Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Effect of sterol composition on the activity of the yeast G-protein-coupled receptor Ste2.

The main sterol of the human cell membrane is cholesterol, whereas in yeast it is ergosterol. In this study, we constructed a cholesterol-producing yeast strain by disrupting the genes related to ergosterol synthesis and inserting the genes related to cholesterol synthesis. The total sterols of the mutant yeast were extracted and the sterol composition was analyzed by GC-MS. We confirmed that cholesterol was produced instead of ergosterol in yeast and subsequently examined the activity of the yeast G-protein-coupled receptor (GPCR) Ste2p. Ste2p signaling was assessed in wild type (WT) with ergosterol and the cholesterol-producing yeast instead of ergosterol to determine whether sterol composition affects the activity of the yeast GPCR. Our results demonstrated that Ste2p could transduce a signal even in the cholesterol-rich membrane, but the maximum signal intensity was weaker than that transduced in the ergosterol-rich original (WT) membrane. This result indicates that sterol composition affects the activity of yeast GPCRs, and thus, this provides new insight into GPCR-mediated transduction using yeast for future fundamental and applied studies on GPCRs from yeast to other organisms.

Functional screening system for yeast-secreted peptides acting on G-protein coupled receptors.

We established a novel functional screening system for peptides acting on G-protein coupled receptors (GPCRs). Peptides are a promising drug scaffold because of their intermediate molecular size between that of therapeutic small molecules and antibodies. They also offer potential advantages of targeting not only membrane proteins but also intracellular protein-protein interactions. Phage display technology has been used for exploring novel peptides acting on GPCRs, but it is unclear whether the identified peptides functionally modulate targets because the technology selects peptides based on binding ability but not functional activity to targets. In a novel screening system that we established, yeast cells were utilized as a peptide producer while mammalian cells stably producing the receptor for glucagon-like peptide 1 (GLP1R) were used as a biosensor for receptor activation. Three kinds of GLP1R agonists secreted by yeasts were successfully detected for their functional activities without any purification and condensation of those peptides. By applying the functional screening system, we were able to identify GLP1R agonist-secreting yeasts based on GLP1R activation from the cell mixture containing a number of background yeasts that produced non-active control peptides. Further applications of this system would include not only activity evaluation of bioactive peptides without chemical synthesis but also discovery of novel peptides activating druggable GPCRs.

Screening of randomly mutagenized glucagon-like peptide-1 library by using an integrated yeast-mammalian assay system.

Glucagon-like peptide-1 (GLP1) is a 30-amino acid peptide hormone activating the GLP1 receptor (GLP1R), a class B G-protein coupled receptor (GPCR), and is considered to be effective for treating diabetes and other metabolic diseases. Phage display is the first innovative technology in order to prepare and screen a large polypeptide library including GLP1R agonists, but this methodology is not as effective in discovering functional peptides such as activators for GPCRs. Here, we report a novel functional screening system for GPCR-acting peptides, which integrates a yeast peptide secretion system into a biological detection system with GPCR-producing mammalian cells. Using this screening system, we found attractive GLP1R agonists with several substitutions from a random mutant GLP1 library which was secreted by yeast, Saccharomyces cerevisiae. This system established here not only enables peptides to be analyzed in the soluble form but also needs no chemical synthesis, purification, and condensation of peptides of interests, and therefore, can be widely applied to the discovery of novel bioactive peptides acting on GPCRs.

Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases.

Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti-viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild-type and drug-resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs-displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug-resistant influenza virus.

Membrane-displayed somatostatin activates somatostatin receptor subtype-2 heterologously produced in Saccharomyces cerevisiae.

The G-protein-coupled receptor (GPCR) superfamily, which includes somatostatin receptors (SSTRs), is one of the most important drug targets in the pharmaceutical industry. The yeast Saccharomyces cerevisiae is an attractive host for the ligand screening of human GPCRs. Here, we demonstrate the utility of the technology that was developed for displaying peptide ligands on yeast plasma membrane, termed "PepDisplay", which triggers signal transduction upon GPCR activation. A yeast strain that heterologously produced human somatostatin receptor subtype-2 (SSTR2) and chimeric Gα protein was constructed along with membrane-displayed somatostatin; somatostatin was displayed on the yeast plasma membrane by linking it to the anchoring domain of the glycosylphosphatidylinositol anchored plasma membrane protein Yps1p. We demonstrate that the somatostatin displayed on the plasma membrane successfully activated human SSTR2 in S. cerevisiae. The methodology presented here provides a new platform for identifying novel peptide ligands for both liganded and orphan mammalian GPCRs.

Zingiberaceous and citrus constituents, 1'-acetoxychavicol acetate, zerumbone, auraptene, and nobiletin, suppress lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 murine macrophages through different modes of action.

In the present study, we explored the suppressive activities of 1'-acetoxychavicol acetate (ACA), auraptene, nobiletin, and zerumbone toward LPS-induced cyclooxygenase (COX)-2 mRNA expression in mouse macrophages and the underlying molecular mechanisms. Pretreatment of RAW264.7 cells with LPS led to the activation of mitogen-activated protein kinase (MAPK)s [p38, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK)1/2] and Akt, together with degradation of the inhibitor of nuclear factor-kappaB (IkappaB)-alpha protein and nuclear translocation of nuclear factor (NF)-kappaB p65, and the resultant activation of activator protein (AP)-1, NF-kappaB, and cAMP-responsive element-binding protein (CREB) transcription factors. ACA abrogated ERK1/2 and JNK1/2, but not p38 MAPK, as well as the activation of those transcription factors. Although it allowed LPS-triggered phosphorylation of those MAPKs and NF-kappaB nuclear translocation, nobiletin suppressed the activation of AP-1, NF-kappaB, and CREB. Zerumbone had no effect on those transcription factors, though it attenuated COX-2 mRNA expression, suggesting that it disrupts the stabilization of COX-2 mRNA. Conversely, zerumbone significantly accelerated spontaneous COX-2 mRNA decay, the potency of which was comparable with that of SB203580, an inhibitor of p38 MAPK, whose activation has key roles in the proinflammatory mRNA stabilization processes. Because SB203580 but not zerumbone suppressed LPS-induced p38 MAPK activation, the molecular targets of zerumbone may be MAPK-activated protein kinase-2 or located downstream. However, auraptene suppressed the expression of COX-2 protein but not mRNA, implying that it targets translation. We propose that these phytochemicals are promising chemopreventive agents for inflammation-associated carcinogenesis. Their use in combination may enhance their efficacy because of their different modes of action.