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In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium-activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100-500 ng/µL of T4 DNA, but not 20 ng/µL, was sufficient for the formation of nucleus-like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA-induced nucleus-like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA.
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The development of a compact and affordable fluorescence microscope can be a formidable challenge for growing needs in on-site testing and detection of fluorescent labeled biological systems, especially for those who specialize in biology rather than in engineering. In response to such a situation, we present an open-source miniature fluorescence microscope using Raspberry Pi. Our fluorescence microscope, with dimensions of 19.2 × 13.6 × 8.2 cm3 (including the display, computer, light-blocking case, and other operational requirements), not only offers cost-effectiveness (costing less than $500) but is also highly customizable to meet specific application needs. The 12.3-megapixel Raspberry Pi HQ Camera captures high-resolution imagery, while the equipped wide-angle lens provides a field of view measuring 21 × 15 mm2. The integrated wireless LAN in the Raspberry Pi, along with software-controllable high-powered fluorescence LEDs, holds potential for a wide range of applications. This open-source fluorescence microscope offers biohybrid sensor developers a versatile tool to streamline unfamiliar mechanical design tasks and open new opportunities for on-site fluorescence detections.
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Microorganisms possess remarkable locomotion abilities, making them potential candidates for micromachine propulsion. Here, the use of Chlamydomonas Reinhardtii (CR) is explored, a motile green alga, as a micromotor by harnessing its propulsive force with microtraps. The objectives include developing the microtrap structure, evaluating trapping efficiency, and investigating the movement dynamics of biohybrid micromachines driven by CR. Experimental analysis demonstrates that trap design significantly influences trapping efficiency, with a specific trap configuration (multi-ring structure with diameters of 7 µm - 10 µm - 13 µm) showing the highest effectiveness. The micromachine empowered with two CRs facing the same direction exhibits complex, random-like motion with yaw, pitch, and roll movements, while the micromachine with four CRs in a circular position each facing the tangential direction of the circle demonstrates controlled rotational motion. These findings highlight the degree of freedom and movement potential of biohybrid micromachines.
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The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.
Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Animais , Cromatina/genética , Expressão Gênica , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/genética , Transcrição Gênica , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The ever-stricter regulations on animal experiments in the field of cosmetic testing have prompted a surge in skin-related research with a special focus on recapitulation of thein vivoskin structurein vitro. In vitrohuman skin models are seen as an important tool for skin research, which in recent years attracted a lot of attention and effort, with researchers moving from the simplest 2-layered models (dermis with epidermis) to models that incorporate other vital skin structures such as hypodermis, vascular structures, and skin appendages. In this study, we designed a microfluidic device with a reverse flange-shaped anchor that allows culturing of anin vitroskin model in a conventional 6-well plate and assessing its barrier function without transferring the skin model to another device or using additional contraptions. Perfusion of the skin model through vascular-like channels improved the morphogenesis of the epidermis compared with skin models cultured under static conditions. This also allowed us to assess the percutaneous penetration of the tested caffeine permeation and vascular absorption, which is one of the key metrics for systemic drug exposure evaluation.
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Epiderme , Pele , Animais , Pele/metabolismo , Epiderme/química , Epiderme/metabolismo , Absorção Cutânea , Cafeína/farmacologia , Cafeína/análise , Cafeína/metabolismo , PerfusãoRESUMO
Even though the physical nature of shear and longitudinal moduli are different, empirical correlations between them have been reported in several biological systems. This correlation is of fundamental interest and immense practical value in biomedicine due to the importance of the shear modulus and the possibility to map the longitudinal modulus at high-resolution with all-optical spectroscopy. We investigate the origin of such a correlation in hydrogels. We hypothesize that both moduli are influenced in the same direction by underlying physicochemical properties, which leads to the observed material-dependent correlation. Matching theoretical models with experimental data, we quantify the scenarios in which the correlation holds. For polymerized hydrogels, a correlation was found across different hydrogels through a common dependence on the effective polymer volume fraction. For hydrogels swollen to equilibrium, the correlation is valid only within a given hydrogel system, as the moduli are found to have different scalings on the swelling ratio. The observed correlation allows one to extract one modulus from another in relevant scenarios.
