Guan Wang

© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte and from five representatives of vascular plants using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.

Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine. In this study, we demonstrate in Arabidopsis stems that targeted expression of AdoMet hydrolase in secondary cell wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl units and a reduction of dimethylated syringyl units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild-type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.

© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons LtdSphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1, which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

© 2016 The Author.Background: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. Results: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. Conclusion: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates.

Background and Objective Pulsed dye laser is a commonly used treatment for Port Wine Stain birthmarks. However, deeper components of PWS are often resistant to PDL. Deeper penetrating lasers, including the long pulsed Neodymium:Yttrium Aluminum Garnet laser have been used, but carry greater risk. This study evaluates the distinct blood vessel thermal responses to visible and near infrared lasers using animal and numerical models. Study Design/Materials and Methods Blood vessels in the rodent dorsal skin chamber were irradiated by a 595 nm PDL and a long-pulsed 1,064 nm Nd:YAG laser. Laser-induced immediate and 1-hour post-structural and functional changes in the vessels were documented. Numerical simulations were conducted using a 1,000 μm depth SD mouse skin fold to simulate experimental conditions. Results PDL irradiation produced immediate blood vessel hemorrhage. Modeling indicated this occurs due to preferential heating of the superior parts of large blood vessels. Nd:YAG irradiation resulted in blood vessel constriction; modeling indicated more uniform heating of vessel walls. Conclusion PDL and Nd:YAG lasers result in distinct tissue responses. This supports different observable clinical treatment end points when using these devices. Vessel constriction associated with the Nd:YAG may be more difficult to observe and is one reason this device may carry greater risk. © 2014 Wiley Periodicals, Inc.

Cellular reprogramming can manipulate the identity of cells to generate the desired cell types1– 3. The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can enforce somatic cell reprogramming to pluripotent stem cells4– 7. By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner8– 10. However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome2,11,12 and reduced plasticity13,14; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans.

Internet-enabled technologies have provided a way for people to communicate and collaborate with each other. The collaboration and communication made crowdsourcing an efficient and effective activity. Crowdsourcing is a modern paradigm that employs cheap labors for accomplishing different types of tasks. The task is usually posted online as an open call, and members of the crowd self-select a task to be carried out. Crowdsourcing involves initiators or crowdsourcers, the crowd, crowdsourcing task, the process, and the crowdsourcing platform where requesters offer various tasks and crowd workers complete these tasks. As the crowdsourcing is carried out in the online environment, it gives rise to certain challenges. The major problem is the selection of crowd that is becoming a challenging issue with the growth in crowdsourcing popularity. Crowd selection has been significantly investigated in crowdsourcing processes. Nonetheless, it has observed that the selection is based only on a single feature of the crowd worker which was not sufficient for appropriate crowd selection. For addressing the problem of crowd selection, a novel “ant colony optimization-based crowd selection method” is presented in this paper that selects a crowd worker based on multicriteria features. By utilizing the proposed model, the efficiency and effectiveness of crowdsourcing activity will be increased.

The cyclooxygenase 2 pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway.The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling.Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers and a concomitant increase in expression of epithelial markers, and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment.Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway.

Lithium-oxygen cells are a promising power source, and carbons are an attractive non-metal catalyst for air electrodes. To improve the electrochemical performance, various carbons are heated in an inert atmosphere. It is found that heat treatment at 900 C can differently improve the electrochemical performance of multiwalled carbon nanotubes, acetylene carbon black and activated carbon, but the improvement of CNTs is the most obvious. After heat treatment, the peak current density of the oxygen reduction reaction and the 1st discharge capacity of CNTs increase ∼30% and ∼125%, respectively, while the charge transfer reaction resistance and the Warburg diffusion resistance decrease ∼7.0% and ∼11.1%, respectively. AC has the highest charge capacities and capacity retention ratio in spite of little influence by heat treatment. The possible mechanism and reasons are analyzed using different techniques. Microstructure is superior to conductivity for enhancing the rechargeability and the cyclability, and heat treatment is effective for some carbon materials in improving the electrochemical performance of Li-O2 cells. © 2014 Elsevier Ltd.