Association of Polymorphisms of the CHI3L1 Gene with Asthma and Atopy: A Populations-Based Study of 6514 Danish Adults

Background

YKL-40 is a chitinase-like glycoprotein encoded by the chitinase 3-like 1 gene, CHI3L1, localized at chromosome 1q32.1. Increased levels of serum YKL-40 have been reported to be a biomarker for asthma and a reduced lung function. Interestingly, the C-allele of the -131 C→G (rs4950928) polymorphism of CHI3L1 has been shown to associate with bronchial hyperresponsiveness and reduced lung function suggesting that variations in CHI3L1 may influence risk of asthma. The objective of the present study was to investigate the association of common variation in the CHI3L1 locus with asthma, atopy and lung function in a large population-based sample of adults.

Methods/Principal Findings

Eleven single nucleotide polymorphisms (SNPs) of CHI3L1 including rs4950928 were genotyped in 6514 individuals. Asthma was defined as self-reported history of physician-diagnosed asthma. Total IgE and specific IgE to inhalant allergens were measured on serum samples. Lung function was measured by spirometry. Homozygosity of the rs4950928 G allele as compared to homozygosity of the C allele was associated with self-reported physician diagnosed asthma (OR 1.5 (95% CI, 1.00–2.26)) and with prevalence of atopic asthma (OR 1.93 (95% CI, 1.21–3.07)) after adjustment for age, sex, smoking status, socio-economic class and BMI. Carriers of rs883125 G allele had a significantly lower prevalence of atopy (OR 0.82 (CI, 0.72; 0.94)) as compared to homozygosity of the C allele. None of the SNPs examined were significantly associated with FEV1. However, two SNPs (rs10399931and rs4950930) appeared to be significantly associated with FEV₁/FVC-ratio. Subgroup analyses of never-smokers did not consistently influence the associations in an either positively og negatively way.

Conclusions

In contrast to previous studies, the rs4950928 G allele, and not the C allele, was found to be associated with asthma. A few other SNPs of the CHI3L1 was found to be significantly associated with atopy and FEV1/FVC ratio, respectively. Thus, more studies seem warranted to establish the role of CHI3L1 gene in asthma and atopy.     

Enhancing Identifications of Lipid-embedded Proteins by Mass Spectrometry for Improved Mapping of Endothelial Plasma Membranes in Vivo

Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits.The plasma membrane provides a fundamental physical interface between the inside and outside of any cell. Beyond creating a protected compartment with a segregated, distinct, and well controlled internal milieu for the cell, it also mediates a wide variety of basic biological functions including signal transduction, molecular transport, membrane trafficking, cell migration, cell-cell interactions, intercellular communication, and even drug resistance. Plasma membrane-associated proteins, especially integral membrane proteins (IMPs)¹ that traverse the lipid bilayer, are key elements mediating these vital biological processes. Consistent with its fundamental importance in both normal cellular functions and pathophysiology, the plasma membrane has also been targeted extensively for biomarker discovery and drug development. In fact, more than two-thirds of known targets for existing drugs are plasma membrane proteins (1).Despite the potential benefits, profiling the proteome of plasma membranes comprehensively using standard large scale methods including MS-based strategies has been limited and technically quite challenging. Intrinsic hydrophobicity, a wide concentration range of proteins, and other factors have hampered IMP resolution and identification using conventional two-dimensional gel electrophoresis. Gel and gel-free protein separations, including combinations of both, have been reported as an alternative to two-dimensional gel electrophoresis (2–9). Yet most such efforts have focused predominantly on identifying rather soluble proteins from body fluids (i.e. plasma, serum, and cerebrospinal fluid), cell lysates, or cytoplasm. These proteins, unlike IMPs, are relatively abundant and readily susceptible to enzymatic digestion in solution. Various attempts have been made to solubilize and enrich for IMPs, including different detergents, solvents, high pH solutions, and affinity purification (10–22). Even when organellar membranes are enriched through isolation by subcellular fractionation, the yield of proteins identified has been below expectation, especially for multipass transmembrane proteins such as G-protein-coupled receptors.Here we systematically characterize four analytical approaches to enhance the identification of proteins, specifically those embedded in plasma membranes isolated directly from vascular endothelium in rat lung. Endothelial cells (ECs) constitute the tissue-blood interface that controls many important physiological functions, including tissue homeostasis, nutrition, vasomotion, and even drug delivery. In vivo mapping of the EC plasma membrane proteome provides unique opportunities for extending basic understanding in vascular biology and for directing the delivery of therapeutic and imaging agents in vivo (23–25). But it also presents distinct challenges beyond those generally associated with extraction, solubilization, and identification of IMPs in cells and tissues. ECs form a thin monolayer lining each blood vessel. They constitute a very small fraction of all the cells existing in tissue, thereby making it difficult to isolate sufficiently pure EC plasma membrane fractions for proteomics analysis using conventional subcellular fractionation techniques. Although relatively simple to isolate from tissue and grow in culture, ECs require cues from the tissue microenvironment to maintain their tissue-specific qualities and thus undergo rapid and considerable phenotypic drift after isolation (26).We have developed a specialized coating procedure using colloidal silica nanoparticles perfused through the blood vessels of the tissue to isolate luminal plasma membranes of the vascular endothelium as they exist natively in tissue (26–28). Our initial survey of these plasma membranes isolated directly from rat lungs used primarily three standard analytical techniques of the time: two-dimensional electrophoresis, Western analysis, and the shotgun method of two-dimensional liquid chromatography-tandem mass spectrometry (24, 26). We identified 450 proteins of which only ∼15% were IMPs. Although at the time this was a notable total number of proteins, more IMPs are expected. In fact, this large scale 2DC study did not identify several well known EC surface marker proteins, including specific enzymes, adhesion molecules, and growth factor receptors.Here we comparatively analyze four different MS-based strategies involving two- and three-dimensional separation by combining protein prefractionation via SDS-PAGE with in-gel digestion to produce peptides separated by one- and two-dimensional nano-HPLC before seamless and continuous MS analysis. Each method used multiple replicate measurements to comprehensively identify proteins, especially IMPs, and in doing so achieved a clear statistical definition of completeness that permits meaningful comparisons. Ultimately this analysis greatly expanded the EC plasma membranome to 1,833 proteins of which nearly 30% are membrane-embedded.     

Pre‐dispersal seed predators and fungi differ in their effect on Luehea seemannii capsule development,seed germination,and dormancy across two Panamanian forests

Pre‐dispersal seed predation can greatly reduce crop size affecting recruitment success. In addition, non‐fatal damage by seed predators may allow infection by fungi responsible for post‐dispersal seed losses. The objectives of this study were (1) to quantify pre‐dispersal seed predation and fungal infection in a Neotropical tree species, Luehea seemannii, that produces dehiscent fruits and wind‐dispersed seeds, and (2) to link pre‐dispersal effects on seed quality to seed survival in the soil. To examine how seed predators and fungi influence seed losses, mesh exclosures, fungicide, and the combination of both treatments were applied to separate branches in the canopy of trees in Gamboa and Parque Natural Metropolitano (PNM), Panama. To determine if treatments affect seed viability and survival in the soil, half of the seeds collected from each treatment were buried for 4 weeks in forest soils and subsequently allowed to germinate before and after the breaking of dormancy. Overall, 24 percent of developing fruit were lost to insect attack. In contrast, fungi infected only 3 percent of seeds at the pre‐dispersal stage. For seeds germinated directly after collection, fungicide significantly increased germination in the wetter site (Gamboa) but decreased germination in the drier site (PNM). The pre‐dispersal insect exclosure treatment increased the fraction of seeds that remained dormant after burial in the soil. This result suggests that exposure to insect predators may cause physical damage to seeds that results in the loss of physical dormancy but does not necessarily increase the susceptibility of seeds to pathogen attack in the soil.     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Behavioural differences underlie toxicity and predation variation in blooms of Prymnesium parvum

