A Robust Protocol to Increase NimbleGen SeqCap EZ Multiplexing Capacity to 96 Samples

Contemporary genetic studies frequently involve sequencing of a targeted gene panel, for instance consisting of a set of genes associated with a specific disease. The NimbleGen SeqCap EZ Choice kit is commonly used for the targeted enrichment of sequencing libraries comprising a target size up to 7 Mb. A major drawback of this commercially available method is the exclusive use of single-indexing, meaning that at most 24 samples can be multiplexed in a single reaction. In case of relatively small target sizes, this will lead to excessive amounts of data per sample. We present an extended version of the NimbleGen SeqCap EZ protocol which allows to robustly multiplex up to 96 samples. We achieved this by incorporating Illumina dual-indexing based custom adapters into the original protocol. To further extend the optimization of cost-efficient sequencing of custom target panels, we studied the effect of higher pre-enrichment pooling factors and show that pre-enrichment pooling of up to 12 samples does not affect the quality of the data. To facilitate evaluation of capture efficiency in custom design panels, we also provide a detailed reporting tool.     

Antioxidant potential of microalgae in relation to their phenolic and carotenoid content

In the past decades, food scientists have been searching for natural alternatives to replace synthetic antioxidants. In order to evaluate the potential of microalgae as new source of safe antioxidants, 32 microalgal biomass samples were screened for their antioxidant capacity using three antioxidant assays, and both total phenolic content and carotenoid content were measured. Microalgae were extracted using a one-step extraction with ethanol/water, and alternatively, a three-step fractionation procedure using successively hexane, ethyl acetate, and water. Antioxidant activity of the extracts varied strongly between species and further depended on growth conditions and the solvent used for extraction. It was found that industrially cultivated samples of Tetraselmis suecica, Botryococcus braunii, Neochloris oleoabundans, Isochrysis sp., Chlorella vulgaris, and Phaeodactylum tricornutum possessed the highest antioxidant capacities in this study and thus could be a potential new source of natural antioxidants. The results from the different types of extracts clearly indicated that next to the well-studied carotenoids, phenolic compounds also contribute significantly to the antioxidant capacity of microalgae.     

Physical and chemical limnology of alpine lakes and pools in the Rwenzori Mountains (Uganda–DR Congo)

This study describes the physical and chemical properties of 17 Afroalpine lakes (>2 m deep) and 11 pools (<2 m deep) in the Rwenzori mountains, Uganda-DR Congo, with the aim to establish the baseline conditions against which to evaluate future environmental and biological changes in these unique tropical ecosystems, and to provide the foundation for lake-based paleoenvironmental studies. Most Rwenzori lakes are located above 3,500 m elevation, and dilute (5–52 μS/cm specific conductance at 25°C) open systems with surface in- and outflow. Multivariate ordination and pairwise correlations between environmental variables mainly differentiate between (1) lakes located near or above 4,000 m (3,890–4,487 m), with at least some direct input of glacial meltwater and surrounded by rocky catchments or alpine vegetation; and (2) lakes located mostly below 4,000 m (2,990–4,054 m), remote from glaciers and surrounded by Ericaceous vegetation and/or bogs. The former group are mildly acidic to neutral clear-water lakes (surface pH: 5.80–7.82; Secchi depth: 120–280 cm) with often above-average dissolved ion concentrations (18–52 μS/cm). These lakes are (ultra-) oligotrophic to mesotrophic (TP: 3.1–12.4 μg/l; Chl-a: 0.3–10.9 μg/l) and phosphorus-limited (mass TN/TP: 22.9–81.4). The latter group are mildly to strongly acidic (pH: 4.30–6.69) waters stained by dissolved organic carbon (DOC: 6.8–13.6 mg/l) and more modest transparency (Secchi-disk depth: 60–132 cm). Ratios of particulate carbon, particulate nitrogen and chlorophyll a in these lakes indicate that organic matter in suspension is primarily derived from the lakes’ catchments rather than aquatic primary productivity. Since key features in the Rwenzori lakes’ abiotic environment are strongly tied to temperature and catchment hydrology, these Afroalpine lake ecosystems can be expected to respond sensitively to climate change and glacier melting. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Leptin Levels,Leptin Receptor Gene Polymorphisms,and Energy Metabolism in Women

