Fabrication of cell plastics composed only of unicellular green alga Chlamydomonas reinhardtii as a raw material.

Cell plastics in this study were fabricated with only unicellular green alga Chlamydomonas reinhardtii as raw materials. The sizes of cell-major axis as structures were 8.4 ± 1.2 µm, and the aspect ratios of those were 1.2 ± 0.1, showing homogeneous particle size. After optimizing extraction condition of intracellular contents, cell plastics were fabricated with the cells as ingredient components and the intracellular contents as matrix components. Those cell plastics were observed with scanning electron microscopy, displaying the smooth surfaces of the cell plastics at a low magnification level. However, the surface, especially exposed surface, were rough at high magnification level. Tensile strength test revealed that increasing the ratio of intracellular contents in the cell plastics until 21% led enhancing mechanical properties of Young's modulus and tensile strength; however, 25% of intracellular contents displayed decreases of those properties. As the optimal point, the cell plastic (21%), which contained 21% (w/w) of intracellular contents in cell plastics, showed 764 ± 100 MPa and 8.6 ± 5.2 MPa of Young's modulus and tensile strength. The cell plastics showed few plastic region and soon fractured, indicating the possibility that cells and intracellular contents could be electrostatically connected. Additionally, cells were shown as a negative charge and displayed the possibility to contribute electrically cell-gathering with intracellular ionic components. Therefore, cells and intracellular contents containing ionic metabolites could be electrostatically connected for giving the mechanical strength to cell plastics. In this study, we successfully demonstrated fabricating cell plastics with only cells for the first time and also showed the high possibility of conjugating each cell with the intracellular contents. KEY POINTS: • Cell plastics are fabricated with unicellular green algal cell directly. • Unicellular cells required to be conjugated for the fabrication with matrix. • Cells were conjugated with intracellular contents for cell-plastic fabrication.

Epigenetic and oncogenic inhibitors cooperatively drive differentiation and kill KRAS-mutant colorectal cancers.

Current treatments for KRAS-mutant colorectal cancers (CRCs) are often limited by cellular plasticity and rewiring responses. Here we describe a promising therapeutic strategy that simultaneously targets epigenetic and oncogenic signals. Specifically, we show that inhibitors of the histone methyltransferase, EZH2, synergize with various RAS pathway inhibitors and promote dramatic tumor regression in vivo. Together these agents cooperatively suppress WNT-driven transcription and drive CRCs into a more differentiated cell state by inducing the Groucho/TLE corepressor, TLE4, along with a network of WNT pathway inhibitors and intestinal differentiation proteins. However, these agents also induce the pro-apoptotic protein BMF, which subsequently kills these more differentiated cells. Accordingly, cell death can be prevented by activating ß-catenin, blocking differentiation, or by ablating BMF expression. Collectively, these studies reveal a new therapeutic approach for treating KRAS-mutant CRCs and illustrate a critical convergence of EZH2 and RAS on oncogenic WNT signals, intestinal differentiation, and apoptosis.

Identification of Cell-Attachment Factors Derived from Green Algal Cells Disrupted by Sonication in Fabrication of Cell Plastics.

Cell plastics which are composed of unicellular green algal cells have been proposed in previous studies. While unicellular green algae can be freely arranged using fabrication processes, a matrix is required to attach the cells together. To date, although the cell contents collected from Chlamydomonas reinhardtii show the possibility of attaching cells, but it is unclear which components can be considered attachment factors. Therefore, in this study, C. reinhardtii cells were disrupted with sonication, and the components were separated and purified with hexane. The cell plastics with only 0.5 wt% of intermediate showed similar mechanical properties to those with 17 wt% and 25 wt% of cell components that were untreated with hexane, meaning that the purified intermediates could function as matrices. The purified intermediate was composed of approximately 60 wt% of protein as the main component, and proteomic analysis was performed to survey the main proteins that remained after hexane treatment. The protein compositions of the cell content and purified intermediate were compared via proteomic analysis, revealing that the existing ratios of 532 proteins were increased in the purified intermediate rather than in the cell content. In particular, the outer structure of each of the 49 proteins-the intensity of which was increased by over 10 times-had characteristically random coil conformations, containing ratios of proline and alanine. The information could suggest a matrix of cell plastics, inspiring the possibility to endow the cell plastics with more properties and functions.

