Because of the temporal limitations of the Nestin promoter and the lack of expression in the CNS using the Gata2 promoter, we generated six additional lines, one for eGFP-IDH1wildtype, eGFP-IDH1R132H, eGFP-IDH1G70D and eGFP, and two for eGFP-IDH1R132C, in which the transgene was expressed under control of a Gfap promoter. Constructs used for these lines are referred to as GfapIDH1wt, GfapR132H, GfapR132C, GfapG70D and GfapGFP. The expression vector (gfap:GFP) has been demonstrated to have glial-specific expression in the zebrafish CNS, detectable during the embryonic stage [34]. The IDH1G70D mutation is an enzymatic null mutation that was included to serve as a non-D2HG-producing control [15, 35]. The IDH1R132C mutation was generated to study potential differences between IDH1R132H and IDH1R132C, and is a mutant with reportedly higher neomorphic enzymatic activity [36]. Expression of the transgenes was confirmed using fluorescent imaging on 1, 3 and 5 dpf (Fig 2A and S1B Fig). GFP was observed in the brain and spinal cord in all the Gfap transgenic zebrafish lines. CNS-specific expression of the transgene was further confirmed by immunohistochemistry using anti-GFP antibodies (Fig 2B). Expression of the transgene on the RNA level was confirmed by RT-QPCR till at least 20 dpf (Fig 2C). There were no obvious differences in results from these assays between IDH1R132H and IDH1R132C transgenic zebrafish. The D2HG levels were markedly elevated in both GfapR132C zebrafish lines (line 84 and 85) on 3 dpf, which are about 8 and 18 times higher compared to the GfapGFP and GfapIDH1wt lines (line 73 and 92, Fig 2D). D2HG levels in GfapG70D zebrafish remained similar as in GfapGFP and GfapIDH1wt lines. L2HG levels of all the IDH1 transgenic lines were similar to the GfapGFP control, confirming that expression of transgene only affects the D2HG level (Fig 2E).When crossing GfapR132C line 85, an average of 21.1% of generated embryos showed abnormal tail development on 1dpf, which was higher than in the GfapGFP (3%) and GfapIDH1wt (0%) control lines (Fig 2F and 2G). The tail defects may be explained by the fact that the first detectable expression of Gfap is at 10 h post fertilization in the developing tail bud [34]. However, no clear (pre-) cancerous lesion was observed in any of the zebrafish lines studies.
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The following cell lines were used for virus isolation in this study: Vero E6 and Huh7 cells, which were cultured in DMEM containing 10% FBS. All cell lines were tested and free of mycoplasma contamination, submitted for species identification and authenticated by morphological evaluation by microscopy. None of the cell lines was on the list of commonly misidentified cell lines (by ICLAC).
It is refreshing to turn from this gloomy picture to the wonderfullydifferent effect produced by the very same circumstances upon the mindof the man who comprehends the facts of the case. It will be observedthat the two have no single emotion in common; in the former case allwas despondency and horror, while in this case we find none but thehighest and most beautiful sentiments. At the base of the thought-formwe find a full expression of deep sympathy, the lighter green indicatingappreciation of the suffering of the mourners and condolence with them,while the band of deeper green shows the attitude of the thinker towardsthe dead man himself. The deep rose-colour exhibits affection towardsboth the dead and the living, while the upper part of the cone and thestars which rise from it testify to the feeling aroused within thethinker by the consideration of the subject of death, the blueexpressing its devotional aspect, while the violet shows the thought of,and the power to respond to, a noble ideal, and the golden stars denotethe spiritual aspirations which its contemplation calls forth. The bandof clear yellow which is seen in the centre of this thought-form is verysignificant, as indicating that the man's whole attitude is based uponand prompted by his intellectual comprehension of the situation, andthis is also shown by the regularity of the arrangement of the coloursand the definiteness of the lines of demarcation between them.
A striking feature in this form is the radical difference between thetwo types of music which occur in it, one producing the angular rockymasses, and the other the rounded billowy clouds which lie between them.Other motifs are shown by the broad bands of blue and rose and greenwhich appear at the base of the bell, and the meandering lines of whiteand yellow which quiver across them are probably produced by a ripplingarpeggio accompaniment.
Then I became frantically hot. The tang of my sweat lodged in my nose. I had last shaved the day before graduation. I fingered the fuzzy hair that had grown in the ten days since and then held my hands under my nose. My sweat was vinegar sharp. Three sticky dried lines demarcated the creases between the rolls of my stomach. I scraped their whitish stuff up with my fingernails and rubbed it between my fingertips until it dissolved into oil.
Alcohol consumption and sport have been inextricably linked since ancient times when alcohol was considered the elixir of life [111]. To some extent that may be true, given that a substantial body of epidemiological evidence shows that moderate ingestion of alcohol may reduce the risk of cardiovascular disease [112]. The link between alcohol consumption and mortality is subject to a J-shaped curve i.e. improved longevity with moderate consumption with increasing intake resulting in greater mortality risk [113]. Indeed, dietary guidelines from the American Heart Association recommend moderation of alcohol intake as it has been associated with a lower risk of cardiovascular events [112].
Finally, there is some evidence to suggest that chronic alcohol consumption may result in a positive energy balance and a potentially obesogenic state. Tremblay et al. [133] reported that chronic alcohol consumption did not result in a reduction in lipid intake and that a dietary regimen that provided a large fraction of energy in the form of alcohol increased the risk for a positive energy balance in a free-living state. It appears that alcohol may have a fat-sparing effect [134] similar to that of carbohydrates and may cause fat gain. Suter et al. [135] suggested that alcohol could result in excess body fat gain especially in the upper body. There is some evidence to suggest that obese individuals may be more susceptible to weight gain and the hyperlipidemic effects of alcohol consumption as compared to lean individuals [136]. This is in contrast to epidemiological studies that report a negative association between alcohol consumption and adiposity [137]. This may be explained by the induction of unregulated futile metabolic cycles that appear to significantly aid in the disposal of excess calories [138]. In general, it appears that the effects of alcohol on body weight are controversial and it is likely that moderate consumption of alcohol that replaces calories from carbohydrates and fat is unlikely to result in weight gain [139]. 2ff7e9595c
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