Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins

Chemical shift assignment is the first step in all established protocols for structure determination of uniformly labeled proteins by NMR. The explosive growth in recent years of magic-angle spinning (MAS) solid-state NMR (SSNMR) applications is largely attributable to improved methods for backbone and side-chain chemical shift correlation spectroscopy. However, the techniques developed so far have been applied primarily to proteins in the size range of 5–10 kDa, despite the fact that SSNMR has no inherent molecular weight limits. Rather, the degeneracy inherent to many 2D and 3D SSNMR spectra of larger proteins has prevented complete unambiguous chemical shift assignment. Here we demonstrate the implementation of 4D backbone chemical shift correlation experiments for assignment of solid proteins. The experiments greatly reduce spectral degeneracy at a modest cost in sensitivity, which is accurately described by theory. We consider several possible implementations and investigate the CANCOCX pulse sequence in detail. This experiment involves three cross polarization steps, from H to CA[i], CA[i] to N[i], and N[i] to C′[i−1], followed by a final homonuclear mixing period. With short homonuclear mixing times (<20 ms), backbone correlations are observed with high sensitivity; with longer mixing times (>200 ms), long-range correlations are revealed. For example, a single 4D experiment with 225 ms homonuclear mixing time reveals ∼200 uniquely resolved medium and long-range correlations in the 56-residue protein GB1. In addition to experimental demonstrations in the 56-residue protein GB1, we present a theoretical analysis of anticipated improvements in resolution for much larger proteins and compare these results in detail with the experiments, finding good agreement between experiment and theory under conditions of stable instrumental performance.     

Isolation of a gene that is involved in Campylobacter jejuni 81116 cytotoxin activation

Cytotoxin fractions were isolated from Campylobacter jejuni 81116 and semi-purified by size-exclusion liquid chromatography. The fraction showing the strongest toxicity was injected into mice to produce antiserum. The antiserum was used to screen a C. jejuni 81116 cosmid library. Nine genes were identified in overlapping cosmid inserts that induced reactivity with the antiserum. One of these genes showed high similarity to a periplasmic protein of unknown function and its isogenic mutant showed decreased toxicity compared to the C. jejuni 81116 wild type. This gene contains a Gram-negative bacterial RTX toxin-activating protein C signature, which suggests it may play a role in C. jejuni 81116 cytotoxin activation.     

Gene expression variation in Down's syndrome mice allows prioritization of candidate genes

Background  

Down's syndrome (DS), or trisomy 21, is a complex developmental disorder that exhibits many clinical signs that vary in occurrence and severity among patients. The molecular mechanisms responsible for DS have thus far remained elusive. We argue here that normal variation in gene expression in the population contributes to the heterogeneous clinical picture of DS, and we estimated the amplitude of this variation in 50 mouse orthologs of chromosome 21 genes in brain regions of Ts65Dn (a mouse model of DS). We analyzed the RNAs of eight Ts65Dn and eight euploid mice by real-time polymerase chain reaction.     

Incorporating indel information into phylogeny estimation for rapidly emerging pathogens

Background  

Phylogenies of rapidly evolving pathogens can be difficult to resolve because of the small number of substitutions that accumulate in the short times since divergence. To improve resolution of such phylogenies we propose using insertion and deletion (indel) information in addition to substitution information. We accomplish this through joint estimation of alignment and phylogeny in a Bayesian framework, drawing inference using Markov chain Monte Carlo. Joint estimation of alignment and phylogeny sidesteps biases that stem from conditioning on a single alignment by taking into account the ensemble of near-optimal alignments.     

Disturbance persistence in managed grasslands: shifts in aboveground community structure and the weed seed bank

The length of time and form in which disturbances persist in systems depends on the intensity and frequency of disturbance and on the abilities of resident species to recover from such events. In grazed grasslands, trampling by large mammalian herbivores can periodically facilitate weed establishment by exposing patches of bare ground but whether an intense soil disturbance event results in a temporary increase in weed abundance or a persistent weed problem remains unclear. In May 2002, cattle trampling following heavy rain caused severe damage to nine-month old, rotationally grazed, cool-season pastures (Midwest USA). In September 2002, we compared the aboveground composition of paddocks (i.e., fenced pasture sections) that were heavily disturbed to those that received no damage. Relative to undisturbed paddocks, forage species relative cover was 17% lower in disturbed paddocks, and weed species and bare ground relative cover was 61% and 100% higher, respectively. By September 2004, paddock types did not differ in all aboveground community components. However, the abundance and species richness of weed seeds in the soil seed bank averaged respectively 82% and 30% higher in disturbed paddocks between 2003 and 2004. These findings indicate that a spatially extensive, intense soil disturbance event may soon become undetectable in components of aboveground pasture structure but can persist as an augmented weed seed bank. Because of high weed seed bank longevity, disturbances to formerly disturbed pastures would likely result in higher weed recruitment, with more species represented, than in those which lack previous disturbance. Disturbance history may thus be a useful predictor of weed community composition following subsequent disturbance. Based on empirical data supporting this proposition, we recommend that grassland managers explicitly incorporate disturbance history into dynamic management planning and do not rely exclusively on aboveground characters to evaluate the invasion status or colonization potential of an area by undesirable plants. We emphasize that the ecological legacies of past soil disturbance events cannot only influence the contemporary patterns and processes of grasslands, but importantly, affect their compositional trajectories following subsequent perturbation.     

Soil Organic Phosphorus Transformations During Pedogenesis

Abstract Long-term changes in soil phosphorus influence ecosystem development and lead to a decline in the productivity of forests in undisturbed landscapes. Much of the soil phosphorus occurs in a series of organic compounds that differ in their availability to organisms, but changes in the relative abundance of these compounds during pedogenesis remain unknown. We used alkaline extraction and solution phosphorus-31 nuclear magnetic resonance spectroscopy to assess the chemical nature of soil organic phosphorus along a 120,000-year post-glacial chronosequence at Franz Josef, New Zealand. Inositol phosphates, DNA, phospholipids, and phosphonates accumulated rapidly during the first 500 years of soil development characterized by nitrogen limitation of biological productivity, but then declined slowly to low concentrations in older soils characterized by intense phosphorus limitation. However, the relative contribution of the various compounds to the total organic phosphorus varied along the sequence in dramatic and surprising ways. The proportion of inositol hexakisphosphate, conventionally considered to be relatively recalcitrant in the environment, declined markedly in older soils, apparently due to a corresponding decline in amorphous metal oxides, which weather to crystalline forms during pedogenesis. In contrast, the proportion of DNA, considered relatively bioavailable in soil, increased continually throughout the sequence, due apparently to incorporation within organic structures that provide protection from biological attack. The changes in soil organic phosphorus coincided with marked shifts in plant and microbial communities, suggesting that differences in the forms and bioavailability of soil organic phosphorus have ecological significance. Overall, the results strengthen our understanding of phosphorus transformations during pedogenesis and provide important insight into factors regulating the composition of soil organic phosphorus.     

Perception versus polysomnographic assessment of sleep in CFS and non-fatigued control subjects: results from a population-based study

Background  

Complaints of unrefreshing sleep are a prominent component of chronic fatigue syndrome (CFS); yet, polysomnographic studies have not consistently documented sleep abnormalities in CFS patients. We conducted this study to determine whether alterations in objective sleep characteristics are associated with subjective measures of poor sleep quality in persons with CFS.