Advancement in human induced pluripotent stem cell (iPSC) neuron and microglial differentiation protocols allow for disease modeling using physiologically relevant cells. However, iPSC differentiation and culturing protocols have posed challenges to maintaining consistency. Here, we generated an automated, consistent, and long-term culturing platform of human iPSC neurons, astrocytes, and microglia. Using this platform we generated a iPSC AD model using human derived cells, which showed signs of Aβ plaques, dystrophic neurites around plaques, synapse loss, dendrite retraction, axon fragmentation, phospho-Tau induction, and neuronal cell death in one model. We showed that the human iPSC microglia internalized and compacted Aβ to generate and surround the plaques, thereby conferring some neuroprotection. We investigated the mechanism of action of anti-Aβ antibodies protection and found that they protected neurons from these pathologies and were most effective before pTau induction. Taken together, these results suggest that this model can facilitate target discovery and drug development efforts.
The stigmatic estolide is a lipid-based polyester constituting the major component of exudate in solanaceous plants. Although the exudate is believed to play important roles in the pollination process, the biosynthetic pathway of stigmatic estolide, including genes encoding the key enzymes, remains unknown. Here we report the cloning and characterization of the cytochrome P450 gene CYP86A22, which encodes a fatty acyl-CoA -hydroxylase involved in estolide biosynthesis in the stigma of Petunia hybrida. A CYP86A22 cDNA was isolated from a developing stigma cDNA library, and the corresponding gene was shown to express predominantly in the developing stigma. Among six P450 genes isolated from this library, only CYP86A22 was implicated in -hydroxylation following RNA interference (RNAi)-mediated suppression. Unlike wild-type plants in which -hydroxy fatty acids (mainly in the form of 18-hydroxy oleic acid and 18-hydroxy linoleic acid) compose 96% of total stigma fatty acids, the -hydroxy fatty acids were essentially absent in the stigmas from 18 of 46 CYP86A22-RNAi transgenic plants and had varying levels of suppression in the remaining 28 plants. Furthermore, lipids in the 18 CYP86A22-RNAi stigmas were predominantly triacylglycerols and diacylglycerols instead of the estolides, which characterize the wild-type stigma. Analyses of recombinant CYP86A22 conclusively demonstrated that this P450 is a -hydroxylase with a substrate preference for both saturated and unsaturated acyl-CoAs rather than free fatty acids. We conclude that the cytochrome P450 enzyme CYP86A22 is the key fatty acyl-CoA -hydroxylase essential for the production of -hydroxy fatty acids and the biosynthesis of triacylglycerol-/diacylglycerol-based estolide polyesters in the petunia stigma.The stigma of Petunia hybrida and other solanaceous species is covered by copious exudate, whereas the stigma of Arabidopsis thaliana and other Brassicaceae has little or no exudate on the surface (1, 2). The exudate is primarily lipidic, although other compounds such as proteins, sugars, and pigments are also present (3-6). The lipidic character of the exudate derives from the presence of a large amount of estolide, a lipid-based polyester. Estolide is rich in -hydroxy fatty acids, i.e. fatty acids containing a hydroxy group on the terminal or omega () carbon. The exudate of petunia stigma contains up to 96% -hydroxy fatty acids mainly in the form of 18-hydroxy-oleic acid and 18-hydroxy-linoleic acid (7,8). These -hydroxy fatty acid monomers are thought to be attached to a glycerol backbone and then iteratively esterified such that the -hydroxyl group of one fatty acid is esterified to a carboxyl group of another to ultimately form the estolide. In tobacco stigma, estolides are formed as tetra-, penta-, hexa-, and heptaacyl glycerides and capped by normal fatty acids (9, 10). The stigmatic exudate has shown significant physiological roles in the pollination process (11)(12)(13)(14). Despite its importance, the biosynthetic pathway of stigmatic estolide, including ge...
Transient gene expression in mammalian cells is an efficient process for producing recombinant proteins for various research applications to support large molecule therapeutics development. For the first time, we report a high throughput small molecule (SM) screen to identify novel compounds that increase antibody titers after polyethylenimine (PEI) transient transfection of a HEK293 cell line. After screening 31,413 SMs in a 50 μL scaled-down process, we validated 164 SMs to improve yields by up to twofold. The titer increase mediated by the SMs varied for different antibodies. SM dose optimizations resulted in almost threefold higher titers. The top 2, structurally distinct SM hits, increased antibody titers more than twofold in a 1 mL production process. Averaged across three antibodies of different expression levels, the compounds enhanced transient productivity by ∼80%. Intriguingly, both compounds arrested cells in the G2/M cell cycle phase leading to a decrease in growth and nutrient consumption, while elevating titer, nuclear plasmid DNA (pDNA) copy numbers, and mRNA levels. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 3:1579-1588, 2017.
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