How Oxygen Availability Affects the Antimicrobial Efficacy of Host Defense Peptides: Lessons Learned from Studying the Copper-Binding Peptides Piscidins 1 and 3

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we performed growth inhibition and time-kill assays against C. difficile showing that both piscidins suppress proliferation of C. difficile by killing bacterial cells. Microscopy experiments show that the peptides accumulate at sites of membrane curvature. We find that both piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. Notably, copper does not enhance piscidin activity against C. difficile. Thus, while antimicrobial activity of piscidin peptides is conserved in aerobic and anaerobic settings, the peptide–copper interaction depends on environmental oxygen to achieve its maximum potency. The development of pharmaceuticals from HDPs such as piscidin will necessitate consideration of oxygen levels in the targeted tissue

Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (−196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral.

Coral reefs are experiencing unprecedented degradation due to human activities, and protecting specific reef habitats may not stop this decline, because the most serious threats are global (i.e., climate change), not local. However, ex situ preservation practices can provide safeguards for coral reef conservation. Specifically, modern advances in cryobiology and genome banking could secure existing species and genetic diversity until genotypes can be introduced into rehabilitated habitats. We assessed the feasibility of recovering viable sperm and embryonic cells post-thaw from two coral species, Acropora palmata and Fungia scutaria that have diffferent evolutionary histories, ecological niches and reproductive strategies. In vitro fertilization (IVF) of conspecific eggs using fresh (control) spermatozoa revealed high levels of fertilization (>90% in A. palmata; >84% in F. scutaria; P>0.05) that were unaffected by tested sperm concentrations. A solution of 10% dimethyl sulfoxide (DMSO) at cooling rates of 20 to 30°C/min most successfully cryopreserved both A. palmata and F. scutaria spermatozoa and allowed producing developing larvae in vitro. IVF success under these conditions was 65% in A. palmata and 53% in F. scutaria on particular nights; however, on subsequent nights, the same process resulted in little or no IVF success. Thus, the window for optimal freezing of high quality spermatozoa was short (∼5 h for one night each spawning cycle). Additionally, cryopreserved F. scutaria embryonic cells had∼50% post-thaw viability as measured by intact membranes. Thus, despite some differences between species, coral spermatozoa and embryonic cells are viable after low temperature (-196°C) storage, preservation and thawing. Based on these results, we have begun systematically banking coral spermatozoa and embryonic cells on a large-scale as a support approach for preserving existing bio- and genetic diversity found in reef systems

Adult and larval forms of <i>A. palmata</i> and <i>F. scutaria</i>.

<p><b>A</b>) Adult and developing <i>A. palmata</i> larvae (inset) at the cornflake stage at ∼24 h. Scale bar = 50 µm. Adult photo by R. Williams, Smithsonian Institution. <b>B</b>) Adult and developing <i>F. scutaria</i> larvae at the swimming stage. Scale bar = 50 µm. Embryos that reached these stages were scored as successfully developed.</p

Cryopreservation of coral sperm (all sperm exposed to freezing in these treatments).

<p><b>A</b>) <i>A. palmata</i> sperm were cryopreserved at cooling rates 20 to 30°C/min using 10% DMSO and PG, and IVF success was assessed and averaged over the single spawning period. This averaged graph revealed no difference between the two cryoprotectants and a mean fertilization success of ∼18%. <b>B</b>) However, if the <i>A. palmata</i> fertilization success during the spawning period was graphed by day, a 65% fertilization success occurred on Day 3 with 10% DMSO, whereas it was 25, 0 and 3% on Day 1, 2 and 4, respectively. For the first 3 nights, the control with fresh sperm held at ∼90%, then fell to 76% on the fourth evening. <b>C</b>) <i>F. scutaria</i> sperm were cryopreserved at rates 20 to 30°C/min using 10% DMSO versus PG, and fertilization success was assessed and averaged over two spawning periods (July and August 2010). Averaging indicated that 10% DMSO was the preferred cryoprotectant, and (as in <i>A. palmata</i>) there was no variability in time in terms of physiological responses during a spawning season. <b>D</b>) Variability in <i>F. scutaria</i> IVF success after cryopreservation over two nights of a single representative month (August 2010). Fresh sperm IVF success held steady at 65%, but sperm cryopreserved with 10% DMSO varied from 52 to 13% on the two evenings. Bars with the same letters were not different (<i>P</i>>0.05; ANOVA), whereas bars with different letters were different (<i>P</i><0.05; ANOVA).</p

Embryonic <i>F. scutaria</i> cells after cryopreservation (all cells exposed to freezing in these treatments).

<p>Mean post-thaw viability of <i>F. scutaria</i> cells was ∼50% for all cryoprotectants tested. Ten thousand events were measured for each sample. Controls (the three left bars) were live-stained and unstained cells and 100% dead cells that produced control data for the flow cytometer (i.e., 100% intact versus 100% dead). Bars with the same letters were not different, whereas bars with different letters were different (P<0.05; Kruskal-Wallis test).</p

Species-specific sperm concentrations were not necessary for successful <i>in vitro</i> fertilization.

<p><b>A</b>) Regardless of the <i>A. palmata</i> sperm concentration used (10⁶ to 10⁸ cells/ml), a successful <i>in vitro</i> fertilization success of >92% was observed regardless of whether the eggs were exposed to the sperm for 5 min (grey bars) or overnight (black bars) (<i>P</i>>0.05; ANOVA). <b>B</b>) Both sperm concentrations for <i>F. scutaria</i> produced uniform IVF results (<i>P</i>>0.05; Mann-Whitney).</p

Experiment II: <i>In vitro</i> fertilization after exposing fresh sperm to various cryoprotectant treatments.

1<p>Sigma-Aldrich, St Louis, MO, USA.</p>2<p>Acros Organics, Fair Lawn, NJ, USA.</p>*<p>Thirty to 50 fresh eggs were used per pooled sperm sample.</p>***<p>An inseminate concentration of 10⁶ sperm/ml was used for each species.</p

The effect of cooling rate for successful <i>F. scutaria</i> spermatozoa cryopreservation (no sperm exposed to freezing in any of these treatments).

<p>Two freezing ranges were examined and two cryoprotectant solutions (<i>F. scutaria</i>, n = 3–7 pooled sperm donors/cooling range and n = 16 egg donors) and then the influence of cooling rate on IVF success. Only the 10% DMSO at a cooling rate greater than 20°C produced reasonable post-thaw fertilization. Bars with the same letter were not different (<i>P</i>>0.05; ANOVA), whereas bars with different letters were different (<i>P</i><0.05; ANOVA).</p