Rab and Arl GTPase family members cooperate in the localization of the golgin GCC185

GCC185 is a large coiled-coil protein at the trans Golgi network that is required for receipt of transport vesicles inbound from late endosomes and for anchoring noncentrosomal microtubules that emanate from the Golgi. Here, we demonstrate that recruitment of GCC185 to the Golgi is mediated by two Golgi-localized small GTPases of the Rab and Arl families. GCC185 binds Rab6, and mutation of residues needed for Rab binding abolishes Golgi localization. The crystal structure of Rab6 bound to the GCC185 Rab-binding domain reveals that Rab6 recognizes a two-fold symmetric surface on a coiled coil immediately adjacent to a C-terminal GRIP domain. Unexpectedly, Rab6 binding promotes association of Arl1 with the GRIP domain. We present a structure-derived model for dual GTPase membrane attachment that highlights the potential ability of Rab GTPases to reach binding partners at a significant distance from the membrane via their unstructured and membrane-anchored, hypervariable domains.     

HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE(65) and PXXP(75). The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met(20). How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE(65)-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP(75) to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2(-/-) mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.     

Estimating prevalence and test accuracy in disease ecology: How Bayesian latent class analysis can boost or bias imperfect test results

1. Obtaining accurate estimates of disease prevalence is crucial for the monitoring and management of wildlife populations but can be difficult if different diagnostic tests yield conflicting results and if the accuracy of each diagnostic test is unknown. Bayesian latent class analysis (BLCA) modeling offers a potential solution, providing estimates of prevalence levels and diagnostic test accuracy under the realistic assumption that no diagnostic test is perfect.
2. In typical applications of this approach, the specificity of one test is fixed at or close to 100%, allowing the model to simultaneously estimate the sensitivity and specificity of all other tests, in addition to infection prevalence. In wildlife systems, a test with near‐perfect specificity is not always available, so we simulated data to investigate how decreasing this fixed specificity value affects the accuracy of model estimates.
3. We used simulations to explore how the trade‐off between diagnostic test specificity and sensitivity impacts prevalence estimates and found that directional biases depend on pathogen prevalence. Both the precision and accuracy of results depend on the sample size, the diagnostic tests used, and the true infection prevalence, so these factors should be considered when applying BLCA to estimate disease prevalence and diagnostic test accuracy in wildlife systems. A wildlife disease case study, focusing on leptospirosis in California sea lions, demonstrated the potential for Bayesian latent class methods to provide reliable estimates under real‐world conditions.
4. We delineate conditions under which BLCA improves upon the results from a single diagnostic across a range of prevalence levels and sample sizes, demonstrating when this method is preferable for disease ecologists working in a wide variety of pathogen systems.

     

Effect of hyperoxidized guanine on DNA primer-template structures: Spiroiminodihydantoin leads to strand slippage

Oxidation of guanine in DNA can lead to mutagenic lesions such as 7-hydro-8-oxoguanine (oG). Upon further oxidation, a more mutagenic lesion, spirominodihydantoin (Sp), can occur. In this study, nuclear magnetic resonance (NMR) investigations were performed to determine the structural features of DNA primer-template models with 5′-GG, 5′-G(oG), 5′-G(Sp) and 5′-T(Sp) templates, that mimic the situation in which the downstream G of the template has been oxidized to oG or hyperoxidized to Sp. Our results show that misalignment occurs only in the 5′-G(Sp) and 5′-T(Sp) templates, providing structural insights into the observed differences in mutagenicity of Sp and oG during DNA replication.     

THE THEORETICAL RESPONSE OF LIVING CELLS TO CONTACT WITH SOLID BODIES

The theoretical behavior of a hypothetical fluid cell in contact with flat and curved solid surfaces is discussed from the point of view of surface tension. An equation is derived for calculating the equilibrium position of the cell on a flat surface in terms of the surface tensions between the cell and the plasma, the plasma and the solid surface, and the solid surface and the cell. It is shown that the same equilibrium is predicted from consideration of the contact angle between the cell and the solid body. The relative surface energy has been calculated at various stages in the ingestion of a solid particle by a fluid cell four times as large in diameter, and it is thus shown that no particle will be ingested until the surface tensions are such that the cell would spread to infinity on a flat surface of the same substance. Here again the same equilibrium is predicted from considerations of the contact angle. The adhesiveness of blood cells to solid substances is shown to be a pure surface tension phenomenon, but in most reactions between living cells and solid bodies the fluidity of the protoplasm is also a factor of prime importance. The frequent occurrence of adhesiveness as a property of cells in contact with solid bodies is due in part to the fact that, by so adhering, the surface area of the cell not touching the solid is decreased.     

Ligninolytic system of Phanerochaete chrysosporium: Inhibition by o-Phthalate

The degradation rate of [synthetic-¹⁴C]-lignin to ¹⁴CO₂ by Phanerochaete chrysosporium in cultures buffered with 0.01 M 2,2-dimethylsuccinate (DMS) was twice that in 0.01 M o-phthalate-buffered cultures. This difference could be totally accounted for by o-phthalate inhibition of the activity of the ligninolytic system. ¹⁴CO₂ production from ring-, sidechain-, and methoxyl-labeled lignins was inhibited, the degree of inhibition being dependent on o-phthalate concentration. Oxidations of ¹⁴C-glucose, ¹⁴C-acetovanillone, and ¹⁴C-apocynol were not inhibited; thus o-phthalate is not a general inhibitor, and might inhibit activities involved in attack of the lignin polymer. DMS is a suitable buffer for the ligninolytic system. Degradation rates of ring-labeled lignin to ¹⁴CO₂ of 10–15% in 24 h were obtained consistently over the pH range 3.6–4.5, with an optimum near pH 4.0.Non-Standard Abbreviations DMS dimethylsuccinate