Structure and Dynamic Studies of the Nuclear Pore Complex at the Single-Molecule Level

Nuclear pore complexes (NPCs) are large macromolecular structures forming the only known direct route across the double bilayer membrane of the nuclear envelope. The NPC structure has been extensively explored in an effort to elucidate the mechanisms by which they control transport. Many of these studies have found the presence of a central mass or plug within the central channel of NPCs, although neither the function nor identity of the central mass were clear. Here, several techniques including electron microscopy, Förster resonance energy transfer (FRET), and high-resolution near-field scanning optical microscopy (NSOM) are utilized to specifically locate vault ribonucleoproteins to NPCs. This interaction, along with several other results, strongly suggests that vaults represent the central mass of NPCs. A single-molecule transport assay was also developed in order to record the translocation of individual fluorescent dextrans through NPCs. Comparison of the single-molecule dwell times under various conditions led to a better understanding of the specific mechanism controlling the non signal-mediated transport of cargo through NPCs

Alien Registration- Dickenson, Wilfred E. (Norridgewock, Somerset County)

https://digitalmaine.com/alien_docs/9095/thumbnail.jp

QED effects in molecules: test on rotational quantum states of H2_2

Quantum electrodynamic effects have been systematically tested in the progression of rotational quantum states in the $X ^{1}\Sigma_{g}^{+}, v=0$ vibronic ground state of molecular hydrogen. High-precision Doppler-free spectroscopy of the $EF ^{1}\Sigma_{g}^{+} - X ^{1}\Sigma_{g}^{+}$ (0,0) band was performed with 0.005 cm$^{-1}$ accuracy on rotationally-hot H$_2$ (with rotational quantum states J up to 16). QED and relativistic contributions to rotational level energies as high as 0.13 cm$^{-1}$ are extracted, and are in perfect agreement with recent calculations of QED and high-order relativistic effects for the H$_2$ ground state.Comment: 4 pages, 3 figures, to be published in Physical Review Letter

The lady vanishes: what's missing from the stem cell debate

Most opponents of somatic cell nuclear transfer and embryonic stem cell technologies base their arguments on the twin assertions that the embryo is either a human being or a potential human being, and that it is wrong to destroy a human being or potential human being in order to produce stem cell lines. Proponents’ justifications of stem cell research are more varied, but not enough to escape the charge of obsession with the status of the embryo. What unites the two warring sides in ‘the stem cell wars’ is that women are equally invisible to both: ‘the lady vanishes’. Yet the only legitimate property in the body is that which women possess in their reproductive tissue and the products of their reproductive labour. By drawing on the accepted characterisation in law of property as a bundle of rights, and on a Hegelian model of contract as mutual recognition, we can lessen the impact of the tendency to regard women and their eggs as merely receptacles and women’s reproductive labour as unimportant

Phosphomimetic Tyrosine Mutations in Spa47 Inhibit Type Three Secretion ATPase Activity and Shigella Virulence Phenotype

Shigella is a highly infectious human pathogen responsible for 269 million infections and 200,000 deaths per year. Shigella virulence is absolutely reliant on the injection of effector proteins into the host cell cytoplasm via its type three secretion system (T3SS). The protein Spa47 is a T3SS ATPase whose activity is essential for the proper function of the Shigella T3SS needle-like apparatus through which effectors are secreted. A phosphoproteomics study recently found several Shigella T3SS proteins, including Spa47, to be tyrosine phosphorylated, suggesting a means of regulating Spa47 enzymatic activity, T3SS function, and overall Shigella virulence. The work presented here employs phosphomimetic mutations in Spa47 to probe the effects of phosphorylation at these targeted tyrosines through in vitro radiometric ATPase assays and circular dichroism as well as in vivo characterization of T3SS secretion activity, erythrocyte hemolysis, and cellular invasion. Results presented here demonstrate a direct correlation between Spa47 tyrosine phosphorylation state, Spa47 ATPase activity, T3SS function, and Shigella virulence. Together, these findings provide a strong foundation that leads the way to uncovering the specific pathway(s) that Shigella employ to mitigate wasteful ATP hydrolysis and effector protein secretion when not required as well as T3SS activation in preparation for host infection and immune evasion

Glen E. Dickenson and M. W. Husband to Governor Ross Barnett, 28 September 1962

Senders discuss the attempted invasions of both Cuba and Mississippi.https://egrove.olemiss.edu/west_union_gov/1057/thumbnail.jp

Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-32P ATP

ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase that supports protein secretion through its specialized type three secretion apparatus (T3SA), supporting infection of human host cells. Characterizing ATPases, such as Spa47, requires a means to accurately determine enzyme activity (ATP hydrolysis) as a function of time, reaction conditions, and potential cofactors, regulators, inhibitors, etc. Here, we describe a detailed protocol for characterizing the enzyme kinetics of Spa47 using a direct α−32P ATPase assay

Centrifuge Modeling of Pile-Supported Wharves for Seismic Hazards

Recent earthquakes have highlighted many seismic hazard concerns for western U.S. ports. Port waterfront structures are commonly constructed utilizing pile-supported wharves in combination with rock dike structures retaining a hydraulically placed backfill. Seismic damage is generally attributed to weak soils that are often prevalent in the marine environment (e.g. liquefiable sands, sensitive cohesive soils). In response to past damage, many ports are instigating soil improvement strategies to eliminate or minimize potential occurrences of liquefaction and to increase the strength of cohesive soils. The design of a seismically resilient wharf requires an understanding of its performance during design level earthquakes. Due to the complex nature of pile-supported wharves, state-of-the-art centrifuge modeling techniques are being used to better understand their seismic performance. The authors used the large-scale centrifuge facility at the University of California at Davis. This paper presents details on the construction, instrumentation, and testing of the models. Results from the tests are also included, such as the seismic pile behavior, effect of soil improvement, and the overall behavior

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from \u3cem\u3eShigella flexneri\u3c/em\u3e

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents