Investigation of a genome wide association signal for obesity: synthetic association and haplotype analyses at the melanocortin 4 receptor gene locus

Background

Independent genome-wide association studies (GWAS) showed an obesogenic effect of two single nucleotide polymorphisms (SNP; rs12970134 and rs17782313) more than 150 kb downstream of the melanocortin 4 receptor gene (MC4R). It is unclear if the SNPs directly influence MC4R function or expression, or if the SNPs are on a haplotype that predisposes to obesity or includes functionally relevant genetic variation (synthetic association). As both exist, functionally relevant mutations and polymorphisms in the MC4R coding region and a robust association downstream of the gene, MC4R is an ideal model to explore synthetic association.

Methodology/Principal Findings

We analyzed a genomic region (364.9 kb) encompassing the MC4R in GWAS data of 424 obesity trios (extremely obese child/adolescent and both parents). SNP rs12970134 showed the lowest p-value (p = 0.004; relative risk for the obesity effect allele: 1.37); conditional analyses on this SNP revealed that 7 of 78 analyzed SNPs provided independent signals (p≤0.05). These 8 SNPs were used to derive two-marker haplotypes. The three best (according to p-value) haplotype combinations were chosen for confirmation in 363 independent obesity trios. The confirmed obesity effect haplotype includes SNPs 3′ and 5′ of the MC4R. Including MC4R coding variants in a joint model had almost no impact on the effect size estimators expected under synthetic association.

Conclusions/Significance

A haplotype reaching from a region 5′ of the MC4R to a region at least 150 kb from the 3′ end of the gene showed a stronger association to obesity than single SNPs. Synthetic association analyses revealed that MC4R coding variants had almost no impact on the association signal. Carriers of the haplotype should be enriched for relevant mutations outside the MC4R coding region and could thus be used for re-sequencing approaches. Our data also underscore the problems underlying the identification of relevant mutations depicted by GWAS derived SNPs.     

Systematic construction of a conceptual minimal model of plasma cholesterol levels based on knockout mouse phenotypes

Elevated plasma cholesterol, a well-known risk factor for cardiovascular diseases, is the result of the activity of many genes and their encoded proteins in a complex physiological network. We aim to develop a minimal kinetic computational model for predicting plasma cholesterol levels. To define the scope of this model, it is essential to discriminate between important and less important processes influencing plasma cholesterol levels. To this end, we performed a systematic review of mouse knockout strains and used the resulting dataset, named KOMDIP, for the identification of key genes that determine plasma cholesterol levels. Based on the described phenotype of mouse knockout models, 36 of the 120 evaluated genes were marked as key genes that have a pronounced effect on the plasma cholesterol concentration. The key genes include well-known genes, e.g., Apoe and Ldlr, as well as genes hardly linked to cholesterol metabolism so far, e.g., Plagl2 and Slc37a4. Based on the catalytic function of the genes, a minimal conceptual model was defined. A comparison with nine conceptual models from literature revealed that each of the individual published models is less complete than our model. Concluding, we have developed a conceptual model that can be used to develop a physiologically based kinetic model to quantitatively predict plasma cholesterol levels.     

Distinct Subcellular Localization of a Group of Synaptobrevin-Like SNAREs in Paramecium tetraurelia and Effects of Silencing SNARE-Specific Chaperone NSF

