Characterisation of a novel Emaravirus identified in mosaic-diseased Eurasian aspen (Populus tremula)

Since Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic-diseased Eurasian aspen (Populus tremula) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA-dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot-associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT-PCR in mosaic-diseased P. tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic-diseased Eurasian aspen by virus-specific RT-PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic-disease is graft-transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft-inoculated seedlings used as rootstocks as well as in the virus-infected scions used for graft-inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic-associated virus.     

Activation of Hypoxia Response in Endothelial Cells Contributes to Ischemic Cardioprotection

Small-molecule inhibition of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) is being explored for the treatment of anemia. Previous studies have suggested that HIF-P4H-2 inhibition may also protect the heart from an ischemic insult. Hif-p4h-2^gt/gt mice, which have 76 to 93% knockdown of Hif-p4h-2 mRNA in endothelial cells, fibroblasts, and cardiomyocytes and normoxic stabilization of Hif-α, were subjected to ligation of the left anterior descending coronary artery (LAD). Hif-p4h-2 deficiency resulted in increased survival, better-preserved left ventricle (LV) systolic function, and a smaller infarct size. Surprisingly, a significantly larger area of the LV remained perfused during LAD ligation in Hif-p4h-2^gt/gt hearts than in wild-type hearts. However, no difference was observed in collateral vessels, while the size of capillaries, but not their number, was significantly greater in Hif-p4h-2^gt/gt hearts than in wild-type hearts. Hif-p4h-2^gt/gt mice showed increased cardiac expression of endothelial Hif target genes for Tie-2, apelin, APJ, and endothelial nitric oxide (NO) synthase (eNOS) and increased serum NO concentrations. Remarkably, blockage of Tie-2 signaling was sufficient to normalize cardiac apelin and APJ expression and resulted in reversal of the enlarged-capillary phenotype and ischemic cardioprotection in Hif-p4h-2^gt/gt hearts. Activation of the hypoxia response by HIF-P4H-2 inhibition in endothelial cells appears to be a major determinant of ischemic cardioprotection and justifies the exploration of systemic small-molecule HIF-P4H-2 inhibitors for ischemic heart disease.     

Facultative Sex Allocation and Sex‐Specific Offspring Survival in Barrow's Goldeneyes

Sex allocation theory predicts that females should bias their reproductive investment towards the sex generating the greatest fitness returns. The fitness of male offspring is often more dependent upon maternal investment, and therefore, high‐quality mothers should invest in sons. However, the local resource competition hypothesis postulates that when offspring quality is determined by maternal quality or when nest site and maternal quality are related, high‐quality females should invest in the philopatric sex. Waterfowl – showing male‐biased size dimorphism but female‐biased philopatry – are ideal for differentiating between these alternatives. We utilized molecular sexing methods and high‐resolution maternity tests to study the occurrence and fitness consequences of facultative sex allocation in Barrow's goldeneyes (Bucephala islandica). We determined how female structural size, body condition, nest‐site safety and timing of reproduction affected sex allocation and offspring survival. We found that the overall sex ratio was unbiased, but in line with the local resource competition hypothesis, larger females produced female‐biased broods and their broods survived better than those of smaller females. This bias occurred despite male offspring being larger and tending to have lower post‐hatching survival. The species shows strong female breeding territoriality, so the benefit of inheriting maternal quality by philopatric daughters may exceed the potential mating benefit for sons of high‐quality females.     

Site fertility drives temporal turnover of vegetation at high latitudes

Experimental evidence shows that site fertility is a key modulator underlying plant community changes under climate change. Communities on fertile sites, with species having fast dynamics, have been found to react more strongly to climate change than communities on infertile sites with slow dynamics. However, it is still unclear whether this generally applies to high‐latitude plant communities in natural environments at broad spatial scales. We tested a hypothesis that vegetation of fertile sites experiences greater changes over several decades and thus would be more responsive under contemporary climate change compared to infertile sites that are expected to show more resistance. We resurveyed understorey communities (vascular plants, bryophytes, and lichens) of four infertile and four fertile forest sites along a latitudinal bioclimatic gradient. Sites had remained outside direct human disturbance. We analyzed the magnitude of temporal community turnover, changes in the abundances of plant morphological groups and strategy classes, and changes in species diversity. In agreement with our hypothesis, temporal turnover of communities was consistently greater on fertile sites compared to infertile sites. However, our results suggest that the larger turnover of fertile communities is not primarily related to the direct effects of climatic warming. Furthermore, community changes in both fertile and infertile sites showed remarkable variation in terms of shares of plant functional groups and strategy classes and measures of species diversity. This further emphasizes the essential role of baseline environmental conditions and nonclimatic drivers underlying vegetation changes. Our results show that site fertility is a key determinant of the overall rate of high‐latitude vegetation changes but the composition of plant communities in different ecological contexts is variously impacted by nonclimatic drivers over time.     

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.     

Chemopreventive effects of hydroxymatairesinol on uterine carcinogenesis in Donryu rats

Hydroxymatairesinol (HMR), obtained from the heartwood of spruce (Picea abies), has been demonstrated to exert chemo-preventive effects on the development of mammary tumors in rats. To examine the influence of HMR on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 11 weeks of age and fed thereafter 0, 200, or 600 ppm HMR mixed in the soy-containing diet until 15 months of age. Incidences of uterine adenocarcinoma in both 200 and 600 ppm HMR-dosed groups were significantly reduced to 11% and 15%, respectively, less than 50% of 0 ppm, at the end of the experiment (P < 0.05). A delay in the start of persistent estrus by HMR was observed at 8 months of age compared with controls given carcinogen alone. From urinalysis, HMR was metabolized mainly to enterolactone and hydroxyenterolactone. These findings suggest that HMR or its metabolites exert chemo-preventive effects in the rat ENNG-uterine carcinogenesis model.     

Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme

Eight human liver UDP-glucuronosyltransferases (UGTs) were expressed in baculovirus-infected insect cells as fusion proteins carrying a short C-terminal extension that ends with 6 histidine residues (His tag). The activity of recombinant UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B15 was almost fully inhibited by 0.2% Triton X-100. In the case of UGT1A9, however, glucuronidation of alpha-naphthol and scopoletin was resistant to such inhibition, whereas glucuronidation of entacapone and several other aglycones was sensitive. His-tagged UGT1A9 was purified by immobilized metal-chelating chromatography (IMAC). Purified UGT1A9 glucuronidated scopoletin at a high rate, whereas its glucuronidation activity toward entacapone was low and largely dependent on phospholipid addition. Recombinant UGT1A9 in which the His tag was replaced by hemagglutinin antigenic peptide (HA tag) was also prepared. Insect cells were co-infected with baculoviruses encoding both HA-tagged and His-tagged UGT1A9. Membranes from the co-infected cells, or a mixture of membranes from separately infected cells, were subjected to detergent extraction and IMAC, and the resulting fractions were analyzed for the presence of each type of UGT1A9 using tag-specific antibodies. In the case of separate infection, the HA-tagged UGT1A9 did not bind to the column. When co-infected with His-tagged UGT1A9, however, part of the HA-tagged enzyme was bound to the column and was eluted by imidazole concentration gradient together with the His-tagged UGT1A9, suggesting the formation of stable dimers that contain one His-tagged and one HA-tagged UGT1A9 monomers.