Precision of cyathostomin luminal worm counts: Investigation of storage duration and fixative.

Essentially all grazing horses are infected with cyathostomin parasites. Adult cyathostomins reside in the large intestine of the horse and larval stages encyst within intestinal mucosa. Manual worm collection from aliquots of intestinal content is the current gold standard for retrieval and enumeration of luminal parasites, however, no research has been conducted to standardize specific parameters for processing and storage of samples. The aims of this study were (1) to evaluate the precision of current standard operating procedures for enumeration of luminal adult cyathostomin populations, (2) investigate the influence of chosen fixative, either 70 % ethanol or 10 % buffered formalin, as well as storage duration, immediately post necropsy vs. stored for eight weeks, on the magnitude and precision of worm counts, and (3) compare the luminal count magnitude between the three intestinal segments (cecum, ventral colon, dorsal colon). Ten miniature horses were enrolled in this study for euthanasia and necropsy over a four-week period. Luminal worm counts were conducted for 2 % aliquots of the cecum, ventral colon, and dorsal colon and samples were allocated to the two fixatives and the two storage durations. Precision was evaluated by coefficient of variation (CV) and was 13.04 % for total cyathostomin counts. Mean CV for large intestinal segments ranged from 15.31 % to 52.50 % irrespective of fixative used or storage duration. cecum worm counts were significantly lower compared to the ventral colon (p = 0.008) and dorsal colon (p = 0.01). Fixative and storage duration were not statistically associated with count precision or magnitude. This study demonstrated moderate to high precision estimates for luminal cyathostomin worm counts but did not identify any effects of fixative and storage duration within the framework of the study. This is the first study to determine cyathostomin worm count precision, and results will be useful for power analyses in the future.

A dynamic loop provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase.

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function.