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Rhodococcus equi is a Gram-positive facultative intracellular pathogen associated with life-threatening bronchopneumonial disease in foals. Key to R. equi’s intracellular survival in host macrophages is the production of virulence associated proteins (Vaps). Numerous vap genes are found on virulence plasmids isolated from different species, and the Vaps share a high degree of sequence identity. VapA has been extensively studied, and although vapK and vapN genes from other R. equi virulence plasmids have been shown to be essential for R. equi intracellular survival, their mode of action is less characterised. We, therefore, examined whether VapK and VapN worked mechanistically in the same way as VapA. Indeed, like VapA, VapK and VapN neutralised lysosomal pH and reduced lysosomal hydrolase activity. A loss of VapA and R. equi virulence could be regained by the presence of either VapK or VapN. The acid-neutralisation activity was also observed to a lesser extent with VapB. There was a differential activity across these virulence-promoting Vaps with the most “acid-neutralising” activity found with VapN, then VapA and K, and finally VapB. These data suggest that VapA production, which is often found in equine infections, can be substituted by VapK and B (produced by plasmids often found in porcine species) or VapN (produced by plasmids often isolated in bovine and human samples). These data imply that the molecular mechanism(s) that VapA uses to neutralise lysosomal acidity should also be seen in VapN and K which will help guide researchers in identifying their precise mode of action and aid the future development of targeted therapeutics.

VAMP7 is a SNARE protein involved in the fusion of endosomes and lysosomes with various cellular membranes. The longin domain of VAMP7 forms autoinhibitory interactions that prevent VAMP7 SNARE assembly. New structural and functional data reveal how the regulatory protein Varp, a known VAMP7 binding partner, kinetically inhibits SNARE complex formation. SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans -SNARE complexes are assembled from glutamine-contributing SNAREs (Q-SNAREs) embedded in one membrane and an arginine-contributing SNARE (R-SNARE) embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE–binding regulator of SNARE complex formation. Varp colocalizes with and binds to VAMP7, an R-SNARE that is involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7–SNARE motif is trapped between Varp and the VAMP7 longin domain, and hence Varp kinetically inhibits the ability of VAMP7 to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as VAMP7, such as Rab32–GTP.

Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family.

Key Points Lysosomes are dynamic organelles that receive membrane traffic input from the secretory, endocytic, autophagic and phagocytic pathways. They can also fuse with the plasma membrane. Live-cell imaging has shown that lysosomes interact with late endosomes by 'kiss-and-run' events and by direct fusion. Fusion results in the formation of hybrid organelles, in which the degradation of endocytosed macromolecules occurs and from which lysosomes are re-formed. The use of yeast genetics and mammalian cell-free systems has identified much of the protein machinery that is involved in the delivery of macromolecules to lysosomes. The fusion of late endosomes with lysosomes involves tethering, the formation of trans -SNARE (soluble N -ethylmaleimide sensitive factor attachment protein receptor) complexes and phospholipid bilayer fusion. Conventional lysosomes may fuse with the plasma membrane in response to a rise in cytosolic Ca 2+ and can provide the additional membrane required for plasma-membrane wound repair. Specialized secretory lysosomes and lysosome-related organelles exist in some cell types. Lysosomes may also fuse with phagosomes and autophagosomes. Some phagocytosed pathogens can prevent or delay phagolysosome biogenesis; others escape their intracellular vacuole by degrading the phagosomal membrane and may evade autophagy or reside in autophagic compartments and delay the formation of autolysosomes. Upregulating autophagic pathways and the formation of autophagolysosomes provides the prospect of therapies for a range of proteinopathies including Huntington's disease and Parkinson's disease. Far from being a static organelle at the end of the endocytic pathway, the lysosome is capable of dynamically fusing with many organelles as well as the plasma membrane. The lysosome provides hydrolytic enzymes for the degradation of macromolecules, has secretory functions and is important for plasma membrane repair. Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.

