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Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo -like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.
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In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method in which can be produced homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.
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Células-Tronco Pluripotentes Induzidas , Animais , Camundongos , Reprogramação Celular , Células-Tronco Embrionárias Murinas , Epigênese GenéticaRESUMO
Magnetoelectric coupling is achieved near room temperature in a spin crossover FeII molecule-based compound, [Fe(1bpp)2 ](BF4 )2 . Large atomic displacements resulting from Jahn-Teller distortions induce a change in the molecule dipole moment when switching between high-spin and low-spin states leading to a step-wise change in the electric polarization and dielectric constant. For temperatures in the region of bistability, the changes in magnetic and electrical properties are induced with a remarkably low magnetic field of 3â T. This result represents a successful expansion of magnetoelectric spin crossovers towards ambient conditions. Moreover, the observed 0.3-0.4â mC m-2 changes in the H-induced electric polarization suggest that the high strength of the coupling obtained via this route is accessible not just at cryogenic temperatures but also near room temperature, a feature that is especially appealing in the light of practical applications.
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The single-molecule magnet {Mn84} is a challenge to theory because of its high nuclearity. We directly compute two experimentally accessible observables, the field-dependent magnetization up to 75 T and the temperature-dependent heat capacity, using parameter-free theory. In particular, we use first-principles calculations to derive short- and long-range exchange interactions and compute the exact partition function of the resulting classical Potts and Ising spin models for all 84 Mn S = 2 spins to obtain observables. The latter computation is made possible by using hyperoptimized tensor network contractions, a technique developed to simulate quantum supremacy circuits. We also synthesize the magnet and measure its heat capacity and magnetization, observing qualitative agreement between theory and experiment and identifying an unusual bump in the heat capacity and a plateau in the magnetization. Our work also identifies some limitations of current theoretical modeling in large magnets, such as sensitivity to small, long-range exchange couplings.
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Organic-inorganic hybrids of halogenoindates(III) are typically represented by one of the zero-dimensional units: InX4-, InX52-, InX63-, or In2X115-. Higher dimensional anionic forms, although not forbidden, have remained almost elusive. Here we report for the first time In3+-based organic-inorganic hybrids, (C4H5N2S)2InCl5 and (C4H5N2S)2InBr5, with 1D anionic chains of trans-halide-bridged InX6 octahedra whose formation is guided by 2-mercaptopyrimidinium cations (C4H5N2S+). The chains are characterized by the significant ease of deformation, which is reflected in the elongation of the bridging bonds or the displacement of In3+ ions. The materials show a robust band gap predominantly governed by C4H5N2S+ cations. Dielectric relaxation processes in (C4H5N2S)2InBr5 arise from the cations' dynamics and suggest the ability of the brominated system to accommodate even larger cations. Our work represents a successful attempt to expand the structural diversity of halogenoindates(III) and opens a pathway to reach multifunctional 1D In3+-based hybrids.
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We combine high field polarization, magneto-infrared spectroscopy, and lattice dynamics calculations with prior magnetization to explore the properties of (NH4)2[FeCl5·(H2O)]âa type II molecular multiferroic in which the mixing between charge, structure, and magnetism is controlled by intermolecular hydrogen and halogen bonds. Electric polarization is sensitive to the series of field-induced spin reorientations, increasing linearly with the field and reaching a maximum before collapsing to zero across the quasi-collinear to collinear-sinusoidal reorientation due to the restoration of inversion symmetry. Magnetoelectric coupling is on the order of 1.2 ps/m for the Pâ¥c, Hâ¥c configuration between 5 and 25 T at 1.5 K. In this range, the coupling takes place via an orbital hybridization mechanism. Other forms of mixing are active in (NH4)2[FeCl5·(H2O)] as well. Magneto-infrared spectroscopy reveals that all of the vibrational modes below 600 cm-1 are sensitive to the field-induced transition to the fully saturated magnetic state at 30 T. We analyze these local lattice distortions and use frequency shifts to extract spin-phonon coupling constants for the Fe-O stretch, Fe-OH2 rock, and NH4+ libration. Inspection also reveals subtle symmetry breaking of the ammonium counterions across the ferroelectric transition. The coexistence of such varied mixing processes in a platform with intermolecular hydrogen- and halogen-bonding opens the door to greater understanding of multiferroics and magnetoelectrics governed by through-space interactions.
