HaploRec: efficient and accurate large-scale reconstruction of haplotypes

Background  

Haplotypes extracted from human DNA can be used for gene mapping and other analysis of genetic patterns within and across populations. A fundamental problem is, however, that current practical laboratory methods do not give haplotype information. Estimation of phased haplotypes of unrelated individuals given their unphased genotypes is known as the haplotype reconstruction or phasing problem.     

Differential direct effects of cyclo-oxygenase-1/2 inhibition on proteoglycan turnover of human osteoarthritic cartilage: an in vitro study

Treatment of osteoarthritis (OA) with nonsteroidal anti-inflammatory drugs (NSAIDs) diminishes inflammation along with mediators of cartilage destruction. However, NSAIDs may exert adverse direct effects on cartilage, particularly if treatment is prolonged. We therefore compared the direct effects of indomethacin, naproxen, aceclofenac and celecoxib on matrix turnover in human OA cartilage tissue. Human clinically defined OA cartilage from five different donors was exposed for 7 days in culture to indomethacin, naproxen, aceclofenac and celecoxib--agents chosen based on their cyclo-oxygenase (COX)-2 selectivity. As a control, SC-560 (a selective COX-1 inhibitor) was used. Changes in cartilage proteoglycan turnover and prostaglandin E2 production were determined. OA cartilage exhibited characteristic proteoglycan turnover. Indomethacin further inhibited proteoglycan synthesis; no significant effect of indomethacin on proteoglycan release was found, and proteoglycan content tended to decrease. Naproxen treatment was not associated with changes in any parameter. In contrast, aceclofenac and, prominently, celecoxib had beneficial effects on OA cartilage. Both were associated with increased proteoglycan synthesis and normalized release. Importantly, both NSAIDs improved proteoglycan content. Inhibition of prostaglandin E2 production indirectly showed that all NSAIDs inhibited COX, with the more COX-2 specific agents having more pronounced effects. Selective COX-1 inhibition resulted in adverse effects on all parameters, and prostaglandin E2 production was only mildly inhibited. NSAIDs with low COX-2/COX-1 selectivity exhibit adverse direct effects on OA cartilage, whereas high COX-2/COX-1 selective NSAIDs did not show such effects and might even have cartilage reparative properties.     

Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures.

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.     

Intra‐ and intermale variability of mature sperm traits analysed in two brackish water populations of the pipefish Syngnathus abaster (Syngnathidae)

Sperm cells are highly diversified in animals, and considerable research effort has focused on variation in sperm morphology among species. Surprisingly, little is known about intraspecific variation in sperm morphology. We analysed within‐ and between‐male variation in mature sperm traits in two brackish water populations of the pipefish Syngnathus abaster. Four morphometric parameters, such as the width and length of the head (including nucleus, and midpiece), length of flagellum and total sperm length were taken into account. The differences in all morphometric parameters analysed between populations were not statistically significant. Moreover, the multidimensional scaling analysis shows that (i) the two populations seem to be indistinguishable based on their spermatozoa and (ii) there is not polymorphism, being sperm not distinguishable into discrete classes both within a single male and between males of each populations. The latter datum does not seem to support the presence of polymorphic sperm in syngnathids. Both populations, however, exhibit a high variation in all sperm traits, both among individual sperm within an ejaculate and among males within each population. The relationship between sperm traits variability and the low selection pressure determined by the absence of postcopulatory sexual selection (i.e. absence of sperm competition) is discussed.     

Using habitat selection theories to predict the spatiotemporal distribution of migratory birds during stopover – a case study of pink‐footed geese Anser brachyrhynchus

Understanding how animals select for habitat and foraging resources therein is a crucial component of basic and applied ecology. The selection process is typically influenced by a variety of environmental conditions including the spatial and temporal variation in the quantity and quality of food resources, predation or disturbance risks, and inter‐ and intraspecific competition. Indeed, some of the most commonly employed ecological theories used to describe how animals choose foraging sites are: nutrient intake maximisation, density‐dependent habitat selection, central‐place foraging, and predation risk effects. Even though these theories are not mutually exclusive, rarely are multiple theoretical models considered concomitantly to assess which theory, or combination thereof, best predicts observed changes in habitat selection over space and time. Here, we tested which of the above theories best‐predicted habitat selection of Svalbard‐breeding pink‐footed geese at their main spring migration stopover site in mid‐Norway by computing a series of resource selection functions (RSFs) and their predictive ability (k‐fold cross validation scores). At this stopover site geese fuel intensively as a preparation for breeding and further migration. We found that the predation risk model and a combination of the density‐dependent and central‐place foraging models best‐predicted habitat selection during stopover as geese selected for larger fields where predation risk is typically lower and selection for foraging sites changed as a function of both distance to the roost site (i.e. central‐place) and changes in local density. In contrast to many other studies, the nutritional value of the available food resources did not appear to be a major limiting factor as geese used different food resources proportional to their availability. Our study shows that in an agricultural landscape where nutritional value of food resources is homogeneously high and resource availability changes rapidly; foraging behaviour of geese is largely a tradeoff between fast refuelling and disturbance/predator avoidance.     

