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These clones displayed a CD4+CD8dim+ (TC5) and CD4+ CD8– (TC7) phenotype and mediated a specific major histocompatibility complex class I-restricted cytotoxic activity toward Cou-LB autologous tumor cell line.
Based on restriction analysis with several four base cutters, the pENTR dTOPO clones derived from each species could be classified into 4 classes whereas the pBINPLUS clones displayed a single digestion pattern.
In correlation with the growth defect, the hFXNI154F expressing clones displayed a very altered morphology, with numerous small grainy rounded cells with a retracted cytoplasm and some elongated spindle-shaped cells (Fig. 3, lines hFXNI154F-1D3 and hFXNI154F-2C1).
Furthermore, while the clones expressing the wild type hFXN presented normal fibroblast morphology (Fig. 3, line hFXN-1D12), the hFXNG130V expressing clones displayed a slightly altered morphology with smaller or less spread out cells (Fig. 3, line hFXNG130V-G4).
These clones displayed a clear tendency to expand within the boundary along the mediolateral (ML) axis (see white arrows in flat mounted-hindbrains of Figure 1B); indeed, all clones that respected boundaries displayed a larger ML than AP length.
Interestingly, both hFXNG130V and hFXNI154F clones displayed a significantly reduced catalase activity (44% and 51%, respectively, Fig. 6B) that correlated with a decrease in catalase expression at the protein level (Fig. 6C) and at the mRNA level (not shown).
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It is of note that the antisense clones displayed an upregulated MIF-sense mRNA.
Unexpectedly, we observed a marked modification in N1E-115 cell size (Fig. 2a and 2c): both HIF2α clones displayed an average 30% increase in cell diameter (p < 0.001), while HIF2α(1 485) cells were 15 to 25% smaller than native or pCDNA3.1-transfected cells (p < 0.001).
Both clones display a mixed serotonergic and dopaminergic phenotype (Baudry et al. 2015).
Therefore, as both the G130V and I154F mutant clones display a stable and spontaneous biochemical phenotype and are able to proliferate in classical culture conditions, these new cellular models will be useful for functional-based large-scale drug screening.
BACs with large insert clones display a lower variation compared with the oligo platform.
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