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Biogas is an environmentally friendly and sustainable energy resource that can substitute or complement conventional fossil fuels. For practical uses, biogas upgrading, mainly through the effective separation of CO2 (0.33 nm) and CH4 (0.38 nm), is required to meet the approximately 90-95% purity of CH4, while CO2 should be concomitantly purified. In this study, a high CO2 perm-selective zeolite membrane was synthesized by heteroepitaxially growing a chabazite (CHA) zeolite seed layer with a synthetic precursor that allowed the formation of all-silica deca-dodecasil 3 rhombohedral (DDR) zeolite (with a pore size of 0.36 × 0.44 nm2). The resulting hydrophobic DDR@CHA hybrid membrane on an asymmetric α-Al2O3 tube was thin (ca. 2 µm) and continuous, thus providing both high flux and permselectivity for CO2 irrespective of the presence or absence of water vapor (the third largest component in the biogas streams). To the best of our knowledge, the CO2 permeance of (2.9 ± 0.3) × 10-7 mol m-2 s-1 Pa-1 and CO2/CH4 separation factor of ca. 274 ± 73 at a saturated water vapor partial pressure of ca. 12 kPa at 50 °C have the highest CO2/CH4 separation performance yet achieved. Furthermore, we explored the membrane module properties of the hybrid membrane in terms of the recovery and purity of both CO2 and CH4 under dry and wet conditions. Despite the high intrinsic membrane properties of the current hybrid membrane, reflected by the high permeance and SF, the corresponding module properties indicated that high-performance separation of CO2 and CH4 for the desired biogas upgrading was achieved at a limited processing capacity. This supports the importance of understanding the correlation between the membrane and module properties, as this will provide guidance for the optimal operating conditions.
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Materiais Biocompatíveis/química , Reatores Biológicos , Dióxido de Carbono/isolamento & purificação , Metano/isolamento & purificação , Zeolitas/química , Dióxido de Carbono/química , Teste de Materiais , Metano/química , Tamanho da PartículaRESUMO
Pinned and mobile ferroelastic domain walls are detected in response to mechanical stress in a Mn3+ complex with two-step thermal switching between the spin triplet and spin quintet forms. Single-crystal X-ray diffraction and resonant ultrasound spectroscopy on [MnIII(3,5-diCl-sal2(323))]BPh4 reveal three distinct symmetry-breaking phase transitions in the polar space group series Cc â Pc â P1 â P1(1/2). The transition mechanisms involve coupling between structural and spin state order parameters, and the three transitions are Landau tricritical, first order, and first order, respectively. The two first-order phase transitions also show changes in magnetic properties and spin state ordering in the Jahn-Teller-active Mn3+ complex. On the basis of the change in symmetry from that of the parent structure, Cc, the triclinic phases are also ferroelastic, which has been confirmed by resonant ultrasound spectroscopy. Measurements of magnetoelectric coupling revealed significant changes in electric polarization at both the Pc â P1 and P1 â P1(1/2) transitions, with opposite signs. All these phases are polar, while P1 is also chiral. Remanent electric polarization was detected when applying a pulsed magnetic field of 60 T in the P1â P1(1/2) region of bistability at 90 K. Thus, we showcase here a rare example of multifunctionality in a spin crossover material where the strain and polarization tensors and structural and spin state order parameters are strongly coupled.
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Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.
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Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Albumina Sérica Humana/farmacologia , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neurais/metabolismo , Oryza/genética , Oryza/metabolismo , Fenótipo , Proteínas de Plantas/farmacologia , Proteínas Recombinantes/farmacologia , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismoRESUMO
A MnIII spin crossover complex with atypical two-step hysteretic thermal switching at 74â K and 84â K shows rich structural-magnetic interplay and magnetic-field-induced spin state switching below 14â T with an onset below 5â T. The spin states, structures, and the nature of the phase transitions are elucidated via X-ray and magnetization measurements. An unusual intermediate phase containing four individual sites, where 1 / 4 are in a pure low spin state, is observed. The splitting of equivalent sites in the high temperature phase into four inequivalent sites is due to a structural reorganization involving a primary and a secondary symmetry-breaking order parameter that induces a crystal system change from orthorhombicâmonoclinic and a cell doubling. Further cooling leads to a reconstructive phase transition and a monoclinic low-temperature phase with two inequivalent low-spin sites. The coupling between the order parameters is identified in the framework of Landau theory.
