RESUMO
High-resolution imaging analysis using various types of cells is an essential tool for dissecting cell functions. Generally, obtaining such images requires the cells to be cultured on glass substrates; however, it often results in the unstable status of cells. In this study, we report that coating the glass substrate using nanosheet composed of hydrophobic polystyrene, with Matrigel, significantly improves the viability of human pluripotent stem cells (hPSCs). Moreover, the nanosheet coating does not affect the transcriptome status of hPSC and enables researchers to perform the high-resolution imaging assay. These results indicate that the nanosheet coating is beneficial to the cells vulnerable to glass substrate culture. Using the nanosheet coating, we revealed that the spreading status of lnc RNA XIST, essential for X-chromosome inactivation (XCI) in female cells, in the nuclei significantly differs in each hPSC line. Taken together, our study provides a novel method to investigate biological questions using high-resolution imaging techniques.
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Células-Tronco Pluripotentes , Humanos , Feminino , Inativação do Cromossomo X , Transcriptoma , Diferenciação CelularRESUMO
OBJECTIVE: Evaluating friction in human skin is important to assess its condition and the effects of skincare cosmetics. In this study, we evaluated the friction dynamics of moisturized skin to show the effects of moisturization on its mechanical properties. METHODS: Friction force was evaluated using a sinusoidal motion friction evaluation system. The skin of the upper arm of 20 subjects was rubbed using a contact probe. The water content of the stratum corneum and the softness of the skin were measured using a Corneometer and a Cutometer, respectively. RESULTS: When human skin was treated with water or 10 wt% glycerol aqueous solution, the friction coefficients increased by 0.23 ± 0.01 and 0.17 ± 0.14, respectively, and the delay times (normalized by calculating the time interval from contact with the probe to the friction response divided by the friction time for one round trip) increased by 0.048 ± 0.034 and 0.055 ± 0.024, respectively. Three different friction profiles were observed: (a) a stable pattern, in which a smooth profile was observed during the sliding process; (b) an oscillation pattern, in which significant oscillation was obtained; and (c) a stick pattern, in which the friction coefficient increased even during the deceleration process. In the case of untreated skin, the oscillation pattern was observed for the majority of subjects. The appearance rate of the stick pattern increased by 80.3% ± 29.4% after treatment with 10 wt% glycerol aqueous solution. These characteristic friction profiles can be explained by a two-step friction model consisting of two modes: (a) friction at the skin surface and (b) the delayed response due to skin deformation. CONCLUSION: Moisturizing the skin with water or 10 wt% glycerol aqueous solution increased the friction coefficient and delay time, dramatically changing the friction profile. These changes were considered to be due to the swelling and softening of the stratum corneum and the increased true contact area between the contact probe and the skin surface.
OBJECTIF: Une évaluation des effets de la friction sur la peau humaine demeure importante dans le but de juger de l'état de la peau ou de l'efficacité des produits cosmétiques pour les soins de la peau. Dans cette étude, nous avons évalué les propriétés d'une peau hydratée soumise à une friction afin d'identifier les effets de l'hydratation sur les propriétés mécaniques de la peau. MÉTHODE: Les forces de friction ont été évaluées grâce à un système d'évaluation du frottement par mouvement sinusoïdal. Une sonde de contact a été utilisée pour frotter la peau sur le haut du bras de 20 participants. La teneur en eau de la couche cornée et la souplesse de la peau ont été mesurées respectivement à l'aide d'un cornéomètre et d'un cutomètre. RÉSULTAT: Le traitement de la peau humaine avec de l'eau ou une solution de glycérol à 10% a entraîné une augmentation du coefficient de friction respectivement de 0.23 ± 0.01 et de 0.17 ± 0.14, ainsi que du délai de réaction (normalisé en divisant l'intervalle de temps entre le contact avec la sonde jusqu'à l'apparition de la réaction à la friction, par le temps de friction pour un aller-retour), de 0.048 ± 0.034 et de 0.055 ± 0.024. Trois profils de friction différents ont également été observés : (1) un modèle stable, (2) un modèle avec une grande oscillation, et (3) un modèle « collé-glissé ¼ où le coefficient de friction augmente même pendant la décélération. Lorsque la peau est sèche, le modèle oscillant a été observé chez la majorité des participants. Le taux d'apparition du modèle « collé-glissé ¼ a augmenté de 80.3 ± 29.4% dans le cas où la peau a été traitée avec une solution de glycérol à 10%. Ces profils caractéristiques de friction ont pu être expliqués à partir d'un modèle de friction composé de deux modes, (a) une friction à la surface de la peau et (b) un délai de réaction dû à la déformation de la peau. CONCLUSION: L'hydratation de la peau avec de l'eau ou une solution de glycérol à 10% a considérablement modifié le profil de friction en raison d'une augmentation du coefficient de friction et du délai de réaction. Nous avons estimé que ces changements sont relatifs au gonflement et à l'assouplissement de la couche cornée, engendrant une augmentation de la surface de contact réel entre la sonde de contact et la surface de la peau.
