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FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels—and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)—by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the “FimV C-terminal domain,” which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV 861–919 , containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV 689 , a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV 689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV 689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB ( fimV cyaB-R456L ), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa . FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

In contrast to Escherichia coli, a model organism for chemotaxis that has 5 chemoreceptors and a single chemosensory pathway, Pseudomonas aeruginosa PAO1 has a much more complex chemosensory network, which consists of 26 chemoreceptors feeding into four chemosensory pathways. While several chemoreceptors were rigorously linked to specific pathways in a series of experimental studies, for most of them this information is not available. Thus, we addressed the problem computationally. Protein–protein interaction network prediction, coexpression data mining, and phylogenetic profiling all produced incomplete and uncertain assignments of chemoreceptors to pathways. However, comparative sequence analysis specifically targeting chemoreceptor regions involved in pathway interactions revealed conserved sequence patterns that enabled us to unambiguously link all 26 chemoreceptors to four pathways. Placing computational evidence in the context of experimental data allowed us to conclude that three chemosensory pathways in P. aeruginosa utilize one chemoreceptor per pathway, whereas the fourth pathway, which is the main system controlling chemotaxis, utilizes the other 23 chemoreceptors. Our results show that while only a very few amino acid positions in receptors, kinases, and adaptors determine their pathway specificity, assigning receptors to pathways computationally is possible. This requires substantial knowledge about interacting partners on a molecular level and focusing comparative sequence analysis on the pathway-specific regions. This general principle should be applicable to resolving many other receptor–pathway interactions., We thank Jacob Pollack for technical assistance and Ariane Briegel for helpful discussions. This work was supported in part by National Institutes of Health Grants GM072295 (to I.B.Z.) and GM122588 (to G.J.J.).

Chemotaxis allows bacteria to more efficiently colonize optimal microhabitats within their larger environment. Chemotaxis in Escherichia coli is the best-studied model system, and a large number of E. coli strains have been sequenced. The Escherichia/Shigella genus encompasses a great variety of commensal and pathogenic strains, but the role of chemotaxis in their association with the host remains poorly understood. Here we show that the core chemotaxis genes are lost in many, but not all, nonmotile strains but are well preserved in all motile strains. The genes encoding the Tar, Tsr, and Aer chemoreceptors, which mediate chemotaxis to a broad spectrum of chemical and physical cues, are also nearly uniformly conserved in motile strains. In contrast, the clade of extraintestinal pathogenic E. coli strains apparently underwent an ancestral loss of Trg and Tap chemoreceptors, which sense sugars, dipeptides, and pyrimidines. The broad range of time estimated for the loss of these genes (1 to 3 million years ago) corresponds to the appearance of the genus Homo .

Bacterial chemosensory arrays are composed of extended networks of chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), the histidine kinase CheA, and the adaptor protein CheW. Models of these arrays have been developed from cryoelectron microscopy, crystal structures of binary and ternary complexes, NMR spectroscopy, mutational, data and biochemical studies. A new 3.2 Å resolution crystal structure of a Thermotoga maritima MCP protein interaction region in complex with the CheA kinase-regulatory module (P4-P5) and adaptor protein CheW provides sufficient detail to define residue contacts at the interfaces formed among the three proteins. As in a previous 4.5 Å resolution structure, CheA-P5 and CheW interact through conserved hydrophobic surfaces at the ends of their β-barrels to form pseudo 6-fold symmetric rings in which the two proteins alternate around the circumference. The interface between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous studies, whereas the related interface between CheW subdomain 1 and P5 subdomain 2 has only been observed in these ring assemblies. The receptor forms an unexpected structure in that the helical hairpin tip of each subunit has "unzipped" into a continuous α-helix; four such helices associate into a bundle, and the tetramers bridge adjacent P5-CheW rings in the lattice through interactions with both P5 and CheW. P5 and CheW each bind a receptor helix with a groove of conserved hydrophobic residues between subdomains 1 and 2. P5 binds the receptor helix N-terminal to the tip region (lower site), whereas CheW binds the same helix with inverted polarity near the bundle end (upper site). Sequence comparisons among different evolutionary classes of chemotaxis proteins show that the binding partners undergo correlated changes at key residue positions that involve the lower site. Such evolutionary analyses argue that both CheW and P5 bind to the receptor tip at overlapping positions. Computational genomics further reveal that two distinct CheW proteins in Thermotogae utilize the analogous recognition motifs to couple different receptor classes to the same CheA kinase. Important residues for function previously identified by mutagenesis, chemical modification and biophysical approaches also map to these same interfaces. Thus, although the native CheW-receptor interaction is not observed in the present crystal structure, the bioinformatics and previous data predict key features of this interface. The companion study of the P5-receptor interface in native arrays (accompanying paper Piasta et al. (2013) Biochemistry, DOI: 10.1021/bi400385c) shows that, despite the non-native receptor fold in the present crystal structure, the local helix-in-groove contacts of the crystallographic P5-receptor interaction are present in native arrays and are essential for receptor regulation of kinase activity.

