROS production and apoptotic cell number was detected between P4 and P19 ESCs only at the 1st subculture after treatment, but no statistical differences between two ESC lines was detected in other observations. CONCLUSION: The results of this study suggest that post-thaw subculture of ESCs under the same environment is recommended for standardizing their cellular activity. The activity of cell proliferation ability and ATP synthesis can be used as parameters for quality control of ESCs.
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We propose and demonstrate an all-solid-state tunable binary phase Fresnel lens with electrically controllable focal length. The lens is composed of a binary phase Fresnel zone plate, a circular acrylic frame, and a dielectric elastomer (DE) actuator which is made of a thin DE layer and two compliant electrodes using silver nanowires. Under electric potential, the actuator produces in-plane deformation in a radial direction that can compress the Fresnel zones. The electrically-induced deformation compresses the Fresnel zones to be contracted as high as 9.1% and changes the focal length, getting shorter from 20.0 cm to 14.5 cm. The measured change in the focal length of the fabricated lens is consistent with the result estimated from numerical simulation.
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In this data article, we developed a Au nanowire injector (Au NWI) for directly delivering plasmid into the 1-cell stage of the mouse embryos designed to successfully attach and detach the plasmid on the Au NWI, highly minimizing physical and chemical damage on the embryos. This data presents that a Au NWI system does not induce detrimental damages on development of embryos and efficiently express the green fluorescence protein in vitro. The data provided herein is in association with the research article related to reduce the occurrence of mosaicism by a Au NWI," Suppressing Mosaicism by Au Nanowire Injector-driven Direct Delivery of Plasmids into Mouse Embryos" (Park et al., 2017 [1]).
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Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.
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Diferenciação Celular , Oócitos/citologia , Oócitos/ultraestrutura , Superovulação/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Gonadotropina Coriônica/farmacologia , Dextranos , Combinação de Medicamentos , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Gonadotropinas Equinas/farmacologia , Cavalos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Oócitos/efeitos dos fármacos , Organelas/efeitos dos fármacos , Organelas/metabolismo , Superovulação/efeitos dos fármacosRESUMO
Although GPR64 has an important role for male fertility, its physiological roles in the female reproductive system are still unknown. In the present study, immunohistochemical analysis reveals a spatiotemporal expression of GPR64 in the uterus during early pregnancy. Observation of remarkable induction of GPR64 expression in uterine decidual cells points to its potential physiological significance on decidualization. The decidualization of uterine stromal cells is a key event in implantation. Progesterone (P4) signaling is crucial for the decidualization of the endometrial stromal cells for successful pregnancy. Therefore, we examined ovarian steroid hormone regulation of GPR64 expression in the murine uterus. P4 induced GPR64 expression in the epithelial and stromal cells of the uterus in ovariectomized wild-type mice, but not in PRKO mice. ChIP analysis confirmed that PGR proteins were recruited on progesterone response element of Gpr64 gene in the uteri of wild-type mice treated with P4. Furthermore, the expression of GPR64 was increased in human endometrial stromal cells (hESCs) during in vitro decidualization. Interestingly, small interfering RNA (siRNA)-mediated knockdown of GPR64 in hESCs remarkably reduced decidualization. These results suggest that Gpr64 has a crucial role in the decidualization of endometrial stromal cells.