Morphology Physiology data_ESM from Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia

Supporting morphology and physiology dat

Microbiome data_ESM from Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia

Supporting microbiome dat

Electronic supplementary material, table S2 from Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia

Relative abundances (%) of bacteria sampled from the hindgut of hibernating and active frog

Electronic supplementary material, table S1 from Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia

Genera qualified as being potentially ureolytic on the basis of finding in the scholarly literature at least one report documenting ureolytic activity or the presence of urease in any member taxo

Effect of pantothenamides on proliferation of <i>P. falciparum</i> and <i>P. falciparum</i> lysate-catalysed [¹⁴C]pantothenate phosphorylation.

<p>The 50% inhibitory concentrations (IC₅₀ values) measured against <i>P. falciparum</i> parasites cultured (for 96 h) in Albumax-complete RPMI containing 1 µM pantothenate, as determined using the SYBR Green I-based growth assay are shown. Assays were performed using Albumax-complete RPMI prepared within 48 h of the assay, stored at 4°C, and incubated at 37°C for a maximum of 1 h (fresh) or Albumax-complete RPMI incubated continuously at 37°C for 40 h immediately after preparation (aged). The IC₅₀ values shown for parasites cultured in fresh Albumax-complete RPMI are averages from between two and eight independent experiments each performed in duplicate or triplicate. Where the IC₅₀ values determined were below 200 µM, they are presented as the mean ± SEM from between three and eight independent experiments. The IC₅₀ values shown for parasites cultures in aged medium are averages from between two and three independent experiments each performed in triplicate. Where the IC₅₀ values determined were below 200 µM, they are presented as the mean ± range/2 or SEM as appropriate. The percentage inhibition of [¹⁴C]pantothenate phosphorylation by PanK in <i>P. falciparum</i> lysate caused by pantothenamides (when tested at a concentration of 100 µM) in the presence of 0.2 µM pantothenate are also shown. The percentage inhibition was calculated from the measured amounts of [¹⁴C]pantothenate phosphorylated during a 10 min incubation in the presence of pantothenamide and in the presence, instead, of the corresponding concentration of DMSO only. Data are averages ± range/2 from two independent experiments, each performed in duplicate. A value of 100 indicates complete inhibition of [¹⁴C]pantothenate phosphorylation was observed in both independent experiments. The amount of [¹⁴C]pantothenate phosphorylated by <i>P. falciparum</i> lysate was significantly lower in the presence of all pantothenamides (P<0.0001, ANOVA).</p

Effect of pantothenate supplementation on pantothenamide and pantothenol potency in fresh and aged Albumax-complete RPMI.

<p>The bars represent the fold-increases in the IC₅₀ values of compound 12, compound 19 and pantothenol in Albumax-complete RPMI following supplementation with 100 µM pantothenate. The fold-increases in IC₅₀ values were calculated by dividing each IC₅₀ value measured against <i>P. falciparum</i> cultured in the presence of 100 µM pantothenate by the corresponding IC₅₀ value measured against <i>P. falciparum</i> cultured in the presence of 1 µM pantothenate. The fold-increases in IC₅₀ values were determined in assays performed using Albumax-complete RPMI stored for a maximum of 48 h at 4°C, and incubated at 37°C for a maximum of 1 h (fresh; open bar) or Albumax-complete RPMI stored for a minimum of one week at 4°C and incubated intermittently at 37°C, or, soon after preparation, incubated continuously at 37°C for up to 40 h (aged; closed bar). The fold-increases in IC₅₀ values are averaged from between two and four independent experiments in which test compounds were tested in Albumax-complete RPMI containing 1 µM pantothenate and Albumax-complete RPMI supplemented with 100 µM pantothenate in parallel. Each experiment was performed in duplicate or triplicate, and error bars represent SEM or range/2. Pantothenol bars are shown with a broken edge to indicate that only a lower limit on the fold-increase in IC₅₀ could be determined. This is because less than 50% inhibition of growth was observed at the highest pantothenol concentration tested (2 mM).</p

Antiplasmodial effect of a pantothenamide and pantothenol in fresh and aged human serum-complete RPMI.

