Helix Dipole Movement and Conformational Variability Contribute to Allosteric GDP Release in Gα i Subunits † , ‡

Heterotrimeric G proteins (Gαβγ) transmit signals from activated G protein coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal α5 helix of Gα subunits participate in Gα-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the α5 helix of the Gα subunit [Oldham et al., Nat. Struct. Mol. Biol., 13: 772-7 (2006)]. Based on this result, an engineered disulfide bond was designed to constrain the α5 helix of Gαi1 into its EPR-measured receptor-associated conformation through the introduction of cysteines at positions 56 in the α1 helix and 333 in the α5 helix (I56C/Q333C Gαi1). A functional mimetic of the EPR-measured α5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino-terminus of this helix, D328R Gαi1. Both proteins exhibit dramatically elevated basal nucleotide exchange. The 2.9 Å resolution crystal structure of the I56C/Q333C Gαi1 in complex with GDP-AlF4− reveals the shift of the α5 helix toward the guanine nucleotide-binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Gαi1 subunit further revealed altered positions for the switch regions and throughout the Gαi1 subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the α5 helix dipole and overall conformational variability during nucleotide release

Towards a wastewater energy recovery system: The utilization of humidified ammonia by a solid oxide fuel cell stack

This study presents the results of investigations on performance and durability of an ammonia-supplied MK352 solid oxide fuel cell stack with electrolyte supported cells and chromium based interconnects. The performance evaluation revealed no significant differences between ammonia and equivalent hydrogen/nitrogen gases as fuel, which was a result of the excellent ammonia conversion rates up to 99.99%. When using high ammonia flow rates, temperature measurements inside the stack revealed a temperature drop due to the endothermic ammonia decomposition of up to 18.8 K, which proceeded preferentially at the fuel inlet region. An 1000 h durability test with humidified ammonia in 80% fuel utilization condition was performed, which resulted in a stack performance degradation rate of about 1.1%/1000 h. Tests with hydrogen/nitrogen fueled reference stacks revealed similar degradation rates during the initial 1000 h. Post-mortem analyses by scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed no significant micro-structural deterioration of the functional layers of the anode, but nitriding effects on the nickel contact meshes and chromium nitrides were found in the material structure of the interconnects. Also, an oxide layer was found between interconnect and contact meshes at the anode, which appears to be the main cause of the performance degradation

Getting a kick out of it: multimodal signalling during male-male encounters in the foot-flagging frog Micrixalus aff. saxicola from the Western Ghats of India

Several anuran species use multimodal signals to communicate in diverse social contexts. Our study describes acoustic and visual behaviours of the Small Torrent Frog (Micrixalus aff. saxicola), a diurnal frog endemic to the Western Ghats of India. During agonistic interactions males display advertisement calls, foot-flagging and tapping (foot lifting) behaviours to signal the readiness to defend perching sites in perennial streams. Results from a quantitative video analysis of male-male interactions indicate that foot-flagging displays were used as directional signals toward the opponent male, but were less abundant than calls. The acoustic and visual signals were not functionally linked. The call of Micrixalus aff. saxicola thereby did not act as an alert signal. Analysis of behavioural transitions revealed that kicking behaviours (physical attacks) significantly elicited kicks from interacting males. We suggest that foot-flagging displays ritualized from this frequently observed fighting technique to reduce physical attacks

Getting a kick out of it: multimodal signalling during male–male encounters in the foot-flagging frog Micrixalus aff. saxicola from the Western Ghats of India

Several anuran species use multimodal signals to communicate in diverse social contexts. Our study describes acoustic and visual behaviours of the Small Torrent Frog (Micrixalus aff. saxicola), a diurnal frog endemic to the Western Ghats of India. During agonistic interactions males display advertisement calls, foot-flagging and tapping (foot lifting) behaviours to signal the readiness to defend perching sites in perennial streams. Results from a quantitative video analysis of male–male interactions indicate that footflagging displays were used as directional signals toward the opponent male, but were less abundant than calls. The acoustic and visual signals were not functionally linked. The call of Micrixalus aff. saxicola thereby did not act as an alert signal. Analysis of behavioural transitions revealed that kicking behaviours (physical attacks) significantly elicited kicks from interacting males. We suggest that foot-flagging displays ritualized from this frequently observed fighting technique to reduce physical attacks

Irreversible Platelet Activation Requires Protease-Activated Receptor 1-Mediated Signaling to Phosphatidylinositol Phosphates

Thrombin induces platelet activation through an early, reversible stage of platelet aggregation, which is followed by a later, irreversible stage of platelet aggregation. Without intervention, events leading to pathological platelet activation can result in vessel occlusion, acute coronary syndrome, and stroke. Therefore, a better understanding of events leading to platelet-mediated clot formation may provide insight into new therapeutic targets. Once activated, protease activated receptors (PARs) are essential in regulating events leading to platelet aggregation. We have determined a signaling cascade through PAR1, which involves phosphatidylinositol (PI) kinases, phosphatidylinositol bisphosphate (PIP2), and Rap1 activation (independent of P2Y12) in the formation of a stable platelet aggregate. The putative phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 was found to reduce basal and PAR-stimulated PIP2 levels by mass spectrometry and to inhibit PAR1-mediated stable platelet aggregation. Rap1 activation in platelets (during time points corresponding to the late, irreversible phase of aggregation) was found to require the PI signaling pathway. Perturbation of PI3K signaling by isoform-selective inhibitors had differential effects on Rap1 activation through PAR1 and PAR4. Hence, it is possible to disrupt lipid signaling pathways involved in stable clot formation without inhibiting early clot formation, offering a new potential target for antiplatelet therapy

Fast fuel variation and identification of SOFC system changes using online health monitoring tools and fault diagnosis

The greatest challenges of the solid oxide fuel cell (SOFC) technology are its reliability and durability, both of them considering a variety of possible fuels. To address those issues, understanding of the ongoing processes in SOFCs is of crucial importance. The ability to monitor electrochemical processes online and to identify fast alternating operating conditions offers a possibility to determine degradation-inducing processes at their preliminary stage. Thus, their early identification would provide timely counteractions and inhibit irreversible SOFC degradation. To guarantee the safe SOFC operation, the present study focuses on: (1) investigation of SOFC behavior during feeding with different fuels, (2) development and application of non-conventional tools for online-monitoring, and (3) identification of failure modes at the early stage