The cytotoxicity of gefitinib on patient­derived induced pluripotent stem cells reflects gefitinib­induced liver injury in the clinical setting.

Gefitinib is a key drug used in the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. Gefitinib therapy is superior to conventional chemotherapy for the progression-free survival rate of patients with EGFR mutations. However, 10-26% of patients develop grade 3 or higher hepatotoxicity during gefitinib treatment; therefore, the development of preclinical tests for hepatotoxicity prior to clinical use is desirable. The present study evaluated the use of induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iPSC-heps), as a platform for preclinical test development. Patient-derived iPSCs were generated by reprogramming peripheral blood mononuclear cells obtained from two groups of gefitinib-treated patients with severe hepatotoxicity [toxicity group (T group)] or mild hepatotoxicity [no clinical toxicity group (N group)]. To examine the hepatotoxicity, the iPSCs from both T and N groups were differentiated into hepatocytes to obtain iPSC-heps. Differentiation was confirmed by measuring the expression levels of hepatocyte markers, such as albumin or α-fetoprotein, via western blotting and quantitative PCR analyses. Cytotoxicity in iPSCs and iPSC-heps after gefitinib treatment was evaluated using a lactate dehydrogenase release assay. The gefitinib-induced cytotoxicity in iPSCs from the T group was significantly higher than that from the N group, whereas there were no significant differences between the groups of iPSC-heps. These results suggested that using iPSCs in preclinical assessment may be a good indicator for the prediction of gefitinib-induced cytotoxicity in clinical use.

Construction of cell-plastics as neo-plastics consisted of cell-layer provided green alga Chlamydomonas reinhardtii covered by two-dimensional polymer.

Green alga Chlamydomonas reinhardtii has gained interest as a sustainable resource because it can be easily grown using CO2 as a carbon source owing to its high CO2 assimilating activity. Although the robustness of the cell wall of C. reinhardtii makes it difficult to extract its intracellular products, such property is beneficial when using the cell as an ingredient to fabricate "cell-plastic" in this study. The cell layer, which is a component of the cell-plastic, was prepared with an intercellular filler to connect each cell because C. reinhardtii is a single-cell strain. The cell layers were then repeatedly piled to increase the strength of the cell-plastic. To avoid slippage between the cell layers, they were covered with a small amount of a two-dimensional polymer to maintain the flat surface structure of the cell-plastic. Based on the evaluation, the cell-plastic has the potential to be a novel, sustainable plastic using ubiquitous green algal cells in nature.

A transcription factor hierarchy defines an environmental stress response network.

Environmental stresses are universally encountered by microbes, plants, and animals. Yet systematic studies of stress-responsive transcription factor (TF) networks in multicellular organisms have been limited. The phytohormone abscisic acid (ABA) influences the expression of thousands of genes, allowing us to characterize complex stress-responsive regulatory networks. Using chromatin immunoprecipitation sequencing, we identified genome-wide targets of 21 ABA-related TFs to construct a comprehensive regulatory network in Arabidopsis thaliana Determinants of dynamic TF binding and a hierarchy among TFs were defined, illuminating the relationship between differential gene expression patterns and ABA pathway feedback regulation. By extrapolating regulatory characteristics of observed canonical ABA pathway components, we identified a new family of transcriptional regulators modulating ABA and salt responsiveness and demonstrated their utility to modulate plant resilience to osmotic stress.

Fisetin Suppresses Lipid Accumulation in Mouse Adipocytic 3T3-L1 Cells by Repressing GLUT4-Mediated Glucose Uptake through Inhibition of mTOR-C/EBPα Signaling.

3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.