Assuntos
Cosméticos , Pele , Água Corporal/fisiologia , Epiderme , Fricção , HumanosRESUMO
Xist RNA, which is responsible for X inactivation, is a key epigenetic player in the embryogenesis of female mammals. Of the several repeats conserved in Xist RNA, the A-repeat has been shown to be essential for its silencing function in differentiating embryonic stem cells. Here, we introduced a new Xist allele into mouse that produces mutated Xist RNA lacking the A-repeat (XistCAGΔ5' ). XistCAGΔ5' RNA expressed in the embryo coated the X chromosome but failed to silence it. Although imprinted X inactivation was substantially compromised upon paternal transmission, allele-specific RNA-seq in the trophoblast revealed that XistCAGΔ5' RNA still retained some silencing ability. Furthermore, the failure of imprinted X inactivation had more significant impacts than expected on genome-wide gene expression. It is likely that dosage compensation is required not only for equalizing X-linked gene expression between the sexes but also for proper global gene regulation in differentiated female somatic cells.
Assuntos
Mecanismo Genético de Compensação de Dose/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Trofoblastos/metabolismo , Alelos , Animais , Células Cultivadas , Mecanismo Genético de Compensação de Dose/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Imunofluorescência , Camundongos , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist(CAG)) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist(CAG) resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist(CAG) in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4- to 8-cell stages when paternally inherited, and Xist(CAG) was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.
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Alelos , Loci Gênicos , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Animais , Blastocisto/metabolismo , Metilação de DNA/genética , Regulação para Baixo/genética , Embrião de Mamíferos/metabolismo , Feminino , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Padrões de Herança/genética , Camundongos , Mutação/genética , Oogênese , Fenótipo , Regulação para Cima/genética , Cromossomo X/genética , Inativação do Cromossomo X/genéticaRESUMO
X chromosome inactivation (X-inactivation) in female mammals is triggered by differential upregulation of the Xist gene on one of the two X chromosomes and subsequent coating of the X in cis with its non-coding transcripts. Although targeted mutation has clearly shown that Xist is essential for X-inactivation in cis, the molecular mechanism by which Xist RNA induces chromosome silencing is largely unknown. Here, we demonstrate that an Xist mutant generated previously in mouse by gene targeting, Xist(IVS), is unique in that it partially retains the capacity to silence the X chromosome. Although Xist(IVS) is differentially upregulated and its mutated transcript coats the X chromosome in cis in embryonic and extra-embryonic tissues, X-inactivation thus initiated does not seem to be fully established. The state of such incomplete inactivation is probably unstable and the mutated X is apparently reactivated in a subset of extra-embryonic tissues and, perhaps, early epiblastic cells. Xist(IVS), which can be referred to as a partial loss-of-function mutation, would provide an opportunity to dissect the molecular mechanism of Xist RNA-mediated chromosome silencing.
Assuntos
RNA não Traduzido/genética , Inativação do Cromossomo X/genética , Alelos , Animais , Northern Blotting , Feminino , Imunofluorescência , Hibridização in Situ Fluorescente , Masculino , Camundongos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Longo não Codificante , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Herein, we evaluated friction dynamics of human skin treated with polyacrylic acid aqueous solutions or gel creams using a sinusoidal motion friction evaluation system to demonstrate the effect of treatment with polymer aqueous solutions on human skin. A polymer aqueous solution or gel cream was applied to the inner forearms of 10 subjects to evaluate temporal changes in friction force under sinusoidal motion. Water content, skin viscoelasticity, and transepidermal water loss were also simultaneously measured to determine the effects on skin conditions. When human skin was treated with the polymer aqueous solution, the friction coefficient immediately after treatment was 0.69-0.99 and the delay time δ, a normalized parameter of the time difference in the delayed response of friction to the movement of the contact probe divided by the friction time T 0 for one round trip, was 0.171-0.179, which was greater than that of untreated skin. This increase was caused by the swelling and softening of the stratum corneum caused by the penetration of water in the polymer aqueous solution, which increased true contact area between the skin and contact probe. A significant difference was observed in the friction coefficient of the skin immediately after treatment with different polymer aqueous solutions. Among polymers (P1-P4), P4, which has a low-salt resistance and low yield stress, had the lowest friction coefficient because of collapsing of the polymer network structures by shearing and reduced viscosity owing to salts on human skin. The skin treated with a gel cream also exhibited a greater friction coefficient than the untreated skin immediately after treatment and 90 min later. This phenomenon can be caused by the occlusive effect of the oil in the gel cream.
