5,725 research outputs found
Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii
ABSTRACT
Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG
I
is plasma membrane associated whereas PKG
II
is cytosolic in
Toxoplasma gondii
. To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in
T. gondii
. By combining AID regulation with genome editing strategies, we determined that PKG
I
is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKG
II
is functionally insufficient and dispensable in the presence of PKG
I
. The difference in functionality mapped to the first 15 residues of PKG
I
, containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in
T. gondii
.
IMPORTANCE
Toxoplasma gondii
is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in
T. gondii
. We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in
T. gondii
.
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Analysis and Design of a Frame-Membrane Habitat Subjected to Extreme Temperatures on the Lunar Surface
During the era of the Apollo missions, there was been a great deal of interest among scientists, engineers, and the general population regarding the Moon and Outer Space. That interest has cycled through the years since but still burns strongly today as evidenced by the National Space Policy of President Barack Obama in 2010. Within this document, six goals were stated dealing with advanced study of the universe, which likely leads to the necessity of lunar colonization; the focus of this thesis.
The lunar environment provides several extreme challenges that may place any long term mission and its crewmembers in severe danger. These challenges include, but not limited to, reduced gravity, lack of a significant atmosphere, high velocity micrometeoroid impacts, extreme high and low temperatures, and dangerous levels of ionizing radiation. This thesis discusses many of these challenges in brief detail but focuses primarily on the topic of extreme temperature variation. By using the principles of heat flow, it was determined that at the lunar equator, the expected surface temperature atop the lunar habitat ranges from 187.5 K at night to 470 K during the day. These temperatures require the use of a strong thermal shield to provide shelter for the crewmembers and shield the habitat itself from the potentially dangerous temperatures. By using a layer of lunar regolith one-meter thick housed within the depth of the aluminum frame, the interior of the habitat is adequately shielded from the surface temperatures. Using a numerical integration-based analysis, the high and low temperatures were found to dissipate within the outermost thirty centimeters, at which point the regolith temperature remained relatively constant throughout the entire lunar cycle.
This behavior is especially important due to the increased stress levels found within the aluminum frame members making up the structure of the habitat. After the application of the cold nighttime temperatures, the maximum Von Mises stress value within the outer chord members increased by 20% seeing the yield strength safety factor reduce from 2.2 prior to the thermal load to 1.82 after its application. Similarly, the high temperatures of the daytime resulted in a 35% stress increase with a safety factor equal to 1.63. The presence of a high quality thermal shield can help to minimize the exposure of the structural elements to this type of loading and therefore reduced the stresses and deflections within the habitat frame.
With respect to the habitat vibration, it was found that the attachment of the inflatable membrane helps to increase the natural frequency of the structure. However, without sufficient pre-stressing or applied pressure, highly localized vibrations with very low natural frequencies were seen within the individual membrane bays. As the temperature was increased this phenomena was less apparent due to the expansion of the frame members stretching and pre-stressing the membrane.
The idea of colonizing the lunar surface is often difficult one to grasp, especially due to the lack of real world knowledge and experience with its challenges. This thesis presents a study with the goal of lunar colonization in mind beginning with the many different design criteria and loading characteristics on the Moon. These are then considered, with a focus on thermal loading, to determine their effect on the lunar surface and ultimately on the structural behavior of a frame-membrane composite structure. The results of this study provide a great deal of valuable information regarding not only frame-membrane composite structures, but also the lunar surface and colonization as well
Green investment strategies: a positive force in cities
Deterioration of urban neighborhoods is known to induce out migration, but how well do public investments to reverse decline actually work? To evaluate Philadelphia’s greening investment, researchers measured property buyers’ willingness to pay more—and found that greening works.Community development
Paper Session II-B - National Spaceport Testbed
The U.S. space industry continues to struggle to turn space business into successful business. Sensing this, both NASA and the state of Florida are exploring ideas for engaging their technological and economic resources in solving this grand challenge. This paper proposes just such an idea: a revolutionary new facility called the National Spaceport Testbed that would be dedicated to testing new space transportation technology. The one-of-a-kind testbed would allow space entrepreneurs to carry out ground and flight tests at reduced costs; allow NASA to apply resources to technical risk reduction; and allow Florida to attract and retain new space business
Multi-heme Cytochromes in Shewanella oneidensis MR-1:Structures, functions and opportunities
Multi-heme cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometers. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-heme cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-heme cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward there are opportunities to engage multi-heme cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-heme cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-heme cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies
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High Spatial Resolution Kelvin Probe Force Microscopy With Coaxial Probes
Kelvin probe force microscopy (KPFM) is a widely used technique to measure the local contact potential difference (CPD) between an AFM probe and the sample surface via the electrostatic force. The spatial resolution of KPFM is intrinsically limited by the long range of the electrostatic interaction, which includes contributions from the macroscopic cantilever and the conical tip. Here, we present coaxial AFM probes in which the cantilever and cone are shielded by a conducting shell, confining the tip-sample electrostatic interaction to a small region near the end of the tip. We have developed a technique to measure the true CPD despite the presence of the shell electrode. We find the behavior of these probes agrees with an electrostatic model of the force, and we observe a factor of 5 improvement in spatial resolution relative to unshielded probes. Our discussion centers on KPFM, but the field confinement offered by these probes may improve any variant of electrostatic force microscopy.Engineering and Applied Science
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