Stable isotopes identify the natal origins of a generalist brood parasite, the brown-headed cowbird Molothrus ater

Identifying the natal origins of brood parasites is a major challenge that usually requires labor-intensive searching for nests of host species. Stable isotope analysis of feathers and other body tissues of parasitic young could be a possible tool for determining natal origins if tissues reflect the isotopic composition of the diet fed to nestlings. We measured the carbon (¹³C) and nitrogen (¹⁵N) isotope compositions of feathers for two age-classes of brown-headed cowbirds Molothrus ater at the Konza Prairie Biological Station near Manhattan, Kansas: nestlings raised by five species of songbird hosts in two different habitats, and juveniles captured after independence. Isotope values from cowbird nestlings did not differ among host species and we were unable to assign juvenile cowbirds to their natal hosts. However, nestlings raised in grassland habitat had feathers that contained significantly higher δ¹³C values and lower δ¹⁵N values than nestlings raised in shrub habitats. In addition, independent juveniles had isotopic signatures that were similar to cowbird nestlings raised on shrub habitats. Although dickcissel Spiza americana comprised the majority of samples from shrub habitats, our conclusions reflect the natural pattern of parasitism at the site and should be representative of cowbirds raised at Konza. We conclude that stable isotope analysis of feathers is effective for determining the natal origins of parasitic young if isotope values from nestlings are isotopically distinct among habitats.     

Habitat‐linked genetic structure for white‐crowned sparrow (Zonotrichia leucophrys): Local factors shape population genetic structure

Ecological, environmental, and geographic factors all influence genetic structure. Species with broad distributions are ideal systems because they cover a range of ecological and environmental conditions allowing us to test which components predict genetic structure. This study presents a novel, broad geographic approach using molecular markers, morphology, and habitat modeling to investigate rangewide and local barriers causing contemporary genetic differentiation within the geographical range of three white‐crowned sparrow (Zonotrichia leucophrys) subspecies: Z. l. gambelii, Z. l. oriantha, and Z. l. pugetensis. Three types of genetic markers showed geographic distance between sampling sites, elevation, and ecosystem type are key factors contributing to population genetic structure. Microsatellite markers revealed white‐crowned sparrows do not group by subspecies, but instead indicated four groupings at a rangewide scale and two groupings based on coniferous and deciduous ecosystems at a local scale. Our analyses of morphological variation also revealed habitat differences; sparrows from deciduous ecosystems are larger than individuals from coniferous ecosystems based on principal component analyses. Habitat modeling showed isolation by distance was prevalent in describing genetic structure, but isolation by resistance also had a small but significant influence. Not only do these findings have implications concerning the accuracy of subspecies delineations, they also highlight the critical role of local factors such as habitat in shaping contemporary population genetic structure of species with high dispersal ability.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

Metabolomic Derangements Are Associated with Mortality in Critically Ill Adult Patients

Objective

To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.

Rationale

Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.

Methods

We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.

Results

We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).

Conclusion

Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.     

Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.