Hygromycin B-selected cell lines from GAL4-regulated pUAST constructs

Here we report the generation of stable, selectable Drosophila S2 cell lines using the UAS-GAL4 system. Cloning of the hygromycin resistance gene into the pUAST vector and cotransfection with other pUAST constructs in S2 cells results in coexpression of up to four different proteins under hygromycin selection. Protein expression is driven by the ubiquitous Actin5C-GAL4 driver and cell cultures are maintained in hygromycin-supplemented, serum-free media to ensure constitutive protein production. Visual comparison of cells cotransfected with GFP and RFP demonstrates a uniform cell population expressing both markers simultaneously, while Western blot analysis shows concurrent expression of MYC3-tagged proteins. In addition, fluorescent cell sorting (FACS) analysis shows that 80% of the total cell population express the GFP marker. Our data indicate that using this technique it is possible to establish stable, selectable cell lines that provide a pool of readily accessible protein. This facilitates protein-based studies and abolishes the need to carry out time-consuming and expensive transfections.     

The PNPLA3 rs738409 G-allele associates with reduced fasting serum triglyceride and serum cholesterol in Danes with impaired glucose regulation

Background and Aim

Non-alcoholic fatty liver disease (NAFLD) is a common condition, associated with hepatic insulin resistance and the metabolic syndrome including hyperglycaemia and dyslipidemia. We aimed at studying the potential impact of the NAFLD-associated PNPLA3 rs738409 G-allele on NAFLD-related metabolic traits in hyperglycaemic individuals.

Methods

The rs738409 variant was genotyped in the population-based Inter99 cohort examined by an oral glucose-tolerance test, and a combined study-sample consisting of 192 twins (96 twin pairs) and a sub-set of the Inter99 population (n = 63) examined by a hyperinsulinemic euglycemic clamp (n _total = 255). In Inter99, we analyzed associations of rs738409 with components of the WHO-defined metabolic syndrome (n = 5,847) and traits related to metabolic disease (n = 5,663). In the combined study sample we elucidated whether the rs738409 G-allele altered hepatic or peripheral insulin sensitivity. Study populations were divided into individuals with normal glucose-tolerance (NGT) and with impaired glucose regulation (IGR).

Results

The case-control study showed no associations with components of the metabolic syndrome or the metabolic syndrome. Among 1,357 IGR individuals, the rs738409 G-allele associated with decreased fasting serum triglyceride levels (per allele effect(β) = −9.9% [−14.4%;−4.0% (95% CI)], p = 5.1×10⁻⁵) and fasting total cholesterol (β = −0.2 mmol/l [−0.3;−0.01 mmol/l(95% CI)], p = 1.5×10⁻⁴). Meta-analyses showed no impact on hepatic or peripheral insulin resistance in carriers of the rs738409 G-allele.

Conclusion

Our findings suggest that the G-allele of PNPLA3 rs738409 associates with reduced fasting levels of cholesterol and triglyceride in individuals with IGR.     

A regime shift in the Southeast Greenland marine ecosystem

Two major oceanographic changes have recently propagated through several trophic levels in coastal areas of Southeast Greenland (SEG). Firstly, the amount of drift-ice exported from the Fram Strait and transported with the East Greenland Current (EGC) has decreased significantly over the past two decades, and a main tipping element (summer sea ice) has virtually disappeared since 2003 leading to a regime shift in oceanographic and ecological conditions in the region. The following 20-year period with low or no coastal sea ice is unique in the 200-year history of ice observations in the region, and the regime shift is also obvious in the volume of ice export through the Fram Strait after 2013. In the same period, the temperature of the EGC south of 73.5 N has increased significantly (>2°C) since 1980. Secondly, the warm Irminger Current, which advects warm, saline Atlantic Water into the region, has become warmer since 1990. The lack of pack ice in summer together with a warming ocean generated cascading effects on the ecosystem in SEG that are manifested in a changed fish fauna with an influx of boreal species in the south and the subarctic capelin further north. At higher trophic levels there has been an increase in the abundance of several boreal cetaceans (humpback, fin, killer, and pilot whales and dolphins) that are either new to this area or occur in historically large numbers. It is estimated that the new cetacean species in SEG are responsible for an annual predation level of 700,000 tons of fish. In addition, predation on krill species is estimated at >1,500,000 tons mainly consumed by fin whales. Simultaneously, there has been a reduction in the abundance and catches of narwhals and walruses in SEG and it is suggested that these species have been impacted by the habitat changes.     

