The Complex Functions of the Receptor-Like Cytoplasmic Kinase BIK1 in Plant Immunity and Development

The sessile plants have evolved a large number of receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) to modulate diverse biological processes, including plant innate immunity, growth and development. Phosphorylation of RLK/RLCK complex constitutes an essential step to initiate the immune signaling. Two Arabidopsis plasma membrane-resident RLKs FLS2 and BAK1 interact with RLCK BIK1 to initiate plant immune responses to bacterial flagellin. Classically defined as a serine/threonine kinase, BIK1 is shown here to possess tyrosine kinase activity with mass spectrometry, immunoblot and genetic analyses. BIK1 is auto-phosphorylated and trans-phosphorylated by BAK1 at multiple tyrosine (Y) residues in addition to serine/threonine residues. BIK1Y150 is likely catalytic important, whereas Y243 and Y250 are more specifically involved in tyrosine phosphorylation. Importantly, the BIK1 tyrosine phosphorylation plays a crucial role in BIK1-mediated plant innate immunity as the transgenic plants carrying BIK1Y150F, Y243F or Y250F (the mutation of tyrosine to phenylalanine) failed to complement the bik1 mutant deficiency in immunity. Together with previous finding of BAK1 as a tyrosine kinase, these results unveiled tyrosine phosphorylation as a common regulatory mechanism that controls membrane-resident receptor signaling in plants and metazoans. BAK1 complexes with the receptor kinase FLS2 in bacterial flagellin-triggered immunity and BRI1 in brassinosteroid (BR)-mediated growth. In contrast to its positive role in plant immunity, we report here that BIK1 acts as a negative regulator in BR signaling. The bik1 mutants display various BR hypersensitive phenotypes accompanied with increased accumulation of de-phosphorylated BES1 proteins and regulation of BZR1 and BES1 target genes. BIK1 associates with BRI1, and is released from BRI1 receptor upon BR treatment, which is reminiscent of FLS2-BIK1 complex dynamics in flagellin signaling. The ligand-induced release of BIK1 from receptor complexes is associated with BIK1 phosphorylation. However, in contrast to BAK1-dependent FLS2-BIK1 dissociation, BAK1 is dispensable for BRI1-BIK1 dissociation. Consequently, unlike FLS2 signaling which depends on BAK1 to phosphorylate BIK1, BRI1 directly phosphorylates BIK1 to transduce BR signaling. Rapid activation of two branches of Mitogen-activated protein kinase (MAPK) cascades consisting of MEKK1-MKK1/2-MPK4 and MEKK1/?-MKK4/5-MPK3/6 is associated with perception of flagellin. There is limited understanding of how the signal transmits from the FLS2-BAK1 receptor complex to MAPK cascades. I have performed a series of genetic studies on the mutants of bik1 and its related family members. Various combinations of higher order of mutants indicate that flagellin-mediated MAPK activation functions downstream of BIK1. I found that the mekk1/2/3 deletion mutant largely restored various growth defects of bik1, and further genetic assays revealed that the alleviated growth defects can mainly be attributed to the mekk1 mutation, but not mekk2. I also demonstrated that BIK1 likely associates with MEKK1 on the plasma membrane, indicating that BIK1 bridges PRR complexes and MAPK cascades to relay immune signaling

Preparation of Polyfunctionalized Grignard Reagents and their Application in Aryne Chemistry

The preparation of polyfunctinalized arynes has been achieved by the controlled elimination of readily generated 2-magnesiated aryl arylsulfonates obtained by a low temperature halogen/Mg-exchange starting from the corresponding iodides. New types of Grignard reagents via the addition of magnesium aryl thiolates, amides and selenides to arynes and heteroarynes have been prepared. An easy access to highly functionalized arylmagnesium reagents prepared by direct magnesiation of carbocyclic aromatic rings with TMPMgClLiCl was developed

The Complex Functions of the Receptor-Like Cytoplasmic Kinase BIK1 in Plant Immunity and Development