Rhodococcus equi (R. equi) is an important pulmonary pathogen in foals that often leads to the death of the horse. The bacterium harbors a virulence plasmid that encodes numerous virulence‐associated proteins (Vaps) including VapA that is essential for intracellular survival inside macrophages. However, little is known about the precise function of VapA. Here, we demonstrate that VapA causes perturbation to late endocytic organelles with swollen endolysosome organelles having reduced Cathepsin B activity and an accumulation of LBPA, LC3 and Rab7. The data are indicative of a loss of endolysosomal function, which leads cells to upregulate lysosome biogenesis to compensate for the loss of functional endolysosomes. Although there is a high degree of homology of the core region of VapA to other Vap proteins, only the highly conserved core region of VapA, and not VapD of VapG, gives the observed effects on endolysosomes. This is the first demonstration of how VapA works and implies that VapA aids R. equi survival by reducing the impact of lysosomes on phagocytosed bacteria. VapA is essential for Rhodococcus equi survival inside cells. Here, we show that VapA disrupts endolysosomes as a mechanism to aid Rhodococcus equi survival.

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans ‐SNARE complex consisting of Q‐SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R‐SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell‐free systems, we confirmed this result, but found that the same Q‐SNAREs can combine with an alternative R‐SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co‐immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N‐terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.

ObjectiveThis study compared the effects of two different warm-up protocols (normal vs. priming) on the oxygen plateau () incidence rate during a ramp test. It also compared the cardiopulmonary responses during the ramp test and subsequent verification phase.MethodsEleven recreational cyclists performed two experimental visits. The first visit required a normal warm-up (cycling at 50 W for 10 min) followed by the ramp test (30 W.min-1) and supramaximal verification phase with 30 min rest between tests. The second visit required a priming warm-up (cycling at 50 W for 4 min increasing to 70% difference between the gas exchange threshold [GET] and maximum work rate [WRmax] for 6 min) followed by the same protocol as in the first visit. Physiological responses were collected during the exercise and compared. Oxygen kinetics ( Kinetics) and incidence rate were determined during the ramp tests for both visits.ResultsAs planned, following the warm-up the priming visit experienced greater physiological response. However, the incidence rate of during the ramp test was the same between visits (73%), and maximal oxygen uptake was not different between visits after the ramp test (normal, 4.0 ± 0.8; primed, 4.0 ± 0.7 L·min−1, p = 0.230) and verification phase (normal, 3.8 ± 0.6; primed, 3.8 ± 0.7 L·min−1, p = 0.924) using a Holm-Bonferroni correction for controlling family-wise error rate. Kinetics were not different between visits during the ramp test (normal, 10.8 ± 1.1; primed, 10.8 ± 1.2 mL·min−1·W-1, p = 0.407). The verification phase confirmed in 100% for both the normal and priming visits.ConclusionOur hypothesis that a priming warm-up facilitates the incidence rate of during a ramp test is not supported by the results. The verification phase remains a prudent option when determining a ‘true’ is required.