Newly identified proviruses in Thermotogota suggest that viruses are the vehicles on the highways of interphylum gene sharing

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date, only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of bacteria belonging to eight Thermotogota genera and induce viral particle production from one of the proviruses. All except an incomplete provirus from Mesotoga fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of Group 1 are found in the genera Geotoga, Kosmotoga, Marinitoga, Thermosipho and Mesoaciditoga and are similar to the previously characterized Marinitoga viruses, while proviruses from Group 2 are distantly related to the Group 1 proviruses, have different genome organization and are found in Petrotoga and Defluviitoga. Genes carried by both groups are closely related to Firmicutes and Firmicutes (pro)viruses in phylogenetic analyses. Moreover, one of the groups show evidence of recent gene exchange and may be capable of infecting cells from both phyla. We hypothesize that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota.     

Harvest mortality in North American mallards: A reply to Sedinger and Herzog

Sedinger and Herzog (2012) evaluated the evidence for the impact harvest mortality may have on North American duck populations. While doing that, they questioned our review (Pöysä et al. 2004 ) and conclusion that harvest mortality in North American mallards (Anas platyrhynchos) may have shifted from compensatory to additive over the period from the 1960s to the 1980s. In this reply, we correct Sedinger and Herzog's misrepresentations of our 2004 paper and argue that our interpretations of the results published at that time have not been invalidated. © 2013 The Wildlife Society.     

Eicosapentaenoic Acid and Docosahexaenoic Acid in Whole Blood Are Differentially and Sex-Specifically Associated with Cardiometabolic Risk Markers in 8–11-Year-Old Danish Children

n-3 long-chain polyunsaturated fatty acids improve cardiovascular risk markers in adults. These effects may differ between eicosapentaenoic acid (EPA, 20∶5n-3) and docosahexaenoic acid (DHA, 22∶6n-3), but we lack evidence in children. Using baseline data from the OPUS School Meal Study we 1) investigated associations between EPA and DHA in whole blood and early cardiometabolic risk markers in 713 children aged 8–11 years and 2) explored potential mediation through waist circumference and physical activity and potential dietary confounding. We collected data on parental education, pubertal stage, 7-day dietary records, physical activity by accelerometry and measured anthropometry, blood pressure, and heart rate. Blood samples were analyzed for whole blood fatty acid composition, cholesterols, triacylglycerol, insulin resistance by the homeostatic model of assessment (HOMA-IR), and inflammatory markers. Whole blood EPA was associated with a 2.7 mmHg (95% CI 0.4; 5.1) higher diastolic blood pressure per weight% EPA, but only in boys. Heart rate was negatively associated with both EPA and DHA status (P = 0.02 and P = 0.002, respectively). Whole blood EPA was negatively associated with triacylglycerol (P = 0.003) and positively with total cholesterol, low density and high density lipoprotein (HDL) cholesterol and HDL:triacylglycerol (all P<0.01) whereas DHA was negatively associated with insulin and HOMA-IR (P = 0.003) and tended to be negatively associated with a metabolic syndrome-score (P = 0.05). Adjustment for waist circumference and physical activity did not change the associations. The association between DHA and HOMA-IR was attenuated but remained after adjustment for fiber intake and none of the other associations were confounded by dietary fat, protein, fiber or energy intake. This study showed that EPA status was negatively associated with triacylglycerol and positively with cholesterols whereas DHA was negatively associated with insulin resistance, and both were inversely associated with heart rate in children. The sex-specific associations with blood pressure confirm our previous findings and warrant further investigation.     

Haematopoietic stem cell niches: new insights inspire new questions

Haematopoietic stem cell (HSC) niches provide an environment essential for life‐long HSC function. Intense investigation of HSC niches both feed off and drive technology development to increase our capability to assay functionally defined cells with high resolution. A major driving force behind the desire to understand the basic biology of HSC niches is the clear implications for clinical therapies. Here, with particular emphasis on cell type‐specific deletion of SCL and CXCL12, we focus on unresolved issues on HSC niches, framed around some very recent advances and novel discoveries on the extrinsic regulation of HSC maintenance. We also provide ideas for possible paths forward, some of which are clearly within reach while others will require both novel tools and vision.     

Individual Differences in Inhibitory Control,Not Non-Verbal Number Acuity,Correlate with Mathematics Achievement

Given the well-documented failings in mathematics education in many Western societies, there has been an increased interest in understanding the cognitive underpinnings of mathematical achievement. Recent research has proposed the existence of an Approximate Number System (ANS) which allows individuals to represent and manipulate non-verbal numerical information. Evidence has shown that performance on a measure of the ANS (a dot comparison task) is related to mathematics achievement, which has led researchers to suggest that the ANS plays a critical role in mathematics learning. Here we show that, rather than being driven by the nature of underlying numerical representations, this relationship may in fact be an artefact of the inhibitory control demands of some trials of the dot comparison task. This suggests that recent work basing mathematics assessments and interventions around dot comparison tasks may be inappropriate.