The sessile plants have evolved a large number of receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) to modulate diverse biological processes, including plant innate immunity, growth and development. Phosphorylation of RLK/RLCK complex constitutes an essential step to initiate the immune signaling. Two Arabidopsis plasma membrane-resident RLKs FLS2 and BAK1 interact with RLCK BIK1 to initiate plant immune responses to bacterial flagellin. Classically defined as a serine/threonine kinase, BIK1 is shown here to possess tyrosine kinase activity with mass spectrometry, immunoblot and genetic analyses. BIK1 is auto-phosphorylated and trans-phosphorylated by BAK1 at multiple tyrosine (Y) residues in addition to serine/threonine residues. BIK1Y150 is likely catalytic important, whereas Y243 and Y250 are more specifically involved in tyrosine phosphorylation. Importantly, the BIK1 tyrosine phosphorylation plays a crucial role in BIK1-mediated plant innate immunity as the transgenic plants carrying BIK1Y150F, Y243F or Y250F (the mutation of tyrosine to phenylalanine) failed to complement the bik1 mutant deficiency in immunity. Together with previous finding of BAK1 as a tyrosine kinase, these results unveiled tyrosine phosphorylation as a common regulatory mechanism that controls membrane-resident receptor signaling in plants and metazoans. BAK1 complexes with the receptor kinase FLS2 in bacterial flagellin-triggered immunity and BRI1 in brassinosteroid (BR)-mediated growth. In contrast to its positive role in plant immunity, we report here that BIK1 acts as a negative regulator in BR signaling. The bik1 mutants display various BR hypersensitive phenotypes accompanied with increased accumulation of de-phosphorylated BES1 proteins and regulation of BZR1 and BES1 target genes. BIK1 associates with BRI1, and is released from BRI1 receptor upon BR treatment, which is reminiscent of FLS2-BIK1 complex dynamics in flagellin signaling. The ligand-induced release of BIK1 from receptor complexes is associated with BIK1 phosphorylation. However, in contrast to BAK1-dependent FLS2-BIK1 dissociation, BAK1 is dispensable for BRI1-BIK1 dissociation. Consequently, unlike FLS2 signaling which depends on BAK1 to phosphorylate BIK1, BRI1 directly phosphorylates BIK1 to transduce BR signaling. Rapid activation of two branches of Mitogen-activated protein kinase (MAPK) cascades consisting of MEKK1-MKK1/2-MPK4 and MEKK1/?-MKK4/5-MPK3/6 is associated with perception of flagellin. There is limited understanding of how the signal transmits from the FLS2-BAK1 receptor complex to MAPK cascades. I have performed a series of genetic studies on the mutants of bik1 and its related family members. Various combinations of higher order of mutants indicate that flagellin-mediated MAPK activation functions downstream of BIK1. I found that the mekk1/2/3 deletion mutant largely restored various growth defects of bik1, and further genetic assays revealed that the alleviated growth defects can mainly be attributed to the mekk1 mutation, but not mekk2. I also demonstrated that BIK1 likely associates with MEKK1 on the plasma membrane, indicating that BIK1 bridges PRR complexes and MAPK cascades to relay immune signaling

Kirenol attenuates experimental autoimmune encephalomyelitis by inhibiting differentiation of Th1 and th17 cells and inducing apoptosis of effector T cells.

Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), is characterized by CNS demyelination mediated by autoreactive T cells. Kirenol, a biologically active substance isolated from Herba Siegesbeckiae, has potent anti-inflammatory activities. Here we investigated effects of kirenol on EAE. Kirenol treatment markedly delayed onset of disease and reduced clinical scores in EAE mice. Kirenol treatment reduced expression of IFN-γ and IL-17A in the serum and proportion of Th1 and Th17 cells in draining lymph nodes. Priming of lymphocytes was reduced and apoptosis of MOG-activated CD4+ T cells was increased in kirenol treated EAE mice. Kirenol treatment of healthy animals did not affect the lymphocytes in these non-immunized mice. Further in vitro studies showed that kirenol inhibited viability of MOG-specific lymphocytes and induced apoptosis of MOG-specific CD4+ T cells in a dose- and time-dependent manner. Kirenol treatment upregulated Bax,downregulated Bcl-2,and increased activation of caspase-3 and release of cytochrome c, indicating that a mitochondrial pathway was involved in kirenol induced apoptosis. Moreover, pretreatment with either a pan-caspase inhibitor z-VAD-fmk or a more specific caspase 3 inhibitor Ac-DEVD-CHO in lymphocytes reduced kirenol induced apoptosis. Our findings implicate kirenol as a useful agent for the treatment of MS

Identifying novel survival mechanisms of Mycobacterium Tuberculosis within its host

Ph.DDOCTOR OF PHILOSOPH

Multi-objective optimization design for a battery pack of electric vehicle with surrogate models

In this investigation, a systematic surrogate-based optimization design framework for a battery pack is presented. An air-cooling battery pack equipped on electric vehicles is first designed. Finite element analysis (FEA) results of the baseline design show that global maximum stresses under x-axis and y-axis transient acceleration shock condition are both above the tensile limit of material. Selecting the panel and beam thickness of battery pack as design variables, with global maximum stress constraints in shock cases, a multi-objective optimization problem is implemented using metamodel technique and multi-objective particle-swarm-optimization (MOPSO) algorithm to simultaneously minimize the total mass and maximize the restrained basic frequency. It is found that 2nd order polynomial response surface (PRS), 3rd order PRS and radial basis function (RBF) are the most accurate and appropriate metamodels for restrained basic frequency, global maximum stresses under x-axis and y-axis shock conditions respectively. Results demonstrate that all the optimal solutions in Pareto Frontier have heavier weight and lower frequency compared with baseline design due to the restriction of global maximum stress response. Finally, two optimal schemes, “Knee Point” and “lightest weight”, satisfied both of the stress constraint conditions, show great consistency with FEA results and can be selected as alternative improved schemes

