Revealing 3D structure of gluten in wheat dough by optical clearing imaging.
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ABSTRACT:
SUBMITTER: Ogawa T
PROVIDER: S-EPMC7969737 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThree-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells.MethodsWe present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models.ResultsEx vivo cleared mouse brain tissues had a >10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma.ConclusionsOptical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.
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Project description:Pulmonary fibrosis, characterized by excessive collagen deposition in the lungs, comprises a key and debilitating component of chronic lung diseases. Methods are lacking for the direct visualization of fibrillar collagen throughout the whole murine lung, a capability that would aid the understanding of lung fibrosis. We combined an optimized organ-level optical clearing (OC) approach with large-scale, label-free multiphoton microscopy (MPM) and second harmonic generation microscopy (SHGM) to reveal the complete network of fibrillar collagen in whole murine lungs. An innate inflammation-driven model based on repetitive poly(I:C) challenge was evaluated. Following OC, mosaic MPM/SHGM imaging with 3D reconstruction and whole organ quantitative analysis revealed significant differences in collagen deposition between PBS and poly(I:C) treated lungs. Airway specific analysis in whole lung acquisitions revealed significant sub-epithelial fibrosis evident throughout the proximal conductive and distal airways with higher collagen deposition in the poly(I:C) group vs PBS group. This study establishes a new, powerful approach based on OC and MPM/SHGM imaging for 3D analysis of lung fibrosis with macroscopic views of lung pathology based on microscopy and providing a new way to analyze the whole lung while avoiding regional sampling bias.
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Project description:Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 μm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols - in particular, for screening purposes - clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.
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Project description:Baking soda (NaHCO3) has critical technological functions in cookie products. Health concern on excessive sodium consumption is increasing; therefore, it is necessary to explore NaHCO3 alternatives, such as KHCO3, for bakery products. This study investigated the impact of KHCO3 on the technological behaviors of cookie dough and end-uses in comparison with control samples prepared with NaHCO3 and explore the changes of physicochemical and conformation properties of soft wheat gluten during the process. Dough rheological measurements demonstrated that addition of KHCO3 reduced dough stickiness, and adding KHCO3 achieved similar dough and baking performances as using NaHCO3, which were partially attributed to the decrease of gliadin to glutenin ratio, changes of secondary structure, and intensive aggregation of gluten by introducing KHCO3. Cookie sensory attributes were also not adversely affected by using KHCO3. Therefore, partially replacing NaHCO3 with KHCO3 in cookie products can be an effective approach for sodium reduction.
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Project description:To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits-tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.
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Project description:Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo-devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo-devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-α-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.
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Project description:BackgroundGliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction.Methodology/principal findingsTransgenic lines of bread wheat with ?-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of ?-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the ?-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of ?-gliadins.Conclusions/significanceTherefore, it is concluded that ?-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten proteins.
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Project description:To better understand the effects of ultrasonic treatment in the whole freezing process (UWF) and the maximum ice crystal formation zone (UMF) on the quality of frozen dough, the textural properties of dough and the structure of gluten were investigated. The results showed that the UWF and UMF treatments improved the textural properties of frozen dough and obtain the best effect at the 60 W/L power densities. Ultrasound-assisted freezing reduced the destructive effect of disulfide bonds on dough, and led to a state of dynamic equilibrium of hydrophobic groups. UWF treatment at 80 W/L and UMF treatment at 40 W/L had positive effects prevented the secondary structure from destruction by freezing. The network of gluten treated by ultrasound-assisted freezing was more uniform and smaller than that of traditional freezing samples, which was similar to the network structure of fresh protein. According to Pearson's correlation analysis, there was a high correlation between SH, α-helix content and springiness. There was a significant positive correlation between β-turn and G', G″, and there was a significant negative correlation between β-turn and hardness. These results suggest that ultrasound-assisted freezing improved the process quality of dough though reducing the damage to gluten structure caused by freezing.
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Project description:Rationale: Intravital optical imaging is a significant method for investigating cerebrovascular structure and function. However, its imaging contrast and depth are limited by the turbid skull. Tissue optical clearing has a great potential for solving this problem. Our goal was to develop a transparent skull window, without performing a craniotomy, for use in assessing cerebrovascular structure and function. Methods: Skull optical clearing agents were topically applied to the skulls of mice to create a transparent window within 15 min. The clearing efficacy, repeatability, and safety of the skull window were then investigated. Results: Imaging through the optical clearing skull window enhanced both the contrast and the depth of intravital imaging. The skull window could be used on 2-8-month-old mice and could be expanded from regional to bi-hemispheric. In addition, the window could be repeatedly established without inducing observable inflammation and metabolic toxicity. Conclusion: We successfully developed an easy-to-handle, large, switchable, and safe optical clearing skull window. Combined with various optical imaging techniques, cerebrovascular structure and function can be observed through this optical clearing skull window. Thus, it has the potential for use in basic research on the physiopathologic processes of cortical vessels.
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Project description:Dental pulp is composed of nerves, blood vessels, and various types of cells and surrounded by a thick and hard enamel-dentin matrix. Due to its importance in the maintenance of tooth vitality, there have been intensive efforts to analyze the complex cellular-level organization of the dental pulp in teeth. Although conventional histologic analysis has provided microscopic images of the dental pulp, 3-dimensional (3D) cellular-level visualization of the whole dental pulp in an intact tooth has remained a technically challenging task. This is mainly due to the inevitable disruption and loss of microscopic structural features during the process of mechanical sectioning required for the preparation of the tooth sample for histological observation. To accomplish 3D microscopic observation of thick intact tissue, various optical clearing techniques have been developed mostly for soft tissue, and their application for hard tissues such as bone and teeth has only recently started to be investigated. In this work, we established a simple and rapid optical clearing technique for intact mouse teeth without the time-consuming process of decalcification. We achieved 3D cellular-level visualization of the microvasculature and various immune cell distributions in the whole dental pulp of mouse teeth under normal and pathologic conditions. This technique could be used to enable diverse research methods on tooth development and regeneration by providing 3D visualization of various pulpal cells in intact mouse teeth.
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