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Because heparin is a HS variant with increased degrees of sulfation and negative charge (Figure 2C), our findings support that the bulk of free energy (G) of the interaction is derived from the entropically favorable displacement of cations from HS upon Hh binding ( FIGURE 2 (B) HS mixtures derived from 2 independent pools of mouse embryos were coupled to FPLC columns and Shh binding to these matrices analyzed

Besides, the combination of smoothing and baseline correction, as well as smoothing and other spectral filters, was the most significant for improving both prediction metrics in PLS (Table 1 and Supplementary material: Tables 1 and 2)

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