Assuntos
Resinas Acrílicas , Fricção , Soluções , Água , Humanos , Viscosidade , Resinas Acrílicas/química , Pele/efeitos dos fármacos , Elasticidade , Adulto , Feminino , Masculino , Polímeros/química , Creme para a Pele/química , Géis , Perda Insensível de Água/efeitos dos fármacosRESUMO
Moisturization causes physiological changes that improve the barrier function of human skin and mechanical changes, including skin friction characteristics. This study evaluated petrolatum- or silicone oil-treated human skin to determine the effect of moisturizing on the friction dynamics. The friction force on the human skin was measured using a contact probe with a sinusoidal motion. The contact probe was used to rub the skin of the upper arm of 20 subjects. The water content of the stratum corneum, softness, and barrier function of the skin were measured using a corneometer, cutometer, and tewameter, respectively. Both oils reduce the frictional force on the human skin. Simultaneously, silicone oil also reduced the delay time δ, which is the standardized time difference between the frictional force response to contact probe movement. Three typical friction patterns were also discovered, which were significantly changed by the treatment with oil. These changes were attributed to the lubrication effect and elimination of adhesion at the true contact point between the skin and the contact probe.
Assuntos
Óleos de Silicone , Pele , Humanos , Fricção , Óleos , EpidermeRESUMO
The mutation of the X-linked protocadherin (PCDH) 19 gene in heterozygous females causes epilepsy. However, because of the erosion of X-chromosome inactivation (XCI) in female human pluripotent stem cells, precise disease modeling often leads to failure. In this study, using a mathematical approach and induced pluripotent stem cells retaining XCI derived from patients with PCDH19 missense mutations, we found that heterotypic conditions, which are composed of wild-type and missense PCDH19, led to significant cell-to-cell proximity and impaired neuronal differentiation, accompanied by the aberrant accumulation of doublecortin, a microtubule-associated protein. Our findings suggest that ease of adhesion between cells expressing either wild-type or missense PCDH19 might lead to aberrant cell aggregation in early embryonic phases, causing poor neuronal development.
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BACKGROUND: Mutation of the SPTAN1 gene, which encodes α-fodrin (non-erythrocyte α-II spectrin), is one of the causes of developmental and epileptic encephalopathies (DEEs). SPTAN1-related DEE is radiologically characterized by cerebral atrophy, especially due to white matter volume reduction, hypomyelination, pontocerebellar hypoplasia, and a thin corpus callosum, however, no neurochemical analysis has been reported. CASE REPORT: A Japanese infant female presented with severe psychomotor delay, tonic spasms, and visual impairment. Whole-exome sequencing revealed a de novo variant of the SPTAN1 gene, leading to a diagnosis of SPTAN1-related DEE. MR spectroscopy at ages 5 months, 11 months, and 1 year and 4 months revealed decreased N-acetylaspartate and choline-containing compounds, and increased glutamate or glutamine. CONCLUSION: The decreased concentrations of N-acetylaspartate and choline-containing compounds may have resulted from neuroaxonal network dysfunction and hypomyelination, respectively. The increased glutamate or glutamine may have reflected a disrupted glutamate-glutamine cycle caused by dysfunction of exocytosis, in which α-fodrin plays an important role. MR spectroscopy revealed neurochemical derangement in SPTAN1-related DEE, which may be a possible pathomechanism and will be useful for its diagnosis.
Assuntos
Epilepsia Generalizada , Epilepsia , Neuroquímica , Colina , Epilepsia/genética , Feminino , Glutamatos , Glutamina , Humanos , Lactente , Espectroscopia de Ressonância MagnéticaRESUMO
The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical progenitor-like states to ultimately generate fetal zone adrenal-cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN, and WNT signaling, and steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro, paving the way for cell-based therapies of adrenal insufficiency.
Assuntos
Córtex Suprarrenal , Células-Tronco Pluripotentes Induzidas , Humanos , Via de Sinalização Wnt , Hormônio Adrenocorticotrópico , EsteroidesRESUMO
Development of the adrenal cortex, a vital endocrine organ, originates in the adrenogonadal primordium, a common progenitor for both the adrenocortical and gonadal lineages in rodents. In contrast, we find that in humans and cynomolgus monkeys, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to-mesenchymal transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages provides a molecular framework for understanding human adrenal and gonadal disorders.