We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named Paramecium tetraurelia synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endo- or phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal. NSF silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan.Paramecium tetraurelia is a unicellular organism that belongs to the ciliated protozoans and, thus, to the phylum Alveolata, which also comprises dinoflagellates and apicomplexans, such as the human pathogens Toxoplasma and Plasmodium. Like those organisms, Paramecium has to perform within one cell all functions that are normally shared between different cell types in multicellular organisms. Accordingly complex are the cytoskeletal anatomy (1), food uptake and processing (20), and membrane trafficking pathways (47). This complexity is mirrored in the mere size of the genome, with ∼39,500 protein-coding genes (8). On this background we shall describe new genes and proteins—SNAREs, as defined below—of a superfamily contributing to specific membrane interactions. Together with previous studies (37, 52, 53) we may have now identified most of the SNARE genes in Paramecium. The large number of putative SNARE genes in Paramecium was unexpected and is similar to that in flowering plants (41) and mammals (39).P. tetraurelia is a freshwater filter feeder that lives on bacteria and other small unicellular organisms. Food particles are transported into the oral cavity, first to the cytostome by action of cilia and then concentrated in the cytopharynx, where they are packaged into the nascent food vacuole. In parts of the oral cavity cilia display special arrangements, such as two peniculi and a quadrulus, and oral fibers emanate as rails for vesicle trafficking (3, 20). Vesicles of different sizes and origins travel close to the oral cavity and are frequently associated with the structures just mentioned. Once the food vacuole reaches a certain size, the nascent food vacuole is pinched off the cytopharynx and takes a defined route through the cytoplasm of the cell, termed cytoplasmic streaming or cyclosis (2), which is supported by specialized microtubule structures (54). Vesicles of an ∼0.8-μm size (acidosomes) situated at the site of food vacuole formation at the cytopharynx fuse with the nascent food vacuole after it has detached from the cytopharynx, and they drastically lower the pH of the phagosome lumen (48). This may kill food bacteria, and it initiates a series of events leading to fusion of the digestive vacuole (phagosome) with lysosomes that deliver digestive enzymes for breakdown of digestible vacuole contents (20). The whole cycle of digestion is completed after ∼20 min. Membranes and digestive enzymes are recycled from the digestive vacuole, and undigested waste products are excreted by fusion of the digestive vacuole at a specialized site on the cell surface, the cytoproct (2, 3). The membrane of the defecated vacuole is retrieved as ∼100-nm discoidal vesicles and transported back along microtubular ribbons to the cytostome (2).The whole cortex of Paramecium is a highly ordered structure with regularly arranged organelles (46). Soluble substances are ingested via permanent, regularly arranged ∼0.1-μm large indentations at the cell surface, called parasomal sacs. These have a clathrin coat on their cytoplasmic side and correspond via small trafficking vesicles with the regularly arranged stationary early endosomes (terminal cisternae) situated beneath each ciliary basal body (3). There, different cargos are sorted into 100-nm vesicles that join the digestive pathways described above.Paramecium also possesses dense core secretory vesicles called trichocysts, which are also regularly arranged in a fusion-competent stage at the cell surface. Each trichocyst docking site is surrounded by cortical calcium stores (alveolar sacs) (46). Trichocysts originate from the endoplasmic reticulum (ER) and undergo several stages of maturation until they achieve exocytosis competence (28).Besides trichocysts and parasomal sacs (which may also participate in constitutive exocytosis [19]), no other sites of membrane delivery to the cell membrane are known up to now, as documented in the electron microscope (EM) image gallery presented by R. D. Allen at the website http:/www5.pbrc.hawaii.edu/allen/.Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are of central importance to all membrane trafficking and have been found in every eukaryotic lineage investigated so far (25, 32, 39).The N-ethylmaleimide sensitive factor (NSF) is a SNARE-specific chaperone (32). SNARE molecules form a quarternary complex (SNARE complex) when they assemble to mediate membrane docking for subsequent fusion, with each one of the SNAREs containing one SNARE domain (synaptobrevin or syntaxin) or two (due to backfolding, as in SNAP-25 or related proteins; see below). The crystal structures of different SNARE complexes revealed conserved features that are now believed to be universal in all SNARE complexes (7, 17, 57, 63). Of the four SNARE helices forming a highly stable SNARE complex, three usually carry a glutamine (Q) residue and one carries an arginine (R) at the center of the SNARE domain; this is known as the “3Q + 1R rule” (17). Accordingly, SNAREs have been classified as Q- and R-SNAREs, and this nomenclature usually reflects the evolutionary origin of different types of SNARE proteins better than the old classification, as v- (vesicle) and t- (target) SNAREs. Q-SNAREs can be further subdivided into Qa-, Qb-, and Qc-SNAREs, with Qa-SNAREs often designated as syntaxins, whereas Qb- and Qc-SNAREs can be distinct proteins or both these SNARE domains can reside within a single protein, as in the case of the synaptosomal-associated protein of 25 kDa (SNAP-25). R-SNAREs, like synaptobrevins or the tetanus toxin-insensitive vesicle-associated membrane protein (TI-VAMP), are often situated on the vesicle side and have been subdivided, referring to their length, into brevins and longins (with a longer N-terminal cytosolic stretch). Because the tetanus toxin-sensitive brevins so far have been found only in metazoans and yeast, the more widespread tetanus toxin-insensitive longins have to be considered the ancestral R-SNAREs. Longins are characterized by their conserved longin domain structure, a fold that is similar to a profilin-like fold (18, 49, 50). As found with Sec22, a longin occurring not only in organisms from yeast to mammals but also in Paramecium (37), the longin domain, depending on its folding state, contributes to vesicle formation in the endoplasmic reticulum and further targeting (44).There are exceptions to some of these rules, e.g., there are SNAREs with a central amino acid other than an R (or Q) residue in the zero layer. Nevertheless, the repetitive arrangement of typical amino acids (heptad repeats, relevant for SNARE complex formation) around the zero layer, as characteristic of a SNARE domain, in combination with additional criteria, still allows one to identify such proteins as SNAREs. We have used a bioinformatic approach in the present work (see below).We previously identified a set of R-SNAREs (53), Q-SNAREs (37), and a SNAP-25 homolog (52) in P. tetraurelia. Here, we identified by sequence homology, either of defined domains or of the overall structure, a group of related synaptobrevin-like SNAREs which we investigated in more detail, including their subcellular localization. In contrast to the Paramecium R-SNAREs previously described (53), those newly described here all have an unorthodox amino acid, Asp or rarely His, in the zero layer of their SNARE domain, and only two of them possess a longin domain. We found that all these new SNAREs show distinct subcellular localizations, and we found that a great number of them are associated with food vacuole processing or endosomal trafficking. Some of the synaptobrevin-like SNAREs investigated here show an identical distribution pattern, as previously found for specific Qa-SNAREs (37), and thus they could be constituents of the same SNARE complexes.     