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The manipulation of mesoscale domain wall phenomena has emerged as a powerful strategy for designing ferroelectric responses in functional devices, but its full potential is not yet realized in the field of magnetism. This work shows a direct connection between magnetic response functions in mechanically strained samples of Mn3 O4 and MnV2 O4 and stripe-like patternings of the bulk magnetization which appear below known magnetostructural transitions. Building off previous magnetic force microscopy data, a small-angle neutron scattering is used to show that these patterns represent distinctive magnetic phenomena which extend throughout the bulk of two separate materials, and further are controllable via applied magnetic field and mechanical stress. These results are unambiguously connected to the anomalously large magnetoelastic and magnetodielectric response functions reported for these materials, by performing susceptibility measurements on the same crystals and directly correlating local and macroscopic data.
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OCT4 and NANOG are core transcription factor genes in self-renewal, differentiation, and reprogramming. Here, we generated an OCT4-EGFP, NANOG-tdTomato dual reporter hiPSC line, KKUi001-A, on the basis of human induced pluripotent stem cells using CRISPR/Cas9 technology. EGFP and tdTomato reporter were inserted into before the stop codon of OCT4 and NANOG, respectively. Simultaneous expression of EGFP and tdTomato was observed when expression of OCT4 and NANOG was changed during differentiation and reprogramming. KKUi001-A hiPSC line will be a useful tool to find initial time point of OCT4 and NANOG expression during reprogramming process and to screen small molecules that promote reprogramming.
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Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Reprogramação Celular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Luminescentes , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
Discovery of robust yet reversibly switchable electric dipoles at reduced dimensions is critical to the advancement of nanoelectronics devices. Energy bands flat in momentum space generate robust localized states that are activated independently of each other. We determined that flat bands exist and induce robust yet independently switchable dipoles that exhibit a distinct ferroelectricity in hafnium dioxide (HfO2). Flat polar phonon bands in HfO2 cause extreme localization of electric dipoles within its irreducible half-unit cell widths (~3 angstroms). Contrary to conventional ferroelectrics with spread dipoles, those intrinsically localized dipoles are stable against extrinsic effects such as domain walls, surface exposure, and even miniaturization down to the angstrom scale. Moreover, the subnanometer-scale dipoles are individually switchable without creating any domain-wall energy cost. This offers unexpected opportunities for ultimately dense unit cell-by-unit cell ferroelectric switching devices that are directly integrable into silicon technology.
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In humans, parthenogenesis and androgenesis occur naturally in mature cystic ovarian teratomas and androgenetic complete hydatidiform moles (CHM), respectively. Our previous study has reported human parthenogenetic induced pluripotent stem cells from ovarian teratoma-derived fibroblasts and screening of imprinted genes using genome-wide DNA methylation analysis. However, due to the lack of the counterparts of uniparental cells, identification of new imprinted differentially methylated regions has been limited. CHM are inherited from only the paternal genome. In this study, we generated human androgenetic induced pluripotent stem cells (AgHiPSCs) from primary androgenetic fibroblasts derived from CHM. To investigate the pluripotency state of AgHiPSCs, we analyzed their cellular and molecular characteristics. We tested the DNA methylation status of imprinted genes using bisulfite sequencing and demonstrated the androgenetic identity of AgHiPSCs. AgHiPSCs might be an attractive alternative source of human androgenetic embryonic stem cells. Furthermore, AgHiPSCs can be used in regenerative medicine, for analysis of genomic imprinting, to study imprinting-related development, and for disease modeling in humans.