Objective: Resting metabolic rate (RMR) is mainly determined by fat‐free mass and additionally by age, sex, hormones, and possibly genetic differences. We evaluated whether leptin levels and polymorphisms in the leptin receptor (LEPR) gene were associated with energy expenditure phenotypes. Methods: RMR, body composition, and leptin levels were measured in 125 overweight and obese women. Three LEPR polymorphisms, Lys109Arg, Gln223Arg, and Lys656Asn, were typed on genomic DNA of another group of 192 women in whom RMR was measured. Fat, protein, and carbohydrate oxidation were calculated for 103 of these subjects. In 38 subjects, glucose‐induced thermogenesis was measured over 3 hours. Results: In the first study group, a negative correlation between RMR and leptin levels was found after controlling for fat and fat‐free mass. In multiple regression analysis, leptin contributed significantly to RMR, independent of body composition. In the second study group, RMR was not associated with LEPR polymorphisms. Differences in substrate oxidation rates were found among genotypes at the Lys656Asn site. In fasting conditions, Lys656Lys showed a trend to oxidize more carbohydrates and less fat than Asn656 carriers, a trend which became significant after the glucose load when carbohydrate oxidation rate in Lys656Lys was 15% higher than in Asn656 carriers (p = 0.04), and fat oxidation rate was 44% lower (p = 0.02). Discussion: These results suggest that DNA sequence variations in the LEPR gene could affect substrate oxidation. We hypothesize that this might be caused by differences in glucose levels, leading to differences in glucose oxidation rates.     

Dimethylarginine Levels in Cerebrospinal Fluid of Hyperacute Ischemic Stroke Patients are Associated with Stroke Severity

We hypothesise that asymmetric and symmetric dimethylarginine (ADMA, SDMA) are released in cerebrospinal fluid (CSF) due to ischemia-induced proteolysis and that CSF dimethylarginines are related to stroke severity. ADMA and SDMA were measured in CSF of 88 patients with ischemic stroke or TIA within 24 h after stroke onset (mean 8.6 h) and in 24 controls. Stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS) score at admission. Outcome was evaluated by institutionalization due to stroke and the modified Rankin scale. Dimethylarginine levels were higher in patients with stroke than in TIA patients, who had higher levels than controls and correlated with the NIHSS. Logistic regression analysis confirmed that dimethylarginines were independently associated with stroke severity. The SDMA/ADMA ratio did not differ significantly between controls and stroke patients. CSF dimethylarginine levels are increased in hyperacute ischemic stroke and are associated with stroke severity. R. Brouns is a research assistant of the Fund for Scientific research Flanders (FWO-Vlaanderen).     

Uptake of horseradish peroxidase in spontaneous adenomas of the rat pituitary

The uptake of horseradish peroxidase was investigated by electron microscopy in 9 non-tumorous adenohypophyses and 13 spontaneous pituitary adenomas of adult female Long-Evans rats. Pituitary adenoma cells retained their ability for endocytosis and exhibited more extensive deposition of horseradish peroxidase than non-tumorous adenohypophysial cells.     

Interspecific hybridization between greater kudu and nyala

Hybridization of wildlife species, even in the absence of introgression, is of concern due to wasted reproductive effort and a reduction in productivity. In this study we detail an accidental mating between a female nyala (Tragelaphus angasii) and a male greater kudu (T. strepsiceros). The hybrid was phenotypically nyala and was identified as such based on mitochondrial DNA. Further genetic analysis based on nine microsatellite markers, chromosome number and chromosome morphology however, confirmed its status as an F1 hybrid. Results obtained from a reproductive potential assessment indicated that this animal does not have the potential to breed successfully and can be considered as sterile.     

Epiplastidial tubules inOedogonium cardiacum

Summary Rope-like structures with a diameter of about 30×50 nm occur at the cytoplasmic site of the chloroplast envelope ofOedogonium cardiacum. They seem to consist of a central tubule with surrounding electron-dense fuzzy material. The possible significance of these structures is discussed.