The three-dimensional coordinates of more than 23,000 atoms in an iron-platinum nanoparticle are determined with 22 picometre precision to correlate chemical order/disorder and crystal defects with magnetic properties. Material properties at the single-atom level FePt nanoparticles have practical potential in fields as diverse as catalysis and magnetic storage media. But far from being pristine crystalline materials, these nanoparticles are structurally heterogeneous with grain boundaries and other crystal defects. In this paper, Jianwei Miao and colleagues reveal the complex atomic-scale structure of a single FePt nanoparticle containing more than 22,000 atoms. They do this by generating a high-resolution tomographic tilt series of 68 images of the nanoparticle and reconstructing it using a new algorithm, achieving resolution with 22 picometre precision. The resulting structure reveals the complexity of the nanoparticle, and the chemistry and crystal structure of the grains within the material. When analysing the order/disorder character, the authors find that the grains are more ordered towards the core of the nanoparticle and less ordered towards the surface. They use data from the boundary between two grains to calculate local magnetocrystalline anisotropy energies using density functional theory, revealing how these energies vary across the grain with order parameter and across a grain boundary. Perfect crystals are rare in nature. Real materials often contain crystal defects and chemical order/disorder such as grain boundaries, dislocations, interfaces, surface reconstructions and point defects 1 , 2 , 3 . Such disruption in periodicity strongly affects material properties and functionality 1 , 2 , 3 . Despite rapid development of quantitative material characterization methods 1 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , correlating three-dimensional (3D) atomic arrangements of chemical order/disorder and crystal defects with material properties remains a challenge. On a parallel front, quantum mechanics calculations such as density functional theory (DFT) have progressed from the modelling of ideal bulk systems to modelling ‘real’ materials with dopants, dislocations, grain boundaries and interfaces 19 , 20 ; but these calculations rely heavily on average atomic models extracted from crystallography. To improve the predictive power of first-principles calculations, there is a pressing need to use atomic coordinates of real systems beyond average crystallographic measurements. Here we determine the 3D coordinates of 6,569 iron and 16,627 platinum atoms in an iron-platinum nanoparticle, and correlate chemical order/disorder and crystal defects with material properties at the single-atom level. We identify rich structural variety with unprecedented 3D detail including atomic composition, grain boundaries, anti-phase boundaries, anti-site point defects and swap defects. We show that the experimentally measured coordinates and chemical species with 22 picometre precision can be used as direct input for DFT calculations of material properties such as atomic spin and orbital magnetic moments and local magnetocrystalline anisotropy. This work combines 3D atomic structure determination of crystal defects with DFT calculations, which is expected to advance our understanding of structure–property relationships at the fundamental level.

Regional climate modeling addresses our need to understand and simulate climatic processes and phenomena unresolved in global models. This paper highlights examples of current approaches to and innovative uses of regional climate modeling that deepen understanding of the climate system. High-resolution models are generally more skillful in simulating extremes, such as heavy precipitation, strong winds, and severe storms. In addition, research has shown that finescale features such as mountains, coastlines, lakes, irrigation, land use, and urban heat islands can substantially influence a region’s climate and its response to changing forcings. Regional climate simulations explicitly simulating convection are now being performed, providing an opportunity to illuminate new physical behavior that previously was represented by parameterizations with large uncertainties. Regional and global models are both advancing toward higher resolution, as computational capacity increases. However, the resolution and ensemble size necessary to produce a sufficient statistical sample of these processes in global models has proven too costly for contemporary supercomputing systems. Regional climate models are thus indispensable tools that complement global models for understanding physical processes governing regional climate variability and change. The deeper understanding of regional climate processes also benefits stakeholders and policymakers who need physically robust, high-resolution climate information to guide societal responses to changing climate. Key scientific questions that will continue to require regional climate models, and opportunities are emerging for addressing those questions.

Although of great interest for science and resource utilization, the Moon's permanently shadowed regions (PSRs) near each pole present difficult targets for remote sensing. The Lyman Alpha Mapping Project (LAMP) instrument on the Lunar Reconnaissance Orbiter (LRO) mission is able to map PSRs at far‐ultraviolet (FUV) wavelengths using two faint sources of illumination from the night sky: the all‐sky Ly α glow produced as interplanetary medium (IPM) H atoms scatter the Sun's Ly α emissions, and the much fainter source from UV‐bright stars. The reflected light from these two sources produces only a few hundred events per second in the photon‐counting LAMP instrument, so building maps with useful signal‐to‐noise (SNR) ratios requires the careful accumulation of the observations from thousands of individual LRO orbits. In this paper we present the first FUV albedo maps obtained by LAMP of the Moon's southern and northern polar regions. The results show that (1) most PSR regions are darker at all FUV wavelengths, consistent with their surface soils having much larger porosities than non‐PSR regions (e.g., ∼70% compared to ∼40% or so), and (2) most PSRs are somewhat “redder” (i.e., more reflective at the longer FUV wavelengths) than non‐PSR regions, consistent with the presence of ∼1–2% water frost at the surface. Key Points New FUV albedo maps of the Moon's poles are presented Most permanently shadowed regions (PSRs) have low FUV albedos Most PSRs are relatively red at long FUV wavelengths