Much of the evolutionary ecology of toxic algal blooms (TABs) remains unclear, including the role of algal toxins in the adaptive ‘strategies’ of TAB-forming species. Most eukaryotic TABs are caused by mixotrophs that augment autotrophy with organic nutrient sources, including competing algae (intraguild predation). We leverage the standing diversity of TABs formed by the toxic, invasive mixotroph Prymnesium parvum to identify cell-level behaviours involved in toxin-assisted predation using direct observations as well as comparisons between genetically distinct low- and high-toxicity isolates. Our results suggest that P. parvum toxins are primarily delivered at close range and promote subsequent prey capture/consumption. Surprisingly, we find opposite chemotactic preferences for organic (prey-derived) and inorganic nutrients between differentially toxic isolates, respectively, suggesting behavioural integration of toxicity and phagotrophy. Variation in toxicity may, therefore, reflect broader phenotypic integration of key traits that ultimately contribute to the remarkable flexibility, diversity, and success of invasive populations.     

Antibody to the ligand for CD40 (gp39) inhibits murine AIDS-associated splenomegaly, hypergammaglobulinemia, and immunodeficiency in disease-susceptible C57BL/6 mice.

Infection of genetically susceptible C57BL/6 mice with the LP-BM5 isolate of murine retroviruses cause profound splenomegaly, hypergammaglobulinemia, lymphadenopathy, and an immunodeficiency syndrome which includes the development of terminal B-cell lymphomas. Because many of these and the other manifestations of LP-BM5 virus-induced disease are similar to those seen in AIDS, this syndrome has been named murine AIDS, or MAIDS. Previous reports have shown that the onset of MAIDS depends on the presence of both CD4+ T cells and B cells and have suggested that CD4+ T-cell-B-cell interactions are important to disease pathogenesis. Here, we assessed the possibility that interactions between CD40 and its ligand on activated CD4+ T cells, CD40 ligand/gp39, are involved in the development of MAIDS. To test this hypothesis, LP-BM5-infected B6 mice were treated in vivo with anti-gp39 monoclonal antibody. As a result, MAIDS-associated splenomegaly, hypergammaglobulinemia, germinal center formation, and the loss of in vitro responsiveness to the T- and B-cell mitogens concanavalin A and lipopolysaccharide were inhibited. Anti-gp39 monoclonal antibody-treated LP-BM5-infected mice were also able to mount essentially normal alloantigen-specific cytolytic T-lymphocyte responses. These results support the possibility that molecular interactions between CD40 and gp39 are critical to the development of MAIDS.     

Population structure and marker�Ctrait association analysis of the US peanut (Arachis hypogaea L.) mini-core collection

Peanut (Arachis hypogaea L.) is one of the most important oilseed and nutritional crops in the world. To efficiently utilize the germplasm collection, a peanut mini-core containing 112 accessions was established in the United States. To determine the population structure and its impact on marker-trait association, this mini-core collection was assessed by genotyping 94 accessions with 81 SSR markers and two functional SNP markers from fatty acid desaturase 2 (FAD2). Seed quality traits (including oil content, fatty acid composition, flavonoids, and resveratrol) were obtained through nuclear magnetic resonance (NMR), gas chromatography (GC), and high-performance liquid chromatography (HPLC) analysis. Genetic diversity and population structure analysis identified four major subpopulations that are related to four botanical varieties. Model comparison with different levels of population structure and kinship control was conducted for each trait and association analyses with the selected models verified that the functional SNP from the FAD2A gene is significantly associated with oleic acid (C18:1), linoleic acid (C18:2), and oleic-to-linoleic (O/L) ratio across this diverse collection. Even though the allele distribution of FAD2A was structured among the four subpopulations, the effect of FAD2A gene remained significant after controlling population structure and had a likelihood-ratio-based R ( 2 ) (R ( LR ) ( 2 ) ) value of 0.05 (oleic acid), 0.09 (linoleic acid), and 0.07 (O/L ratio) because the FAD2A alleles were not completely fixed within subpopulations. Our genetic analysis demonstrated that this peanut mini-core panel is suitable for association mapping. Phenotypic characterization for seed quality traits and association testing of the functional SNP from FAD2A gene provided information for further breeding and genetic research.