Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.     

Combinatorial Approach for Large-scale Identification of Linked Peptides from Tandem Mass Spectrometry Spectra

The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein–protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides.The study of protein–protein interactions is crucial to understanding how cellular systems function because proteins act in concert through a highly organized set of interactions. Most cellular processes are carried out by large macromolecular assemblies and regulated through complex cascades of transient protein–protein interactions (1). In the past several years numerous high-throughput studies have pioneered the systematic characterization of protein–protein interactions in model organisms (2–4). Such studies mainly utilize two techniques: the yeast two-hybrid system, which aims at identifying binary interactions (5), and affinity purification combined with tandem mass spectrometry analysis for the identification of multi-protein assemblies (6–8). Together these led to a rapid expansion of known protein–protein interactions in human and other model organisms. Patche and Aloy recently estimated that there are more than one million interactions catalogued to date (9).But despite rapid progress, most current techniques allow one to determine only whether proteins interact, which is only the first step toward understanding how proteins interact. A more complete picture comes from characterizing the three-dimensional structures of protein complexes, which provide mechanistic insights that govern how interactions occur and the high specificity observed inside the cell. Traditionally the gold-standard methods used to solve protein structures are x-ray crystallography and NMR, and there have been several efforts similar to structural genomics (10) aiming to comprehensively solve the structures of protein complexes (11, 12). Although there has been accelerated growth of structures for protein monomers in the Protein Data Bank in recent years (11), the growth of structures for protein complexes has remained relatively small (9). Many factors, including their large size, transient nature, and dynamics of interactions, have prevented many complexes from being solved via traditional approaches in structural biology. Thus, the development of complementary analytical techniques with which to probe the structure of large protein complexes continues to evolve (13–18).Recent developments have advanced the analysis of protein structures and interaction by combining cross-linking and tandem mass spectrometry (17, 19–24). The basic idea behind this technique is to capture and identify pairs of amino acid residues that are spatially close to each other. When these linked pairs of residues are from the same protein (intraprotein cross-links), they provide distance constraints that help one infer the possible conformations of protein structures. Conversely, when pairs of residues come from different proteins (interprotein cross-links), they provide information about how proteins interact with one another. Although cross-linking strategies date back almost a decade (25, 26), difficulty in analyzing the complex MS/MS spectrum generated from linked peptides made this approach challenging, and therefore it was not widely used. With recent advances in mass spectrometry instrumentation, there has been renewed interest in employing this strategy to determine protein structures and identify protein–protein interactions. However, most studies thus far have been focused on purified protein complexes. With today''s mass spectrometers being capable of analyzing tens of thousands of spectra in a single experiment, it is now potentially feasible to extend this approach to the analysis of complex biological samples. Researchers have tried to realize this goal using both experimental and computational approaches. Indeed, a plethora of chemical cross-linking reagents are now available for stabilizing these complexes, and some are designed to allow for easier peptide identification when employed in concert with MS analysis (20, 27, 28). There have also been several recent efforts to develop computational methods for the automatic identification of linked peptides from MS/MS spectra (29–36). However, because of the lack of large annotated training data, most approaches to date either borrow fragmentation models learned from unlinked, linear peptides or learn the fragmentation statistics from training data of limited size (30, 37), which might not generalize well across different samples. In some cases it is possible to generate relatively large training data, but it is often very labor intensive and involves hundreds of separate LC-MS/MS runs (36). Here, employing disulfide-bridged peptides as an example, we propose a novel method that uses a combinatorial peptide library to (a) efficiently generate a large mass spectral reference dataset for linked peptides and (b) use these data to automatically train our new algorithm, MXDB, which can efficiently and accurately identify linked peptides from MS/MS spectra.     