<p>The concentration-response curves show the effect of increasing concentrations of compound <b>12 (A)</b> and pantothenol (<b>B</b>) on the proliferation of <i>P. falciparum</i> parasites cultured (for 96 h) in human serum-complete RPMI as measured using the SYBR Green I-based growth assay. Assays were performed using human serum-complete RPMI prepared immediately prior to experimentation (fresh; open circles) or human serum-complete RPMI heated at 37°C for 40 h immediately following preparation (aged; closed circles). The data obtained with parasites cultured in fresh human serum-complete RPMI are from three independent experiments, each performed in duplicate or triplicate, and error bars represent SEM. The data obtained with parasites cultured in aged human serum-complete RPMI are from two independent experiments, each performed in duplicate or triplicate, and error bars represent range/2. For clarity, in <b>B</b>, concentration-response curves represented by the closed circles are shown with positive error bars only, and the concentration-response curves represented by the open circles are shown with negative error bars only. Where not shown, error bars are smaller than the symbol.</p

Effect of pantetheinase supplementation on the potency of a pantothenamide in aged Albumax-complete RPMI.

<p>The concentration-response curves show the effect of increasing concentrations of compound <b>12</b> on proliferation of <i>P. falciparum</i> parasites cultured (for 96 h) in Albumax-complete RPMI as measured using the SYBR Green I-based growth assay. Assays were performed using (i) Albumax-complete RPMI incubated immediately after preparation at 37°C for 40 h (aged; closed circles); (ii) aged Albumax-complete RPMI supplemented with recombinant human pantetheinase (100 ng/mL) immediately prior to the assay (aged+pantetheinase; open circles); (iii) aged Albumax-complete RPMI supplemented with recombinant human pantetheinase (100 ng/mL) and heated at 37°C for 40 h immediately prior to the assay (aged+pantetheinase+heat; grey circles); and (iv) aged Albumax-complete RPMI heated at 37°C for 40 h before being supplemented with recombinant human pantetheinase (100 ng/mL) immediately prior to the assay (aged+heat+pantetheinase; grey squares). The data are from three independent experiments, each performed in triplicate, and error bars represent SEM. For clarity, the time-courses represented by the open circles are shown with negative error bars only, and the time-courses represented by the grey squares are shown with positive error bars only. Where not shown, error bars are smaller than the symbol.</p

Chemical structures of pantothenate and related compounds.

<p>The hydroxy analogue of pantothenate, pantothenol, inhibits growth of <i>P. falciparum in vitro</i> and <i>in vivo</i>. Amide analogues of pantothenate, pantothenamides (for which the core structure is shown), possess antibacterial activity <i>in vitro</i>. Pantetheine, a naturally occurring pantothenate derivative, is hydrolyzed to pantothenate (and cysteamine) by the enzyme pantetheinase.</p

Antiplasmodial effect of pantothenamides and pantothenol in fresh and aged Albumax-complete RPMI.

<p>The concentration-response curves show the effect of increasing concentrations of compound <b>3</b> (<b>A</b>), compound <b>12</b> (<b>B</b>), compound <b>19</b> (<b>C</b>), and pantothenol (<b>D</b>) on proliferation of <i>P. falciparum</i> parasites cultured (for 96 h) in Albumax-complete RPMI containing 1 µM pantothenate, as measured using the SYBR Green I-based growth assay. Assays were performed using Albumax-complete RPMI stored for a maximum of 48 h at 4°C, and incubated at 37°C for a maximum of 1 h (fresh; open circles) or Albumax-complete RPMI incubated continuously at 37°C for 40 h immediately after preparation (aged; closed circles). The data obtained with parasites cultured in fresh Albumax-complete RPMI are from between three and eight independent experiments performed in duplicate or triplicate and error bars represent SEM. The data obtained with parasites cultured in aged Albumax-complete RPMI are from between two and three independent experiments performed in duplicate or triplicate and error bars represent range/2 or SEM. For clarity, in <b>D</b>, the concentration-response curves represented by the closed circles are shown with negative error bars only, and the concentration-response curves represented by the open circles are shown with positive error bars only. Where not shown, error bars are smaller than the symbol.</p