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The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5 Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.
Assuntos
Nucléolo Celular , Proteínas Ribossômicas , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Humanos , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we establish a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells differentiated from human induced pluripotent stem cells are further induced into M-prospermatogonia-like cells and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enables us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.
Assuntos
Células Germinativas Embrionárias/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Animais , Diferenciação Celular , Epigenômica , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Elementos Reguladores de Transcrição , Análise de Sequência de RNA , Espermatogônias/citologia , Espermatozoides , Testículo/citologia , TranscriptomaRESUMO
In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , RNA não Traduzido/genética , Linhagem Celular , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Loci Gênicos , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Conformação de Ácido Nucleico , Estabilidade Proteica , RNA não Traduzido/químicaRESUMO
There are two modes of X chromosome inactivation (XCI) in the mouse. One mode is imprinted XCI: it is initiated at around the four-cell stage in favor of the paternal X chromosome, and is maintained in the extraembryonic tissues. The other mode is random XCI, which takes place in the epiblast lineage at the periimplantation stage. X-linked noncoding Xist RNA, which becomes upregulated on the X chromosome to be inactivated at the onset of XCI and plays a critical role in both imprinted and random XCI, and its accumulation in the nucleus have been referred to as one of the hallmarks of the presence of the inactivated X chromosome. RNA-FISH has therefore been an invaluable method for the study of XCI. As XCI status changes dynamically during periimplantation development in the mouse, analysis using samples from these developmental stages is absolutely necessary for elucidation of the molecular basis of XCI mechanisms. However, dissection of the embryos at around the periimplantation stages is not easy, and this impedes in vivo analysis of the kinetics of XCI. Here, we describe our methods for dissecting the periimplantation stage embryo and subsequent procedures for RNA-FISH and immunostaining.
Assuntos
Embrião de Mamíferos/metabolismo , Hibridização in Situ Fluorescente/métodos , RNA Longo não Codificante/análise , Inativação do Cromossomo X , Animais , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Epigenômica/métodos , Regulação da Expressão Gênica no Desenvolvimento , CamundongosRESUMO
Coenzyme Q(10) (CoQ(10)) has been widely commercially available in Japan as a dietary and health supplement since 2001 and is used for the prevention of lifestyle-related diseases induced by free radicals and aging. We evaluated CoQ(10) supplements to ensure that these supplements can be used effectively and safely. Commercially available products were selected and assessed by the quality control tests specified in the Japanese Pharmacopoeia XV. When the disintegration time of CoQ(10) supplements was measured, a few tested supplements did not completely disintegrate even after incubation in water for an hour at 37 degrees C. In the content test, many samples were well controlled. However, a few supplements showed low recovery rates of CoQ(10) as compared to manufacturer's indicated contents. Among soft capsule and liquid supplements, the reduced form of CoQ(10) (H(2)CoQ(10)), as well as the oxidized form, was detected by HPLC with electrochemical detector. The results for experimental formulated CoQ(10) supplements demonstrated that H(2)CoQ(10) was produced by the interaction of CoQ(10) with vitamins E and/or C. From these results, we concluded that quality varied considerably among the many supplement brands containing CoQ(10). Additionally, we also demonstrated that H(2)CoQ(10) can be detected in some foods as well as in CoQ(10) supplements.
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A hepatoprotective peptide, pyroglutamyl leucine (pyroGlu-Leu), was identified in wheat gluten hydrolysate through an in vivo activity-guided fractionation approach based on D-galactosamine-induced acute hepatitis in rats and fractionation of peptides with large-scale preparative ampholine-free isoelectric focusing. The active acidic fraction predominantly consisted of pyroglutamyl peptides and free pyroglutamic acid. Pyroglutamyl peptides were derivatized with phenyl isothiocyanate after removal of a pyroglutamyl residue by pyroglutamate aminopeptidase. The derivatives were purified by reversed-phase HPLC and subjected to sequence analysis. The active fraction contained pyroGlu-Ile, pyroGlu-Leu, pyroGlu-Gln, pyroGlu-Gln-Gln, and free pyroGlu. Ingestion of pyroGlu-Leu at 20 mg/kg body weight significantly decreased serum aspartate and alanine aminotransferases to approximately 30% and 20% of those values of the vehicle group, respectively, which were near the normal levels. Thirty minutes after ingestion of pyroGlu-Leu at 20 mg/kg, the concentration of pyroGlu-Leu in portal blood plasma increased to approximately 2 µM.