Online Robot Introspection via Wrench-based Action Grammars

Robotic failure is all too common in unstructured robot tasks. Despite well-designed controllers, robots often fail due to unexpected events. How do robots measure unexpected events? Many do not. Most robots are driven by the sense-plan act paradigm, however more recently robots are undergoing a sense-plan-act-verify paradigm. In this work, we present a principled methodology to bootstrap online robot introspection for contact tasks. In effect, we are trying to enable the robot to answer the question: what did I do? Is my behavior as expected or not? To this end, we analyze noisy wrench data and postulate that the latter inherently contains patterns that can be effectively represented by a vocabulary. The vocabulary is generated by segmenting and encoding the data. When the wrench information represents a sequence of sub-tasks, we can think of the vocabulary forming a sentence (set of words with grammar rules) for a given sub-task; allowing the latter to be uniquely represented. The grammar, which can also include unexpected events, was classified in offline and online scenarios as well as for simulated and real robot experiments. Multiclass Support Vector Machines (SVMs) were used offline, while online probabilistic SVMs were are used to give temporal confidence to the introspection result. The contribution of our work is the presentation of a generalizable online semantic scheme that enables a robot to understand its high-level state whether nominal or abnormal. It is shown to work in offline and online scenarios for a particularly challenging contact task: snap assemblies. We perform the snap assembly in one-arm simulated and real one-arm experiments and a simulated two-arm experiment. This verification mechanism can be used by high-level planners or reasoning systems to enable intelligent failure recovery or determine the next most optima manipulation skill to be used.Comment: arXiv admin note: substantial text overlap with arXiv:1609.0494

Engineering DNA polymerases for application in DNB-based sequencing technology

DNA polymerases serve as the core engine to afford sequence information in sequencing technologies that have revolutionized modern biological research. For application in the DNB-based sequencing platform, an assemblage of DNA polymerases was engineered to catalyze the requisite biochemical reaction. In the process, naturally occurring polymerases were tapped into through deep-learning algorithms for constraints between individual protein residues to narrow down the protein sequence space and to annotate protein sequences in light of their catalytic properties. And the constraints were subsequently applied in designing potential polymerase candidates with the guidance of the sequence annotations. Additionally, ancestral protein sequences were estimated to expand the candidate repertoire. Furthermore, the candidates were subjected to in silico screening before examined by an HTS methodology based on fluorescence signal. Finally, the resulting proteins were expressed and purified for testing in the DNB-based sequencing platform. Our sequencing data suggested that these proteins behave better than their existing counterparts

A DCT-Based Driving Cycle Generation Method and Its Application for Electric Vehicles

Nowadays, many widely used driving cycle (DC) representing and generating methods are designed for traditional vehicles with internal combustion engines (ICE). The real-world driving is viewed as a sequence of acceleration, cruise, deceleration, and idle modes. The emission and fuel consumption in each period should be taken into account carefully. However, for electric vehicles (EVs), most of them are powered by low or zero-emission renewable energy sources. The working status and energy management algorithms of them are very different from traditional vehicles. To facilitate the EV design, we proposed a novel DC representing and construction method to generate DCs for EVs. The whole driving route is divided into several length-fixed segments and each of these segments is converted into a frequency sequence. After doing that, we can adjust the frequency and amplitude of the generated driving cycle directly. The experiment results showed that the proposed method was effective and convenient

Flavonoids activate pregnane × receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

<p>Abstract</p> <p>Background</p> <p>The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is regulated by the pregnane × receptor (PXR), which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk) pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks). Flavonoids have also been shown to induce <it>CYPs </it>expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and <it>CYP </it>expression.</p> <p>Results</p> <p>In a cell-based screen designed to identify compounds that activate PXR-mediated <it>CYP3A4 </it>gene expression in HepG2 human carcinoma cells, we identified several flavonoids, such as luteolin and apigenin, as PXR activators. The flavonoids did not directly bind to PXR, suggesting that an alternative mechanism may be responsible for flavonoid-mediated PXR activation. Consistent with the Cdk5-inhibitory effect of flavonoids, Cdk5 and p35 (a non-cyclin regulatory subunit required to activate Cdk5) were expressed in HepG2. The activation of Cdk5 attenuated PXR-mediated <it>CYP3A4 </it>expression whereas its downregulation enhanced it. The Cdk5-mediated downregulation of <it>CYP3A4 </it>promoter activity was restored by flavonoids, suggesting that flavonoids activate PXR by inactivating Cdk5. <it>In vitro </it>kinase assays showed that Cdk5 directly phosphorylates PXR. The Cdk kinase profiling assay showed that apigenin inhibits multiple Cdks, suggesting that several Cdks may be involved in activation of PXR by flavonoids.</p> <p>Conclusions</p> <p>Our results for the first time link the stimulatory effect of flavonoids on <it>CYP </it>expression to their inhibitory effect on Cdks, through a PXR-mediated mechanism. These results may have important implications on the pharmacokinetics of drugs co-administered with herbal remedy and herbal-drug interactions.</p