Structural, Biochemical, and Cell Biological Characterization of Rab7 Mutants That Cause Peripheral Neuropathy

Coordinated trafficking of intracellular vesicles is of critical importance for the maintenance of cellular health and homeostasis. Members of the Rab GTPase family serve as master regulators of vesicular trafficking, maturation, and fusion by reversibly associating with distinct target membranes and recruiting specific effector proteins. Rabs act as molecular switches by cycling between an active, GTP-bound form and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Rab7 specifically regulates the trafficking and maturation of vesicle populations that are involved in protein degradation including late endosomes, lysosomes, and autophagic vacuoles. Missense mutations of Rab7 cause a dominantly-inherited axonal degeneration known as Charcot-Marie-Tooth type 2B (CMT2B) through an unknown mechanism. Patients with CMT2B present with length-dependent degeneration of peripheral sensory and motor neurons that leads to weakness and profound sensory loss. To gain insight into the pathogenesis of CMT2B, we undertook extensive characterization of two disease-causing Rab7 mutants, L129F and V162M. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. We further demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect as previously suggested, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Using an unbiased proteomics approach, we characterize effector interactions in wild-type and mutant Rab7 and identify several novel Rab7 interactors. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that increased activation of mutant Rab7 is counterbalanced by unregulated, GTP-hydrolysis-independent membrane cycling. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease by misregulation of native Rab7 activity. Future experiments will address the impact of Rab7 misregulation on neuronal trafficking and trophic signaling

Triangle Tea

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Letter to Wes Daniels regarding award of a professional development Scholarship, February 5, 1991

A letter from Joyce A. McCray Pearson to Wes Daniels thanking the Scholarship Committee for awarding her a Scholarship for professional development

Supernova Remnant 1987A: Opening the Future by Reaching the Past

We report an up-turn in the soft X-ray light curve of supernova remnant (SNR) 1987A in late 2003 (~6200 days after the explosion), as observed with the Chandra X-ray Observatory. Since early 2004, the rapid increase of the 0.5-2 keV band X-ray light curve can no longer be described by the exponential density distribution model with which we successfully fitted the data between 1990 and 2003. Around day ~6200, we also find that the fractional contribution to the observed soft X-ray flux from the decelerated shock begins to exceed that of the fast shock and that the X-ray brightening becomes "global" rather than "spotty". We interpret these results as evidence that the blast wave has reached the main body of the dense circumstellar material all around the inner ring. This interpretation is supported by other recent observations, including a deceleration of the radial expansion of the X-ray remnant, a significant up-turn in the mid-IR intensities, and the prevalence of the optical hot spots around the entire inner ring, all of which occur at around day 6000. In contrast to the soft X-ray light curve, the hard band (3-10 keV) X-ray light curve increases at a much lower rate which is rather similar to the radio light curve. The hard X-ray emission may thus originate from the reverse shock where the radio emission is likely produced. Alternatively, the low increase rate of the hard X-rays may simply be a result of the continuous softening of the overall X-ray spectrum.Comment: AASTex preprint style 12 pages including 1 table and 4 figures, Accepted by ApJ

From phenotype to genotype: issues in navigating the available information resources

pre-printObjectives-As part of an investigation of connecting health professionals and the lay public to both disease and genomic information, we assessed the availability and nature of the data from the Human Genome Project relating to human genetic diseases. Methods-We focused on a set of single gene diseases selected from main topics in MEDLINEplus, the NLM's principal resource focused on consumers. We used publicly available websites to investigate specific questions about the genes and gene products associated with the diseases. We also investigated questions of knowledge and data representation for the information resources and navigational issues. Results-Many online resources are available but they are complex and technical. The major challenges encountered when navigating from phenotype to genotype were (1) complexity of the data, (2) dynamic nature of the data, (3) diversity of foci and number of information resources, and (4) lack of use of standard data and knowledge representation methods. Conclusions-Three major informatics issues arise from the navigational challenges. First, the official gene names are insufficient for navigation of these web resources. Second, navigational inconsistencies arise from difficulties in determining the number and function of alternate forms of the gene or gene product and maintaining currency with this information. Third, synonymy and polysemy cause much confusion. These are severe obstacles to computational navigation from phenotype to genotype, especially for individuals who are novices in the underlying science. Tools and standards to facilitate this navigation are sorely needed