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Hidrogéis , Polímeros , Hidrogéis/química , Polímeros/química , Análise Espectral , Modelos Teóricos , ViscosidadeRESUMO
Recently, microfluidic bioprinting methods, which utilize microfluidic devices as printheads to deposit microfilaments, have improved printing resolution. Despite the precise placement of cells, current efforts have not succeeded in forming densely cellularized tissue within the printed constructs, which is highly desired for the biofabrication of solid-organ tissues with firm tissue consistency. In this paper, we presented a microfluidic bioprinting method to fabricate three dimension tissue constructs consisting of core-shell microfibers where extracellular matrices and cells can be encapsulated within the core of the fibers. Using the optimized printhead design and printing parameters, we demonstrated the bioprinting of core-shell microfibers into macroscale constructs and checked the viability of cells after printing. After culturing the printed tissues using the proposed dynamic culture methods, we analyzed the morphology and function of the tissues bothin vitroandin vivo. The confluent tissue morphology in the fiber cores indicates the establishment of intensive cell-cell contacts in the fiber cores, which also leads to the upregulation of the albumin-secretion function compared to the cells cultured in a 2D format. Analysis on the cell density of the confluent fiber cores indicate the formation of densely cellularized tissues with a similar level of cell density ofin-vivosolid organ tissues. In the future, better culture techniques with improved perfusion design are anticipated to enable further the fabrication of thicker tissues, which can be used as thick tissue models or implantation grafts for cell therapy.
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Bioimpressão , Alicerces Teciduais , Engenharia Tecidual/métodos , Bioimpressão/métodos , Microfluídica , Impressão TridimensionalRESUMO
In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.
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Actinas , Peixe-Zebra , Animais , Cromossomos/genética , Mitose , Citoesqueleto de ActinaRESUMO
Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.
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Corantes Fluorescentes , Organelas , Fluorescência , Viscosidade , LisossomosRESUMO
The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells.1-3 How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggest that the clustering of transcription factors dictates the formation of transcription bodies.
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Fatores de Transcrição , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Desenvolvimento Embrionário/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Transcrição Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/metabolismoRESUMO
A superfluid helium insert was developed for cryogenic microscopy of millimeter-sized specimens. An optical-interferometric position sensor, cryogenic objective mirror, and piezo-driven cryogenic stage were fixed to an insert holder that was immersed in superfluid helium. The single-component design stabilized the three-dimensional position of the sample, with root-mean-square deviations of (x, lateral) 0.33 nm, (y, lateral) 0.29 nm, and (z, axial) 0.25 nm. Because of the millimeter working range of the optical sensor, the working range of the sample under the active stabilization was (x, y) 5 mm and (z) 3 mm in superfluid helium at 1.8 K. The insert was used to obtain the millimeter-sized fluorescence image of cell nuclei at 1.8 K without a sample exchange.
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Biohybrid odorant sensors that directly integrate a biological olfactory system have been increasingly studied and are suggested to be the next generation of ultrasensitive sensors by taking advantage of the sensitivity and selectivity of living organisms. In this review, we provide a detailed description of the recent developments of biohybrid odorant sensors, especially considering the requisites for their perspective of on-site applications. We introduce the methodologies to effectively capture the biological signals from olfactory systems by readout devices, and describe the essential properties regarding the gaseous detection, stability, quality control, and portability. Moreover, we address the recent progress on multiple odorant recognition using multiple sensors as well as the current screening approaches for pairs of orphan receptors and ligands necessary for the extension of the currently available range of biohybrid sensors. Finally, we discuss our perspectives for the future for the development of practical odorant sensors.
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Técnicas Biossensoriais , Receptores Odorantes , Odorantes , Receptores Odorantes/genéticaRESUMO
Cell therapy using human-stem-cell-derived pancreatic beta cells (hSC-ßs) is a potential treatment method for type 1 diabetes mellitus (T1D). For therapeutic safety, hSC-ßs need encapsulation in grafts that are scalable and retrievable. In this study, we developed a lotus-root-shaped cell-encapsulated construct (LENCON) as a graft that can be retrieved after long-term hSC-ß transplantation. This graft had six multicores encapsulating hSC-ßs located within 1 mm from the edge. It controlled the recipient blood glucose levels for a long-term, following transplantation in immunodeficient diabetic mice. LENCON xenotransplanted into immunocompetent mice exhibited retrievability and maintained the functionality of hSC-ßs for over 1 year after transplantation. We believe that LENCON can contribute to the treatment of T1D through long-term transplantation of hSC-ßs and in many other forms of cell therapy.
