214 research outputs found
Express your LOV: an engineered flavoprotein as a reporter for protein expression and purification
No full textIn this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for Esp
Identification and Characterization of a Bacterial Catalytic Scaffold with Specificity for Host Endomembrane Traffic
No full textThe fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signaling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher order signaling networks through enzyme scaffolding, and the identity of the effectors or their mechanisms of action are poorly understood. Here, we used a functional screen to identify the EHEC O157:H7 type III effector EspG as a regulator of endomembrane trafficking and we report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAK) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GAP-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signaling inhibition at membrane organelles. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAK are organized on adjacent surfaces of EspG, suggesting its dual role as a “catalytic scaffold” that effectively reprograms cellular events through the functional assembly of GTPase-kinase signaling complex. Bidirectional vesicular transport between ER and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood, in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E.coli Secreted Protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi Intermediate Compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. Mechanistic insights presented in this study establish the effectiveness of a small bacterial catalytic scaffold in studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen
Neural models for monitoring and control with applications in automotive domain
No full textDevelopment of the state-of-the-art cyber-physical systems (CPS), which incorporate physical and computational components, poses new challenges for research and industry. In order for CPS serve its purpose to make human lives safer, easier, more enjoyable and convenient, both academia and industry needs to develop new methods for control and monitoring of such systems. Neural models are a prospective direction for design of CPS controllers and monitors, and in the thesis we first show, how neural models can be applied in CPS control to quantify the uncertainty of the system. We then show, how digital spiking neural model, called TrueNorth, can be used for runtime monitoring of temporal logic specifications of mission-critical properties. To be able to deliver not only qualitative verdict, but also reason quantitatively, we propose an approach to use the model for computation of arithmetic functions and implement neural monitors for semantics of temporal logic based on circular convolution. In the applied part of the work we show how runtime monitoring can speed up verification and validation phase in automotive electronic development. We identify phases, where runtime monitoring can facilitate both concept and post-silicon verification and testing. To build runtime monitors that are capable to keep up with the speed of the physical sensor, we developed an approach to convert formalized requirements to hardware monitors, which are then synthesized in FPGA. Reuse of the monitors from concept to post-silicon verification phase using high-level synthesis speeds up the testing process and enables long-term requirements evaluation. We illustrated our approach by formalizing, creating hardware monitors and evaluating the results in the lab environment for electrical and timing requirements of industrial SENT and SPC protocols
Self-healing by property-guided structural adaptation
No full textSelf-healing is an increasingly popular approach ensuring resiliency, that is, a proper adaptation to failures, in cyber-physical systems (CPS). A very promising way of achieving self-healing is through structural adaptation (SHSA), by replacing a failed component with a substitute component. We present a knowledge base modeling relations among system variables given that certain implicit redundancy exists in the system and show how to extract a substitute from that knowledge base using guided search. The result of our search, i.e., the substitute, is optimal w.r.t. a user-defined utility function considering properties of the system variables (e.g., accuracy). We demonstrate our approach - Self-Healing by Property-Guided Structural Adaptation (SH-PGSA) - by deploying it in a real-world CPS prototype of a rover whose sensors are susceptible to failure. We further show the increased runtime performance to find the optimal substitute by comparing it to related work
Activation of PAK by a bacterial type III effector EspG reveals alternative mechanisms of GTPase pathway regulation.
No full textTargeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy
No full textShiga toxin (STx) produced by Shigella and closely related Shiga toxin 1 and 2 (STx1 and STx2) synthesized by Shiga toxin-producing Escherichia coli (STEC) are bacterial AB5 toxins. All three toxins target kidney cells and may cause life-threatening renal disease. While Shigella infections can be treated with antibiotics, resistance is increasing. Moreover, antibiotic therapy is contraindicated for STEC, and there are no definitive treatments for STEC-induced disease. To exert cellular toxicity, STx, STx1, and STx2 must undergo retrograde trafficking to reach their cytosolic target, ribosomes. Direct transport from early endosomes to the Golgi apparatus is an essential step that allows the toxins to bypass degradative late endosomes and lysosomes. The essentiality of this transport step also makes it an ideal target for the development of small-molecule inhibitors of toxin trafficking as potential therapeutics. Here, we review the recent advances in understanding the molecular mechanisms of the early endosome-to-Golgi transport of STx, STx1, and STx2, as well as the development of small-molecule inhibitors of toxin trafficking that act at the endosome/Golgi interface
Applying Runtime Monitoring for Automotive Electronic Development
No full textThis paper shows how runtime monitoring can be applied at different phases of electronic-product development in automotive industry. Starting with concept development, runtime monitors are generated from the product requirements and then embedded in a chip simulation to track the specification compliance at an early stage. In the later phase when a prototype or a product is available, the runtime monitors from the concept development are reused for synthesis into FPGA for monitoring the implementation correctness of the product/system during runtime tests at real-time speeds. This is advantageous for long-term test scenarios where simulation becomes impractical or where evaluation of large amounts of data is required. For example, about 480 K frames/min are exchanged between a sensor and an ECU. This is beyond the capability of an engineer to check the specification conformance of every frame even for one minute of the system run. We embed monitors in a real-world industrial case study, where we runtime-check the requirements of an automotive sensor interface both in simulation and for the test chip
Selective Protection of an ARF1-GTP Signaling Axis by a Bacterial Scaffold Induces Bidirectional Trafficking Arrest
No full textSummaryBidirectional vesicular transport between the endoplasmic reticulum (ER) and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated in order to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E. coli secreted protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi intermediate compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. The mechanistic insights presented here establish the effectiveness of a small bacterial catalytic scaffold for studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen
MAES Analyzers for a Grand Spectrometer with Improved Performance in the Range of 258 - 269 nm
No full textGenome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking
No full textShiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor–guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors.</jats:p
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