Interspecific interactions influence contrasting spatial genetic structures in two closely related damselfly species

Spatial genetic structure (SGS) is largely determined by colonization history, landscape and ecological characteristics of the species. Therefore, sympatric and ecologically similar species are expected to exhibit similar SGSs, potentially enabling prediction of the SGS of one species from that of another. On the other hand, due to interspecific interactions, ecologically similar species could have different SGSs. We explored the SGSs of the closely related Calopteryx splendens and Calopteryx virgo within Finland and related the genetic patterns to characteristics of the sampling localities. We observed different SGSs for the two species. Genetic differentiation even within short distances in C. splendens suggests genetic drift as an important driver. However, we also observed indication of previous gene flow (revealed by a negative relationship between genetic differentiation and increasing potential connectivity of the landscape). Interestingly, genetic diversity of C. splendens was negatively related to density of C. virgo, suggesting that interspecific interactions influence the SGS of C. splendens. In contrast, genetic differentiation between C. virgo subpopulations was low and only exhibited relationships with latitude, pointing to high gene flow, colonization history and range margin effects as the drivers of SGS. The different SGSs of the two ecologically similar species caution indirect inferences of SGS based on ecologically similar surrogate species.     

Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans

Development in marine invertebrate species can take place through a variety of modes and larval forms, but within a species, developmental mode is typically uniform. Poecilogony refers to the presence of more than one mode of development within a single species. True poecilogony is rare, however, and in some cases, apparent poecilogony is actually the result of variation in development mode among recently diverged cryptic species. We used a phylogenetic approach to examine whether poecilogony in the marine polychaete worm, Pygospio elegans, is the result of cryptic speciation. Populations of worms identified as P. elegansooded, and intermediate larvae; these modes are found both within and among populations. We examined sequence variation among partial mitochondrial cytochrome c oxidase subunit I sequences obtained for 279 individual worms sampled across broad geographic and environmental scales. Despite a large number of unique haplotypes (121 haplotypes from 279 individuals), sequence divergence among European samples was low (1.7%) with most of the sequence variation observed within populations, relative to the variation among regions. More importantly, we observed common haplotypes that were widespread among the populations we sampled, and the two most common haplotypes were shared between populations differing in developmental mode. Thus, our results support an earlier conclusion of poecilogony in P elegans. In addition, predominantly planktonic populations had a larger number of population-specific low-frequency haplotypes. This finding is largely consistent with interspecies comparisons showing high diversity for species with planktonic developmental modes in contrast to low diversity in species with brooded developmental modes.     

Inbreeding rate modifies the dynamics of genetic load in small populations

The negative fitness consequences of close inbreeding are widely recognized, but predicting the long-term effects of inbreeding and genetic drift due to limited population size is not straightforward. As the frequency and homozygosity of recessive deleterious alleles increase, selection can remove (purge) them from a population, reducing the genetic load. At the same time, small population size relaxes selection against mildly harmful mutations, which may lead to accumulation of genetic load. The efficiency of purging and the accumulation of mutations both depend on the rate of inbreeding (i.e., population size) and on the nature of mutations. We studied how increasing levels of inbreeding affect offspring production and extinction in experimental Drosophila littoralis populations replicated in two sizes, N = 10 and N = 40. Offspring production and extinction were measured over 25 generations concurrently with a large control population. In the N = 10 populations, offspring production decreased strongly at low levels of inbreeding, then recovered only to show a consistent subsequent decline, suggesting early expression and purging of recessive highly deleterious alleles and subsequent accumulation of mildly harmful mutations. In the N = 40 populations, offspring production declined only after inbreeding reached higher levels, suggesting that inbreeding and genetic drift pose a smaller threat to population fitness when inbreeding is slow. Our results suggest that highly deleterious alleles can be purged in small populations already at low levels of inbreeding, but that purging does not protect the small populations from eventual genetic deterioration and extinction.     

Isolation, bulk cultivation, and characterization of coronary microvascular pericytes: the second most frequent myocardial cell type in vitro