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In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.
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Blástula/metabolismo , Cromatina/metabolismo , Lamina Tipo B/metabolismo , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Núcleo Celular/metabolismo , Humanos , Ligação Proteica , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Receptor de Lamina BRESUMO
Salt is an essential nutrient; however, excessive salt intake is a prominent public health concern worldwide. Various physiological functions are associated with circadian rhythms, and disruption of circadian rhythms is a prominent risk factor for cardiovascular diseases, cancer, and immune disease. Certain nutrients are vital regulators of peripheral circadian clocks. However, the role of a high-fat and high-salt (HFS) diet in the regulation of circadian gene expression is unclear. This study aimed to investigate the effect of an HFS diet on rhythms of locomotor activity, caecum glucocorticoid secretion, and clock gene expression in mice. Mice administered an HFS diet displayed reduced locomotor activity under normal light/dark and constant dark conditions in comparison with those administered a normal diet. The diurnal rhythm of caecum glucocorticoid secretion and the expression levels of glucocorticoid-related genes and clock genes in the adrenal gland were disrupted with an HFS diet. These results suggest that an HFS diet alters locomotor activity, disrupts circadian rhythms of glucocorticoid secretion, and downregulates peripheral adrenal gland circadian clock genes.
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Ritmo Circadiano/fisiologia , Dieta Hiperlipídica , Glucocorticoides/biossíntese , Atividade Motora/fisiologia , Cloreto de Sódio na Dieta , Glândulas Suprarrenais/metabolismo , Animais , Relógios Circadianos/fisiologia , Masculino , Camundongos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismoRESUMO
Bioengineering with utilization of cells as one of the components of devices has been expected to advance developments of medical and pharmaceutical technologies. When cells are engineered, it is important to establish means for maintaining the activity of the cells, enhancing cell functions, and controlling cell responses. This review summarizes researches for cell encapsulation using synthetic phospholipid polymers composed of 2-methacryloyloxyethyl phosphorylcholine unit, which make hydrogel spontaneously in a cell culture environment and then cells are preserved in situ. The phospholipid polymer hydrogels show no adverse effects on the cell culture process and the mechanical properties of the hydrogels can regulate for controlling the function of cells. It also introduces molecular designs that can be easily recovered from the hydrogel matrix after the encapsulated cells have differentiated. Furthermore, the application of these hydrogels to a microdevice also describes advanced utilization of cultured cells. Phospholipid polymer hydrogels can exhibit its function even when they are applied in vivo, and as one application, introduces the prevention of adhesion with other tissues in the tissue healing process. That is, the potential application of the phospholipid polymer hydrogels in cell engineering are described.
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Hidrogéis , Polímeros , Engenharia Celular , Linhagem Celular , FosfolipídeosRESUMO
Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.
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Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Transcrição Gênica , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Zigoto/metabolismo , Acetilação/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Código das Histonas/efeitos dos fármacos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/efeitos dos fármacosRESUMO
Double-stranded RNA (dsRNA) is well characterized as an inducer of anti-viral interferon responses. We previously reported that dsRNA extracted from a specific edible plant possesses an immune-modulating capacity to confer, in mice, resistance against respiratory viruses, including the H1N1 strain of the influenza A virus (IAV). We report here that the systemic immune-activating capacity of the plant-derived dsRNA protected mice from infection by a highly virulent H5N1 strain of the IAV. In addition, subcutaneous inoculation of the dsRNA together with the inactivated virion of the H5N1 strain of the IAV suppressed the lethality of the viral infection as compared with individual inoculation of either dsRNA or HA protein, suggesting its potential usage as a vaccination adjuvant. Moreover, intra-peritoneal inoculation of the dsRNA limited the growth of B16-F10 melanoma cells through the activation of NK cells in murine models. Taken together, this study demonstrated the systemic immune-modulating capacity of a plant-derived dsRNA and its potential for nucleic acid-based clinical applications.
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Capsicum/química , RNA de Cadeia Dupla/imunologia , Animais , Capsicum/imunologia , Células Cultivadas , Interferon Tipo I/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA de Cadeia Dupla/isolamento & purificação , RNA de Cadeia Dupla/metabolismo , Ribonucleases/metabolismoRESUMO
To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.