A fundamentally new family of thermal spray processes has emerged. These new processes, collectively known as very low pressure plasma spray or VLPPS, differ from traditional thermal spray processes in that coatings are deposited at unusually low chamber pressures, typically less than ~800 Pa (6 Torr). Depending upon the specific process, deposition may be in the form of very fine molten droplets, vapor phase deposition, or a mixture of vapor and droplet deposition. Resulting coatings are similar in quality to coatings produced by alternative coating technologies, such as physical vapor deposition (PVD) or chemical vapor deposition (CVD), but deposition rates can be roughly an order of magnitude higher with VLPPS. With these new process technologies modified low pressure plasma spray (LPPS) systems can now be used to produce dense, high quality coatings in the 1 to 100 micron thickness range with lamellar or columnar microstructures. A history of pioneering work in VLPPS technology is presented, deposition mechanisms are discussed, potential new applications are reviewed, and challenges for the future are outlined.

Cellular receptors usually contain a designated sensory domain that recognizes the signal. Per/Arnt/Sim (PAS) domains are ubiquitous sensors in thousands of species ranging from bacteria to humans. Although PAS domains were described as intracellular sensors, recent structural studies revealed PAS-like domains in extracytoplasmic regions in several transmembrane receptors. However, these structurally defined extracellular PAS-like domains do not match sequence-derived PAS domain models, and thus their distribution across the genomic landscape remains largely unknown. Here we show that structurally defined extracellular PAS-like domains belong to the Cache superfamily, which is homologous to, but distinct from the PAS superfamily. Our newly built computational models enabled identification of Cache domains in tens of thousands of signal transduction proteins including those from important pathogens and model organisms. Furthermore, we show that Cache domains comprise the dominant mode of extracellular sensing in prokaryotes., Author Summary Cell-surface receptors control multiple cellular functions and are attractive targets for drug design. These receptors often have dedicated extracellular domains that bind signaling molecules, such as hormones and nutrients. Computational identification of these ligand-binding domains in genomic sequences is a pre-requisite for their further experimental characterization. Using available three-dimensional structures of several bacterial cell-surface receptors, we built computational models that enabled identification of the Cache domain, as the most common extracellular sensor module in prokaryotes, including many important pathogens. We also demonstrated that the Cache domain is homologous to, but sufficiently different from the most common intracellular sensor module, the PAS domain. These findings provide a unified view on molecular principles of signal recognition by extra- and intracellular receptors.

We describe a tool, the GRaph-Based Interface Language tool (GRBIL), in which developers can easily design, construct, and evaluate system interfaces. The GRBIL system allows a designer to graphically define a system interface and walk through a set of operator goals for using the interface. To evaluate the interface, the system automatically generates a cognitive model of a system operator (built in the ACT-R cognitive architecture), which provides metrics of time on task and potential errors. In addition, the system allows the easy incorporation of dynamic models of the external world in order to evaluate interfaces that involve continually changing environments. We discuss the on-going validation of GRBIL as it is used to evaluate a robotics operator control unit.

There is ample evidence that computer users often do not progress from novice to expert levels of performance, particularly when efficiency is included in the definition of performance. This paper describes a theoretical argument that one of the pieces missing in the understanding of this process is an accurate assessment of how people calculate a cost/benefit analysis (CBA) of learning and using new techniques and strategies. While there are many explanations for why people often do not use accurate methods of calculating this ratio, there is little discussion describing why some people do. We suggest that an important and predictable influence on whether an individual uses an accurate CBA is the observation of others using efficient techniques. We propose that with a more full understanding of the CBA calculation process, it will be possible to predict when users will and will not utilize a more efficient technique.

Despite the fact that keyboard issued commands (KICs) are more efficient than other command methods, experienced users often do not adopt them. In order to examine the factors underlying this phenomenon, a study is presented which investigated the relationships between users' level of knowledge with Microsoft Word, the importance they placed on the costs and benefits of using KICs, and how these factors related to the use of KICs in Microsoft Word. Results indicate that benefits are more strongly associated with the actual use of KICs than costs. The application of these findings to the human factors domain and the implication of the results to facilitate the adoption of efficient techniques and behaviors are discussed.

As computer technology has become ubiquitous in nature, designers must develop text-entry methods that can accommodate a variety of devices, cultures, and languages. This paper presents a study evaluating a One-HAnded Input (OHAI) device developed for mobile text-entry in Chinese. The device is a chorded keyboard system on which text is entered in Chinese using the pinyin system. Participants trained with the device for 10, 1-hour sessions. Their text-entry rates and their rate of input for individual chords were measured after each session. The Power Law of Practice was used to predict input rates after 100 hours of training. Predicted input rates approximate 16 characters per minute, approaching pinyin input rates on conventional keyboards. Input rates for individual chords were also measured in order that future iterations of the software may associate the most commonly used characters with the fastest-to-execute chords. The study also suggests several potential future improvements to the OHAI system, such as augmenting the software with an autocompletion system.