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Células-Tronco Pluripotentes Induzidas/citologia , Herança Paterna , Diferenciação Celular , Células Cultivadas , Metilação de DNA , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Impressão Genômica , Humanos , Mola Hidatiforme/genética , Mola Hidatiforme/metabolismo , Mola Hidatiforme/fisiopatologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Gravidez , Reprodução AssexuadaRESUMO
Spermatogonial stem cells (SSCs) are unipotent adult stem cells, capable of differentiating into sperm cells. SSCs can be cultured in vitro for a long time. SSCs expressing Oct4, a pluripotency marker, and are the only adult cells which pluripotency can be induced under defined culture conditions. However, because 2D culture imposes limitations in cell junction formation, cell shape, metabolism, response to stimuli, and cell interface with medium, mechanistic studies on reprogramming of SSCs using feeder cells still have many challenges. Recent studies have shown that a culture system using a bio-matrix can be used in long-term feeder-free SSCs culture and for induction of pluripotency in SSCs. However, the bio-matrix cannot be the optimal micro-environment in mechanistic studies because it creates a physical barrier to growth factors and other signaling molecules. To overcome this effect of the matrix, we reprogrammed SSCs into pluripotent ESC-like cells, so-called germline-derived pluripotent stem cells (gPSCs) by using a 3D scaffold, in which cells are less responsive to external stimuli than in 2D cultures. Thus, we confirm the possibility of SSC reprogramming in the spheroidal state and suggest the utility of 3D scaffolds as a tool for studying the mechanism of SSC reprogramming into gPSCs without a bio-matrix.
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Modern computer technology is based on the binary logic system. However, the slowdown of its development calls for transition to multivalued logic (MVL) systems. MVL can yield a denser logic implementation on the same chip area at low cost. More information can be transmitted with the same digits over fewer interconnections, thereby reducing power dissipation. Here, we suggest a novel nonvolatile balanced ternary memory based on the multiferroelectric material GeSnTe2. Two different directions and quantities of electric polarization are found to be stable in atomic-thick two-dimensional structures. The balanced ternary data set of {-1, 0, +1} can be implemented in the two-dimensional material on the nanometer scale. One-shot read/write processes are suggested.
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BACKGROUND AND OBJECTIVES: Several recent studies have claimed that cancer cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, in most cases, cancer cells seem to be resistant to cellular reprogramming. Furthermore, the underlying mechanisms of limited reprogramming in cancer cells are largely unknown. Here, we identified the candidate barrier genes and their target genes at the early stage of reprogramming for investigating cancer reprogramming. METHODS: We tried induction of pluripotency in normal human fibroblasts (BJ) and both human benign (MCF10A) and malignant (MCF7) breast cancer cell lines using a classical retroviral reprogramming method. We conducted RNA-sequencing analysis to compare the transcriptome of the three cell lines at early stage of reprogramming. RESULTS: We could generate iPSCs from BJ, whereas we were unable to obtain iPSCs from cancer cell lines. To address the underlying mechanism of limited reprogramming in cancer cells, we identified 29 the candidate barrier genes based on RNA-sequencing data. In addition, we found 40 their target genes using Cytoscape software. CONCLUSIONS: Our data suggest that these genes might one of the roadblock for cancer cell reprogramming. Furthermore, we provide new insights into application of iPSCs technology in cancer cell field for therapeutic purposes.
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Chabazite (CHA)-type zeolites are promising for the separation of CO2 from larger molecules, such as N2 (relevant to postcombustion carbon capture) and CH4 (relevant to natural gas/biogas upgrading). In particular, the pore size of CHA zeolites (0.37 × 0.42 nm2) can recognize slight molecular size differences between CO2 (0.33 nm) and the larger N2 (0.364 nm) or CH4 (0.38 nm) molecules, thus allowing separation in favor of CO2 through CHA membranes. Furthermore, the siliceous constituents in the CHA zeolite can reduce the adsorption capacity toward the smaller H2O molecule (0.265 nm) and, thus, the H2O permeation rate. This is highly desirable for securing good molecular sieving ability with CO2 permselectivity in the presence of H2O vapor. Indeed, a siliceous CHA film obtained with a nominal Si/Al ratio of 100 (CHA_100) showed high CO2/N2 and CO2/CH4 separation performance, especially in the presence of H2O vapor; â¼13.4 CO2/N2 and â¼37 CO2/CH4 separation factors (SFs) at 30 °C. These SFs were higher than the corresponding values (â¼5.2 CO2/CH4 SFs and â¼31 CO2/CH4 SFs) under dry conditions; such improvement could be ascribed to defect blocking by physisorbed water molecules. Finally, the contribution of molecular transport through zeolitic and nonzeolitic parts was quantitatively analyzed by combining information extracted from image processing of fluorescence confocal optical microscopy images with a one-dimensional permeation model. It appears that â¼19 and â¼20% of the total CO2 permeance for CHA_100 were reduced due to transport inhibition by the physisorbed water molecules on the membrane surface and defect, respectively.
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Generation of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.
