Skeletal muscle proteomics: carbohydrate metabolism oscillates with seasonal and torpor-arousal physiology of hibernation

The physiology of small mammalian hibernators shifts profoundly over a year, from summer homeothermy to winter heterothermy. Torpor-arousal cycles define high-amplitude tissue activity fluctuations in winter, particularly for skeletal muscle, which contributes to the energetically demanding rewarming process via shivering. To better understand the biochemistry underlying summer-winter and torpor-arousal transitions, we applied two-dimensional gel electrophoresis coupled with liquid chromatography/mass spectrometry/mas spectrometry to the soluble proteins from hindlimb muscle of 13-lined ground squirrels (Ictidomys tridecemlineatus) in two summer and six winter states. Two hundred sixteen protein spots differed by sampled state. Significantly, intrawinter protein adjustment was a minor component of the dataset despite large discrepancies in muscle activity level among winter states; rather, the bulk of differences (127/138 unequivocally identified proteins spots) occurred between summer and winter. We did not detect any proteomic signatures of skeletal muscle atrophy in this hibernator nor any differential seasonal regulation of protein metabolism. Instead, adjustments to metabolic substrate preferences dominated the detected proteomic differences. Pathways of carbohydrate metabolism (glycolysis and gluconeogenesis) were summer enriched, whereas the winter proteome was enriched for fatty acid β-oxidation. Nevertheless, our data suggest that some reliance on carbohydrate reserves is maintained during winter. Phosphoglucomutase (PGM1), which reversibly prepares glucose subunits for either glycolysis or glycogenesis, showed apparent winter state-specific phosphorylation. PGM1 was phosphorylated during rewarming and dephosphorylated by interbout arousal, implying that glucose supplements lipid fuels during rewarming. This, along with winter elevation of TCA cycle enzymes, suggests that hindlimb muscles are primed for rapid energy production and that carbohydrates are an important fuel for shivering thermogenesis.     

Spatial Autocorrelation of Genotypes under Directional Selection

The spatial distributions of genetic variation under selection-mutation equilibrium within populations that have limited dispersal are investigated. The results show that directional selection with moderate strength rapidly reduces the amount of genetic structure and spatial autocorrelations far below that predicted for selectively neutral loci. For the latter, homozygotes are spatially clustered into separate areas or patches, each consisting of several hundred homozygotes. When selection is added the patches of the deleterious homozygotes are much smaller, in the range of 25 to 50 individuals. Selection also reduces temporal correlations. Also investigated are the effects of random replacement processes, such as mutation, immigration, and long-distance migration, on spatial and temporal correlations. The detection of natural selection through spatial pattern analysis is discussed, and applied to data from populations of the morning glory, Ipomoea purpurea.     

Estimating dispersal from short distance spatial autocorrelation

A series of theoretical studies has formed a strong connection between spatial statistics observed in populations and summary measures of the amount of dispersal. Synthesized, these developments allow dispersal to be indirectly estimated from standing spatial patterns of genetic variation under a range of conditions broad enough to be likely met in most populations of either plants or animals. The spatial correlations at the shortest distances are particularly robust to range of conditions and have disproportionately high statistical power. This review integrates theoretical results in a way that maximizes robustness and flexibility in the use of short distance autocorrelation to estimate Wright's neighborhood size, or the total variance in dispersal distances. Empirical guidelines are developed that are meant to be as practical and broad as possible. The guidelines focus on Moran's I-statistics for diploid genotypes converted to allele frequencies, but are also extended to or compared with several other approaches.     

Mutation at high rates reduces spatial structure within populations

The question of whether or not the high rates (mu) of mutation that occur for some hypervariable markers can affect commonly used empirical measures of spatial structure of genetic variation within populations is addressed. The results show that values of these measures are approximately halved when mu is 10(-2). Finest spatial-scale correlations, measured by either Moran's I-statistics or conditional kinship, are reduced by 30%-50%. When the mutation rate is 10 times lower, much smaller reductions result, e.g. averaging 7% for the finest scale correlations. Still smaller orders of magnitude of mu cause negligible changes in spatial structure, where any effects normally would not be detectable. The reductions are caused by forward mutations, and when the reductions are measured as percentages, they are nearly independent of the amount of structure produced sans mutation, except when dispersal is nearly minimal. The percent reductions are also nearly independent of the number of alleles and of back mutations, hence of the nature of the mutation process (e.g. stepwise or not). The results demonstrate that some hypervariable loci should have reduced spatial structuring, and that marker choice may affect the values observed in experimental surveys. Moreover, if fine-scale correlations are used to indirectly estimate dispersal distances, then mutation at high rates could inflate estimates, easily up to two- to three-fold.     