We present a computational model of closed-loop, pilot-displays-aircraft system designed to evaluate the impact of the addition of a synthetic vision system (SVS) to a commercial airliner cockpit. The NASA-run empirical study revealed that while pilots rarely looked out the window during most phases of approach and landing, they devoted a substantial proportion of their gaze toward the SVS. A model pilot implemented in ACT-R was connected to a commercial flight simulator package in an attempt to model pilots’ attention allocation behaviors. Based on metrics such as transitions from one display to another, the model provides a good approximation to pilot behaviors and should allow us to perform virtual experiments on the impact of changes to the SVS. Evaluation of new technology for the commercial aircraft cockpit is an expensive and time-consuming process. The pool of potential subjects is small and consists of individuals with extensive training who are both relatively difficult and expensive to access. Thus, the typical design-test-modify iteration cycle is generally both slow and costly. One approach with

Previous research (Lane, Napier, Peres, & Sándor, in press) has shown that despite the fact that it typically takes half as much time to issue a command to a computer application using that command's keyboard shortcut, most people issue a particular command by clicking an icon on a toolbar or by selecting the command from a pull-down menu. This study examined reasons why that might be the case with a web survey that focused on demographic characteristics of people who do and do not use keyboard shortcuts, as well as social factors of computer use that might influence use of keyboard shortcuts. Participants' shortcut usage was influenced by social factors, such as working in an environment with other shortcut users and experiential factors, primarily the hours spent using a computer per week.

This paper reports the results of a research project to study the nature and structure of phone messages and the design characteristics of phone messaging systems that would optimize the retrieval of relevant message information. of particular concern with respect to the structure of a phone message was the location of a phone number left in the message. in order to replay any portion of a message, most messaging systems require the user to listen to the message from its beginning. Because it was found that phone numbers are typically left towards the end of a message, such a system is quite inefficient. One proposed solution is the implementation of a “back” button. A simple equation is employed to optimize the length of time encompassed by the back button, which would result in substantial time savings to users at least 17 seconds per message in which it was used. The ramifications of such time savings are discussed.

Plasma spray coating techniques allow unique control of electrolyte microstructures and properties as well as facilitating deposition on complex surfaces. This can enable significantly improved solid oxide fuel cells (SOFCs), including non-planar designs. SOFCs are promising because they directly convert the oxidization of fuel into electrical energy. However, electrolytes deposited using conventional plasma spray are porous and often greater than 50 microns thick. One solution to form dense, thin electrolytes of ideal composition for SOFCs is to combine suspension plasma spray (SPS) with very low pressure plasma spray (VLPPS). Increased compositional control is achieved due to dissolved dopant compounds in the suspension that are incorporated into the coating during plasma spraying. Thus, it is possible to change the chemistry of the feed stock during deposition. In the work reported, suspensions of sub-micron diameter 8 mol.% Y2O3-ZrO2 (YSZ) powders were sprayed on NiO-YSZ anodes at Sandia National Laboratories (SNL) Thermal Spray Research Laboratory (TSRL). These coatings were compared to the same suspensions doped with scandium nitrate at 3 to 8 mol%. The pressure in the chamber was 2.4 torr and the plasma was formed from a combination of argon and hydrogen gases. The resultant electrolytes were well adhered to the anodemore » substrates and were approximately 10 microns thick. The microstructure of the resultant electrolytes will be reported as well as the electrolyte performance as part of a SOFC system via potentiodynamic testing and impedance spectroscopy.« less

We present an experiment that compares how people perform search tasks in a degree-of-interest browser and in a Windows-Explorer-like browser. Our results show that, whereas users do attend to more information in the DOI browser, they do not complete the task faster than in an Explorer-like browser. However, in both types of browser, users are faster to complete high information scent search tasks than low information scent tasks. We present an ACT-R computational model of the search task in the DOI browser. The model describes how a visual search strategy may combine with semantic aspects of processing, as captured by information scent. We also describe a way of automatically estimating information scent in an ontological hierarchy by querying a large corpus (in our case, Google's corpus).

HIV-1 infection among IVDUs is increasing at a rapid rate in the U.S. Because many Emergency Departments experience many visits from patients with this risk factor, the impact on emergency services can be considerable. Because many of these patients currently have asymptomatic infection, knowledge of clinical presentations of HIV-1 infection is valuable, as the Emergency Department may be the site for the initial presentation of complications related to HIV-1. Finally, Emergency Departments may play a vital role in preventing further progression of this deadly and destructive disease among this risk group.