High-Resolution X-ray Spectroscopy of SNR 1987A: Chandra LETG and HETG Observations in 2007

We present an extended analysis of the deep Chandra LETG and HETG observations of the supernova remnant 1987A (SNR 1987A) carried out in 2007. The global fits to the grating spectra show that the temperature of the X-ray emitting plasma in the slower shocks in this system has remained stable for the last three years, while that in the faster shocks has decreased. This temperature evolution is confirmed by the first light curves of strong X-ray emission lines and their ratios. On the other hand, bulk gas velocities inferred from the X-ray line profiles are too low to account for the post-shock plasma temperatures inferred from spectral fits. This suggests that the X-ray emission comes from gas that has been shocked twice, first by the blast wave and again by shocks reflected from the inner ring of SNR 1987A. A new model that takes these considerations into account gives support to this physical picture.Comment: 36 pages, 10 figures, Accepted for publication in Ap

Planned adaptation in risk regulation: An initial survey of US environmental, health, and safety regulation

In principle, we want regulatory programs to be based on current realities, as reflected for example in the best knowledge of relevant experts. That would imply that old rules now on the books should be consistent with today's knowledge base, not just what was known when a rule or standard was originally set. This paper reports on a survey of US programs, examining how often existing rules are actually updated in light of better knowledge, and identifies five programs that attempt to make policy routinely adaptive. These programs exhibit what we term Planned Adaptation: they both revise rules when relevant new knowledge appears, and take steps to produce such improved knowledge. While Planned Adaptation is rare, it is used in several nationally prominent programs, including air pollution, airplane safety, and drug safety. Planned Adaptation is a policy tool that deserves more attention

Water quality impacts of the mountain pine beetle infestation in the Rocky Mountain west

November 2014.Includes bibliographical references.The Mountain Pine Beetle (MPB) is the primary cause of insect-induced mortality in pine forests in western North America where some lodgepole forests have experienced more than 90% tree mortality. The implications of MPB infestation on water resources are particularly important in the Rocky Mountains, which serve as the source-water region for more than 60 million people. Two important potential watershed impacts are changes in the hydrologic cycle and water quality. While impacts on the hydrologic cycle have received some attention, the interconnection between these changes and the impacts of the widespread infestation on water quality are not well understood. This study uses a combination of field sample analysis and modeling based in Rocky Mountain National Park to address two potential MPB-driven effects on water quality: increased metal concentrations with ecotoxicological and human health ramifications and the changes in source water contributions to streamflow with possible implications for metal and carbon transport to downstream drinking water supplies. Previous work from the research team at Colorado School of Mines identified increased potential for disinfection byproduct formation at water treatment plants receiving water from heavily MPB-killed forests. These increases exhibited surprising seasonal trends that suggest that the transport of carbon to streams, and thus the flowpaths of water, may be different in MPB-killed forests. The first question was investigated by sequentially extracting trace metals from soils under trees with vary levels of impact, and using geochemical models to identify important process-level drivers of changes in metal mobility. Laboratory results identify redistribution of metals in soils under beetle-killed trees with greater mobilization potential for cadmium, and increases in zinc and copper, likely related to fluxes from needle leachate. Results also align with geochemical models and identify changes in organic carbon inputs as the primary driver of increased metal mobility. The second questions was addressed using a chemical hydrograph separation approach to partition streamwater into the fractions derived from groundwater, rain, and snow. Results demonstrate that fractional late-summer groundwater contributions from impacted watersheds are approximately 30 ± 15% greater after infestation and when compared with a neighboring watershed that experienced earlier and less-severe attack. Water budget analysis compared to published sap flux and remotely sensing studies reveals that this change is consistent with expected increases in groundwater from loss of transpiration across the watershed. A predictive statistical model (calibrated to observations within and around Rocky Mountain National Park) suggests that dissolved organic carbon concentrations in streams will be higher in areas where tree mortality is higher. Although, a strong statistical correlation was not found with the method used. Ultimately, this study identifies process-level hydrologic and biogeochemical changes that improve understanding of the vulnerability of Rocky Mountain water supplies to MPB outbreaks