Densely arranged pericytes engird the endothelial tube of all coronary microvessels. Since the experimental access to these abundant cells in situ is difficult, a prerequisite for broader investigation is the availability of sufficient numbers of fully differentiated pericytes in homogenous culture. To reach this goal, we applied strictly standardized cell isolation techniques, optimized culture methods and specific histological staining. Approximately 1,000-fold enriched pericytes were proteolytically detached from highly purified coronary microvascular networks (density gradient centrifugation) of eight mammalian species including human. Addition of species-autologous fetal or neonatal serum (10-20% vol/vol) was a precondition for longer term survival of homogenous pericyte cultures. This ensured optimal growth (doubling time <14 h) and full expression of pericyte-specific markers. In 3-mo, 10(10) pericytes (15 g) could be cultivated from 1 bovine heart. Pericytes could be stored in liquid N(2), recultured, and passaged repeatedly without loss of typical features. In cocultures with EC or vascular smooth muscle cells, pericytes transferred fluorescent calcein to each other and to EC via their antler-like extensions, organized angiogenetic sprouting of vessels, and rapidly activated coagulation factors X and II via tissue factor and prothrombinase. The interconnected pericytes of the coronary system are functionally closely correlated with the vascular endothelium and may play key roles in the adjustment of local blood flow, the regulation of angiogenic processes, and the induction of procoagulatory processes. Their successful bulk cultivation enables direct experimental access under defined in vitro conditions and the isolation of pericyte specific antigens for the production of specific antibodies.     

Wall structures of myocardial precapillary arterioles and postcapillary venules reexamined and reconstructed in vitro for studies on barrier functions

The barrier functions of myocardial precapillary arteriolar and postcapillary venular walls (PCA or PCV, respectively) are of considerable scientific and clinical interest (regulation of blood flow and recruitment of immune defense). Using enzyme histochemistry combined with confocal microscopy, we reexamined the cell architecture of human PCA and PVC and reconstructed appropriate in vitro models for studies of their barrier functions. Contrary to current opinion, the PCA endothelial tube is encompassed not by smooth muscle cells but rather by a concentric layer of pericytes cocooned in a thick, microparticle-containing extracellular matrix (ECM) that contributes substantially to the tightness of the arteriolar wall. This core tube extends upstream into the larger arterioles, there additionally enwrapped by smooth muscle. PCV consist of an inner layer of large, contractile endothelial cells encompassed by a fragile, wide-meshed pericyte network with a weakly developed ECM. Pure pericyte and endothelial cell preparations were isolated from PCA and PCV and grown in sandwich cultures. These in vitro models of the PCA and PCV walls exhibited typical histological and functional features. In both plasma-like (PLM) and serum-containing (SCM) media, the PCA model (including ECM) maintained its low hydraulic conductivity (L(P) = 3.24 ± 0.52·10(-8)cm·s(-1)·cmH(2)O(-1)) and a high selectivity index for transmural passage of albumin (SI(Alb) = 0.95 ± 0.02). In contrast, L(P) and SI(Alb) in the PCV model (almost no ECM) were 2.55 ± 0.32·10(-7)cm·s(-1)·cmH(2)O(-1) and 0.88 ± 0.03, respectively, in PLM, and 1.39 ± 0.10·10(-6)cm·s(-1)·cmH(2)O(-1) and 0.49 ± 0.04 in SCM. With the use of these models, systematic, detailed studies on the regulation of microvascular barrier properties now appear to be feasible.     

A Noninvasive Method for Estimating Nitrogen Balance in Free-Ranging Primates

The vital role of body protein as an energy reserve has received little focus in studies of wild primates. Owing to the relatively low protein content of fruit, some frugivorous primates could face a protein deficit if body protein is catabolized for energy during periods of low fruit availability. Such an imbalance can be detected if fatty acids, amino acids, and nitrogen (N) catabolites are reincorporated or recycled back to tissues. Here we describe a method to quantify protein recycling by measuring standardized urea concentration and N isotope signatures from urine samples collected from wild Bornean orangutans (Pongo pygmaeus wurmbii). Our overall goal was to explore if concentrations of urea and ??¹⁵N values could be used as indicators of the amount of protein consumed and the degree of protein recycling, respectively, in wild, free-ranging primates. We examine the relationships between urea concentration, ??¹⁵N values, protein intake, and fruit availability. Urea concentration increased with fruit availability, reflecting a slight increase in protein consumption when fruit was abundant. However, we found no relationship between ??¹⁵N values and fruit availability, suggesting that orangutans avert a negative protein balance during periods of low fruit availability. These noninvasive methods complement recent advances in primate energy balance research and will contribute to our understanding of adaptations of primates during periods of fruit shortage.