Symmetric array of the urothelium surface controlled by the lipid lattice composition

Proteins on the luminal surface of the mammalian membrane urothelium form an hexagonal two-dimensional (2D) lattice of approximately 12nm particles embedded in a lipid matrix. The establishment and/or maintenance of this two-dimensional lattice have not yet been elucidated. To understand the lipid involvement in these effects, which may have a potential role on the permeability barrier, the lattice structural changes induced by different dietary fatty acids were analyzed. Image analysis of the uranyl formate stained urothelial membrane from mice fed with a commercial diet (control) or a formula containing 5% (w/w) corn oil (CO) or olein (O) and our previous data of fluorescence anisotropy indicated that the olein diet profoundly alters the two-dimensional particle network. Besides the increase from 15nm (control) to 17nm center-to-center particle, a statistically significant increase of the particle size was induced by the olein diet. The same parameters on urothelial membranes from animals fed with a corn diet differed on the long-range super-array from the control values. A specific structural change of the urothelial plaque particle organization as a response to changes of lipid composition is described for the first time. These results are a starting point for further research of the lipid implications on the permeability barrier.     

Differential induction of electrophile-responsive element-regulated genes by n-3 and n-6 polyunsaturated fatty acids

In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.     

Application of a continuous bioreactor cascade to study the effect of linoleic acid on hybridoma cell physiology

The aim of the present study is to demonstrate the use of controlled bioreactors for toxicological studies. As a model system the effect of linoleic acid on hybridoma cells is studied in two well-controlled continuously operated bioreactors placed in series. In the first reactor the effect on rapid proliferating cells can be studied, while in the second reactor a special steady state is created, which allows studying the effect on apoptotic cells. Experiments are done at 0, 25, and 50 microM linoleic acid. At the end of the experiment with 50 microM linoleic acid, the concentration of linoleic acid is increased stepwise to determine the cytotoxic level. For rapid proliferating cells exposed to 25 and 50 microM stimulation of growth was observed. At 50 microM there was at the same time an increase in cell death through apoptosis. For stressed apoptotic cells linoleic acid caused partial growth inhibition at 25 and 50 microM and arrest of cell proliferation in the G(2)/M phase at 50 microM. For both, rapid proliferating cells and stressed apoptotic cells, complete growth inhibition occurred at 85 microM, with cells being arrested in the G(2)/M phase and dying mainly through necrosis. Cells in the bioreactor system appeared to be more sensitive towards linoleic acid than cells grown in multi-well plates. (IC(50) = 300 microM; IC(100) = 400 microM). Altogether the results of the present study reveal that the biostat experiments allow detailed analysis of the effect of a bioactive ingredient on cell physiology and behavior.     

Stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria

Stable carbon isotope fractionation during dichloromethane (DCM) degradation by methylotrophic bacteria was investigated under aerobic and nitrate-reducing conditions. The strains studied comprise several Hyphomicrobium strains, Methylobacterium, Methylopila, Methylophilus and Methylorhabdus spp. that are considered to degrade DCM by a glutathione (GSH)-dependent dehalogenase enzyme system in the initial step. The stable carbon isotope fractionation factors (alphaC) of the strains varied under aerobic conditions between 1.043 and 1.071 and under nitrate-reducing conditions between 1.048 and 1.065. Comparison of isotope fractionation under aerobic and nitrate-reducing conditions by individual strains revealed only minor to no differences. The variability in isotope fractionation among strains was found to be related to the polymorphism of the functional genes encoding the DCM dehalogenase.     

The minotaur proteome: Avoiding cross‐species identifications deriving from bovine serum in cell culture models

Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5–15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC‐MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented.     

Integrating diversity of smallholder coffee cropping systems in environmental analysis

The International Journal of Life Cycle Assessment - Coffee represents an important trade asset internationally. Around 70% of global coffee production is provided by 25 million smallholders...     

Cortisol Awakening Response Is Linked to Disease Course and Progression in Multiple Sclerosis

Objectives

Dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis has frequently been reported in multiple sclerosis (MS). So far, HPA axis function in MS has predominantly been studied under pharmacological stimulation which is associated with a series of methodological caveats. Knowledge of circadian cortisol patterns and cortisol awakening response (CAR) is still limited.

Methods

A total of 77 MS patients (55 relapsing-remitting MS (RRMS)/22 secondary-progressive MS (SPMS)) as well as 34 healthy control (HC) subjects were enrolled. Diurnal cortisol release was assessed by repeated salivary cortisol sampling. Neurological disability was rated by the Kurtzke’s Expanded Disability Status Scale (EDSS). Depressive symptoms and perceived stress were assessed by self-report measures.

Results

RRMS but not SPMS patients differed in circadian cortisol release from HC subjects. Differences in cortisol release were restricted to CAR. Treated and treatment naïve RRMS patients did not differ in CAR. In a RRMS follow-up cohort (nine months follow-up), RRMS patients with EDSS progression (≥0.5) expressed a significantly greater CAR compared to HC subjects. RRMS patients with a stable EDSS did not differ from HC subjects. Neither depressive symptoms nor perceived stress ratings were associated with CAR in RRMS patients. In a step-wise regression analysis, EDSS at baseline and CAR were predictive of EDSS at follow-up (R² = 67%) for RRMS patients.

Conclusions

Circadian cortisol release, in particular CAR, shows a course specific pattern with most pronounced release in RRMS. There is also some evidence for greater CAR in RRMS patients with EDSS progression. As a consequence, CAR might be of predictive value in terms of neurological disability in RRMS patients. The possible role of neuroendocrine-immune interactions in MS pathogenesis is further discussed.