Global urban environmental change drives adaptation in white clover

Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale

Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing

Abstract Background Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification. Results This method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples. Conclusions The results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection

The Stapled AKAP Disruptor Peptide STAD-2 Displays Antimalarial Activity through a PKA-Independent Mechanism

<div><p>Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between <i>Hs</i>PKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against <i>Plasmodium falciparum</i> blood stage parasites <i>in vitro</i>. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC₅₀ ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.</p></div

Stapled Peptides.

<p>*Length in amino acids</p><p>Stapled Peptides.</p

STAD-2 uptake is largely independent of the PSAC.

<p>(<b>A</b>) Late-stage iRBC were treated with 1 μM STAD-2 in the presence of 200 μM furosemide following pre-treatment with complete culture medium (Fur/STAD-2) or 200 μM furosemide (Fur/Fur/STAD-2). Treatment of late-stage iRBC with STAD-2 in the presence of furosemide demonstrated a visible, yet insignificant, decrease in STAD-2 uptake only when iRBC were pre-treated with furosemide (two-tailed t test, p = 0.0557, n = 3, mean ± S.E.). (<b>B</b>) Late-stage iRBC were treated with 1 μM STAD-2 in the presence of 5% D-Sorbitol (PSAC solute), 130 mM glycerol (AQP3 solute), or 6 μM AgNO₃ (AQP1 inhibitor). Treatment with STAD-2 in the presence of 5% D-Sorbitol yielded a decrease in STAD-2 uptake similar to that seen in (<b>A</b>) while treatment in the presence of glycerol or AgNO₃ did not differ from STAD-2 alone (n = 2, mean ± S.E.).</p

STAD-2 is uniquely permeable to iRBC.

<p>iRBC were treated for 6 hours with 1 μM stapled peptides of varying charges (<b>A</b>) or variants of STAD-2 (<b>B</b>), analyzed by flow cytometry, and reported as median fluorescence intensity (<b>C</b>, n = 2–6, mean ± S.E.). Of the various stapled peptides analyzed, only STAD-2 was clearly permeable to iRBC.</p

STAD-2 synthesis and function.

<p>(<b>A</b>) STAD-2 and STAD-2 scramble were synthesized by substituting S-pentenyl alanine (S₅, shown as *) into positions that are opposite to the binding surface targeting PKA-R. A helical wheel represents the STAD-2 secondary structure wherein hydrophobic residues are shown in red, S₅ in black, and other residues in grey. (<b>B</b>) Fmoc chemistry was used to synthesize STAD-2 peptides containing the non-natural S₅ residues at <i>i</i>, <i>i</i>+4 positions. Ring-closing metathesis was performed using Grubbs I catalyst to generate the hydrocarbon staple. (<b>C</b>) The interaction between the <i>Hs</i>PKA D/D domain (pale cyan) and the docking sequence of an AKAP (orange) can be inhibited by the stapled disruptor peptide STAD-2 (red). STAD-2 mimics the docking sequence of an AKAP and disrupts binding to the regulatory subunit of PKA. Images were created using the crystal structure of PKA-RII (PDB access code: 2HWN [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129239#pone.0129239.ref041" target="_blank">41</a>]).</p

STAD-2 peptides are selectively permeable to <i>Plasmodium</i>-infected red blood cells.

<p>(<b>A</b>) <i>Plasmodium</i>-iRBC were treated for 6 hours with 1 μM FITC-conjugated STAD-2 and analyzed by flow cytometry. iRBC showed selective permeability to STAD-2 relative to uRBC. (<b>B, C</b>) Treatment of synchronous ring-stage (black bars) or late-stage (grey bars) cultures with 1 μM FITC-conjugated STAD-2, unstapled STAD-2 parent, or STAD-2 scramble demonstrated significantly increased uptake of STAD-2 by late-stage relative to ring-stage iRBC. However, both ring-stage and late-stage iRBC were minimally permeable to STAD-2 parent and STAD-2 scramble controls (2way ANOVA with Bonferroni posttest, p<0.001, n = 3–6, mean ± S.E.).</p

STAD-2 rapidly localizes within the parasitophorous vacuole.

<p>(<b>A</b>) 3D7 iRBC were treated with 1 μM STAD-2 or STAD-2 scramble, stained with 2 μg/mL Hoechst 33342, and analyzed by fluorescence microscopy. STAD-2 peptides consistently localized within the intracellular parasite and at much higher levels than its scrambled control. (<b>B</b>) iRBC treated for 20 minutes, 3 hours, or 6 hours with 1 μM STAD-2 showed that STAD-2 traffics to the parasitophorous vacuole by 20 minutes post-treatment.</p

STAD-2 does not associate with PKA.

<p>(<b>A</b>) Late-stage iRBC were treated with 1 μM FITC-conjugated STAD-2 for 2 hours and probed for <i>P</i>. <i>falciparum</i> PKA-R (top panel) or <i>H</i>. <i>sapiens</i> PKA-RII (bottom panel). STAD-2 did not show clear colocalization with either of the regulatory subunits. (<b>B</b>) Late-stage iRBC were treated with 1 μM STAD-2, 30 μM H89 (small molecule inhibitor of PKA), or 0.001% DMSO and analyzed by light microscopy at 48 hours post-treatment. H89-treated iRBC demonstrated clear absence of parasite digestive vacuoles while STAD-2 treated iRBC were indistinguishable from DMSO controls.</p