7α-hydroxy-3-oxo-4-cholestenoic acid in cerebrospinal fluid reflects the integrity of the blood-brain barrier

There is a continuous flux of the oxysterol 27-hydroxycholesterol (27-OHC) from the circulation across the blood-brain barrier (BBB) into the brain. The major metabolite of 27-OHC in the brain is 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). We confirm a recent report describing the presence of this metabolite in cerebrospinal fluid (CSF) at a relatively high concentration. A simple and accurate method was developed for assay of 7-HOCA in CSF based on isotope dilution-mass spectrometry and use of ²H₄-labeled internal standard. The concentration of this metabolite was found to be markedly increased in CSF from patients with a dysfunctional BBB. There was a high correlation between the levels of 7-HOCA in CSF and the CSF/serum albumin ratio. The concentration of 7-HOCA in CSF was not significantly affected by neurodegeneration. Our findings suggest that 7-HOCA could be used as a diagnostic marker for conditions with a dysfunctional BBB.     

Microbial players involved in the decline of filamentous and colonial cyanobacterial blooms with a focus on fungal parasitism

In the forthcoming decades, it is widely believed that the dominance of colonial and filamentous bloom‐forming cyanobacteria (e.g. Microcystis, Planktothrix, Anabaena and Cylindrospermopsis) will increase in freshwater systems as a combined result of anthropogenic nutrient input into freshwater bodies and climate change. While the physicochemical parameters controlling bloom dynamics are well known, the role of biotic factors remains comparatively poorly studied. Morphology and toxicity often – but not always – limit the availability of cyanobacteria to filter feeding zooplankton (e.g. cladocerans). Filamentous and colonial cyanobacteria are widely regarded as trophic dead‐ends mostly inedible for zooplankton, but substantial evidence shows that some grazers (e.g. copepods) can bypass this size constraint by breaking down filaments, making the bloom biomass available to other zooplankton species. A wide range of algicidal bacteria (mostly from the Alcaligenes, Flavobacterium/Cytophaga group and Pseudomonas) and viruses (Podoviridae, Siphoviridae and Myoviridae) may also contribute to bloom control, via their lytic activity underpinned by a diverse array of mechanisms. Fungal parasitism by the Chytridiomycota remains the least studied. While each of these biotic factors has traditionally been studied in isolation, emerging research consistently point to complex interwoven interactions between biotic and environmental factors.     

Characterisation of COPD heterogeneity in the ECLIPSE cohort

Background

Chronic obstructive pulmonary disease (COPD) is a complex condition with pulmonary and extra-pulmonary manifestations. This study describes the heterogeneity of COPD in a large and well characterised and controlled COPD cohort (ECLIPSE).

Methods

We studied 2164 clinically stable COPD patients, 337 smokers with normal lung function and 245 never smokers. In these individuals, we measured clinical parameters, nutritional status, spirometry, exercise tolerance, and amount of emphysema by computed tomography.

Results

COPD patients were slightly older than controls and had more pack years of smoking than smokers with normal lung function. Co-morbidities were more prevalent in COPD patients than in controls, and occurred to the same extent irrespective of the GOLD stage. The severity of airflow limitation in COPD patients was poorly related to the degree of breathlessness, health status, presence of co-morbidity, exercise capacity and number of exacerbations reported in the year before the study. The distribution of these variables within each GOLD stage was wide. Even in subjects with severe airflow obstruction, a substantial proportion did not report symptoms, exacerbations or exercise limitation. The amount of emphysema increased with GOLD severity. The prevalence of bronchiectasis was low (4%) but also increased with GOLD stage. Some gender differences were also identified.

Conclusions

The clinical manifestations of COPD are highly variable and the degree of airflow limitation does not capture the heterogeneity of the disease.     

Increased Levels of Markers of Microbial Exposure in Homes with Indoor Storage of Organic Household Waste

As part of environmental management policies in Europe, separate collection of organic household waste and nonorganic household waste has become increasingly common. As waste is often stored indoors, this policy might increase microbial exposure in the home environment. In this study we evaluated the association between indoor storage of organic waste and levels of microbial agents in house dust. The levels of bacterial endotoxins, mold β(1→3)-glucans, and fungal extracullar polysaccharides (EPS) of Aspergillus and Penicillium species were determined in house dust extracts as markers of microbial exposure. House dust samples were collected in 99 homes in The Netherlands selected on the basis of whether separated organic waste was present in the house. In homes in which separated organic waste was stored indoors for 1 week or more the levels of endotoxin, EPS, and glucan were 3.2-, 7.6-, and 4.6-fold higher, respectively (all P < 0.05), on both living room and kitchen floors than the levels in homes in which only nonorganic residual waste was stored indoors. Increased levels of endotoxin and EPS were observed, 2.6- and 2.1-fold (P < 0.1), respectively, when separated organic waste was stored indoors for 1 week or less, whereas storage of nonseparated waste indoors had no effect on microbial agent levels (P > 0.2). The presence of textile floor covering was another major determinant of microbial levels (P < 0.05). Our results indicate that increased microbial contaminant levels in homes are associated with indoor storage of separated organic waste. These increased levels might increase the risk of bioaerosol-related respiratory symptoms in susceptible people.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {type:entrez-nucleotide,attrs:{text:LY294002,term_id:1257998346,term_text:LY294002}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {type:entrez-nucleotide,attrs:{text:U73122,term_id:4098075,term_text:U73122}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.