The 10DM24 deoxyribozyme can site-specifically label RNAs with fluorophore-GTP conjugates; however, the 2′,5′-branched RNA linkage is readily cleaved by debranchase. To prevent loss of labels upon cleavage, we synthesized phosphorothioate-modified, fluorescent GTP derivatives and elaborated conditions for their incorporation by 10DM24. RNAs labeled with fluorescent derivatives of Sp-GTPS were found to be resistant to debranchase.
In an ongoing effort to propose biologically protective nutrient criteria, we examined how total nitrogen (TN) and its forms were associated with macroinvertebrate communities in wadeable streams of Maryland. Taxonomic and functional metrics of an index of biological integrity (IBI) were significantly associated with multiple nutrient measures; however, the highest correlations with nutrients were for ammonia-N and nitrite-N and among macroinvertebrate measures were for Beck's Biotic Index and its metrics. Since IBI metrics showed comparatively less association, we evaluated how macroinvertebrate taxa related to proposed nutrient criteria previously derived for those same streams instead of developing nutrient-biology thresholds. We identified one tolerant and three intolerant taxa whose occurrence appeared related to a TN benchmark. Individually, these taxa poorly indicated whether streams exceeded the benchmark, but combining taxa notably improved classification rates. We then extracted major physiochemical gradients using principal components analysis to develop models that assessed their influence on nutrient indicator taxa. The response of intolerant taxa was predominantly influenced by a nutrient-forest cover gradient. In contrast, habitat quality had a greater effect on tolerant taxa. When taxa were aggregated into a nutrient sensitive index, the response was primarily influenced by the nutrient-forest gradient. Multiple lines of evidence highlight the effects of excessive nutrients in streams on macroinvertebrate communities and taxa in Maryland, whose loss may not be reflected in metrics that form the basis of biological criteria. Refinement of indicator taxa and a nutrient-sensitive index is warranted before thresholds in aquatic life to water quality are quantified.
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