PAHs,OCPs and PCBs in sediments from three catchments in Durban,South Africa

Polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were analysed from sediment samples collected in 2012 from rivers, surface runoff canals and estuaries in three highly urbanised catchments in durban, KwaZulu-Natal, South Africa. PAHs were ubiquitous, at concentrations between 36–6 800 ng g^?1 dry mass (dm). Congener ratio diagnosis suggests the PAHs were derived predominantly from pyrogenic sources. Four OCPs and/or their metabolites were detected at varying frequencies and concentrations. Of these, dichlorodiphenyltrichloroethane (ddT) and metabolites were most frequently detected and were at a high concentration in sediment in some systems. Toxaphene was detected at a high concentration at some sites. The total PCB concentration varied widely, from below the method detection limit to 110 ng g^?1 (dm). Based on the comparison of chemical concentrations to international sediment quality guidelines, PAH, OCP and PCB concentrations in most sediment samples posed a low risk to sediment-dwelling organisms. However, in some instances the risk was moderate or high. It is recommended that these compounds be monitored more frequently and comprehensively in aquatic ecosystems to better understand the ecological and human health implications.     

Conservation Genetics of an Endangered Herb, Hanabusaya asiatica (Campanulaceae)

Abstract: Hanabusaya asiatica (Nakai) Nakai (Campanulaceae), a bee- pollinated, perennial herb, is restricted to the mountainous regions of the eastern-central Korean peninsula. Allozyme analyses for 348 individuals assessed the levels of genetic diversity for five populations. Spatial autocorrelation statistics were also used to examine the spatial distribution of allozyme polymorphisms. The species maintains high levels of allozyme diversity ( H _eS = 0.217) and it exhibits low allozyme differentiation among populations ( G _ST = 0.132) compared with other endemics (mean H _e = 0.096, G _ST = 0.248). There is an apparent pattern of isolation by distance among populations. These results suggest that H. asiatica is at a genetic equilibrium. A considerable deficit in numbers of heterozygotes suggests mating among relatives in populations. At least three populations of H. asiatica should be sampled or conserved to capture or maintain > 99 % of the genetic diversity in the species as a whole. Within local populations, individuals are distributed in a structured, isolation by distance, manner. Approximate genetic patch width in the populations of H. asiatica examined is 5 - 8 m. For conservation purposes, it is suggested that, in general, the sampling of H. asiatica should be conducted at intervals in order to efficiently sample the genetic diversity across an entire population.     

Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics.     

Environmental Nontuberculous Mycobacteria in the Hawaiian Islands

Lung disease caused by nontuberculous mycobacteria (NTM) is an emerging infectious disease of global significance. Epidemiologic studies have shown the Hawaiian Islands have the highest prevalence of NTM lung infections in the United States. However, potential environmental reservoirs and species diversity have not been characterized. In this cross-sectional study, we describe molecular and phylogenetic comparisons of NTM isolated from 172 household plumbing biofilms and soil samples from 62 non-patient households and 15 respiratory specimens. Although non-uniform geographic sampling and availability of patient information were limitations, Mycobacterium chimaera was found to be the dominant species in both environmental and respiratory specimens. In contrast to previous studies from the continental U.S., no Mycobacterium avium was identified. Mycobacterium intracellulare was found only in respiratory specimens and a soil sample. We conclude that Hawai’i’s household water sources contain a unique composition of Mycobacterium avium complex (MAC), increasing our appreciation of NTM organisms of pulmonary importance in tropical environments.     

High Resolution Mapping of Modafinil Induced Changes in Glutamate Level in Rat Brain

Modafinil is marketed in the United States for the treatment of narcolepsy and daytime somnolence due to shift-work or sleep apnea. Investigations of this drug in the treatment of cocaine and nicotine dependence in addition to disorders of executive function are also underway. Modafinil has been known to increase glutamate levels in rat brain models. Proton magnetic resonance spectroscopy (¹HMRS) has been commonly used to detect the glutamate (Glu) changes in vivo. In this study, we used a recently described glutamate chemical exchange saturation transfer (GluCEST) imaging technique to measure Modafinil induced regional Glu changes in rat brain and compared the results with Glu concentration measured by single voxel ¹HMRS. No increases in either GluCEST maps or ¹HMRS were observed after Modafinil injection over a period of 5 hours. However, a significant increase in GluCEST (19±4.4%) was observed 24 hours post Modafinil administration, which is consistent with results from previous biochemical studies. This change was not consistently seen with ¹HMRS. GluCEST mapping allows regional cerebral Glu changes to be measured and may provide a useful clinical biomarker of Modafinil effects for the management of patients with sleep disorders and addiction.