Cytometrical analysis of the adverse effects of indican, indoxyl, indigo, and indirubin on rat thymic lymphocytes

Many businesses thrive by producing health supplements from agricultural products, as exemplified by the production of functional (or health) food using plants traditionally cultivated in the rural areas. Dyes, such as indican, indigo, indoxyl, and indirubin, present in dye plants, possess antibacterial, antifungal, and antiproliferative activities. However, these effects may also lead to cytotoxicity. Thus, studies in normal mammalian cells are necessary to identify cytotoxicity and prevent adverse effects of functional foods that contain these dyes. In this study, the effects of indican, indigo, indoxyl, and indirubin were evaluated by flow cytometry using appropriate fluorescent probes in rat thymic lymphocytes. Among the dyes analyzed, indirubin exerted distinct cellular activities. Treatment with indirubin (10–30 μM) increased the population of shrunken dead cells. The side scatter, but not forward scatter, increased in indirubin-treated living cells. It increased the population of annexin V-bound living and dead cells and that of dead cells without annexin V. Indirubin elevated intracellular Ca2+, but not Zn2+ levels. The cellular content of superoxide anions and increased glutathione decreased. Indirubin depolarized the cellular plasma and mitochondrial membranes. It did not potentiate or attenuate the cytotoxicity of A23187 (Ca2+ overload) and H2O2 (oxidative stress). The results suggested that indirubin induces both apoptotic and non-apoptotic cell death. It may be difficult to predict and prevent adverse effects of indirubin due to its diverse activities on normal mammalian cells. Therefore, indirubin should be removed from products that contain dye plant extracts

Machine learning-based estimation of spatial gene expression pattern during ESC-derived retinal organoid development

Abstract Organoids, which can reproduce the complex tissue structures found in embryos, are revolutionizing basic research and regenerative medicine. In order to use organoids for research and medicine, it is necessary to assess the composition and arrangement of cell types within the organoid, i.e., spatial gene expression. However, current methods are invasive and require gene editing and immunostaining. In this study, we developed a non-invasive estimation method of spatial gene expression patterns using machine learning. A deep learning model with an encoder-decoder architecture was trained on paired datasets of phase-contrast and fluorescence images, and was applied to a retinal organoid derived from mouse embryonic stem cells, focusing on the master gene Rax (also called Rx), crucial for eye field development. This method successfully estimated spatially plausible fluorescent patterns with appropriate intensities, enabling the non-invasive, quantitative estimation of spatial gene expression patterns within each tissue. Thus, this method could lead to new avenues for evaluating spatial gene expression patterns across a wide range of biology and medicine fields

Overexpression of Pla2g3 reduces IDE expression.

<p><i>A</i>, <i>B</i>, Quantitative RT-PCR result of IDE in TR-AST cells and rat primary astrocytes. TR-AST cells and primary astrocytes were treated with hydrogen peroxide in indicated conditions. Fold changes to the non-treated cells as controls are indicated. <i>C</i>, Quantitative PCR results of IDE in Pla2g3 transfected HEK293 cells. Human Pla2g3 was transiently expressed and cells were harvested after 48 hours of transfection. Fold changes to the mock control are indicated. *p<0.05, ***p<0.001.</p

Increased expression of Pla2g3 in frontal cortex of AD patients.

<p><i>A</i>, Pla2g3 expression in normal control brain and AD brain. The images are representative of 6 cases in each group. Frontal cortex was stained with anti-Pla2g3 antibody. <i>B</i>, Analysis of Pla2g3-positive cell numbers in frontal cortex. *p<0.05 <i>C-H</i>, Double immunostaining of Pla2g3 and GFAP in normal control brain (C-E) and AD brain (F-H). Arrowheads indicate the intensive staining of Pla2g3 in astrocytes of AD brain. Scale bars: <i>A</i>, 300 μm; <i>C</i>, 50 μm. Abbreviation used; NC; normal control, AD; Alzheimer’s disease patient, I; Molecular layer, II; External granular layer.</p

Microarray analysis of cerebrum specific genes regulated by oxidative stress and aging.

<p><i>A</i>, Each circle is indicating the numbers of either up or down regulated genes by oxidative stress, aging in the cerebrum and in the cerebellum. Five genes were up-regulated and four genes were down regulated by both oxidative stress and aging in the cerebrum without significant changes in the cerebellum. <i>B</i>, The heat maps of five genes up-regulated and <i>C</i>, four genes down-regulated by oxidative stress and aging. Red indicates up-regulated genes and blue indicates down-regulated genes, while white indicates no significant change in expression. The color gradients indicate the intensity of expression. The data shows the results of four mice per each group. Abbreviation used; OS; oxidative stress.</p

Induction of Pla2g3 expression in cerebrum by oxidative stress.

<p><i>A</i>, <i>B</i>, Quantitative RT-PCR results of Pla2g3 in cerebrum and cerebellum are shown. Fold changes to the aged wild-type mice on normal diet are indicated. n = 4 in each group. <i>C</i>, Western blots for Pla2g3 and alpha-tubulin are shown. Abbreviation used; WT normal; wild type 29 months old mice fed on normal diet, WT deficient; 29 months old wild type mice fed on vitamin E deficient diet, ttpKO deficient; 29 months old <i>Ttpa</i>^<i>-/-</i> mice fed on vitamin E deficient diet. *p<0.05.</p