Parallel Evolution of Tetrodotoxin Resistance in Three Voltage-Gated Sodium Channel Genes in the Garter Snake Thamnophis sirtalis

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav’s, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1–1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood–brain barrier. We also report the exon–intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations

[Pemetrexed + Sorafenib] lethality is increased by inhibition of ERBB1/2/3-PI3K-NFκB compensatory survival signaling

In the completed phase I trial NCT01450384 combining the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib it was observed that 20 of 33 patients had prolonged stable disease or tumor regression, with one complete response and multiple partial responses. The pre-clinical studies in this manuscript were designed to determine whether [pemetrexed + sorafenib] –induced cell killing could be rationally enhanced by additional signaling modulators. Multiplex assays performed on tumor material that survived and re-grew after [pemetrexed + sorafenib] exposure showed increased phosphorylation of ERBB1 and of NFκB and IκB; with reduced IκB and elevated G-CSF and KC protein levels. Inhibition of JAK1/2 downstream of the G-CSF/KC receptors did not enhance [pemetrexed + sorafenib] lethality whereas inhibition of ERBB1/2/4 using kinase inhibitory agents or siRNA knock down of ERBB1/2/3 strongly promoted killing. Inhibition of ERBB1/2/4 blocked [pemetrexed + sorafenib] stimulated NFκB activation and SOD2 expression; and expression of IκB S32A S36A significantly enhanced [pemetrexed + sorafenib] lethality. Sorafenib inhibited HSP90 and HSP70 chaperone ATPase activities and reduced the interactions of chaperones with clients including c-MYC, CDC37 and MCL-1. In vivo, a 5 day transient exposure of established mammary tumors to lapatinib or vandetanib significantly enhanced the anti-tumor effect of [pemetrexed + sorafenib], without any apparent normal tissue toxicities. Identical data to that in breast cancer were obtained in NSCLC tumors using the ERBB1/2/4 inhibitor afatinib. Our data argue that the combination of pemetrexed, sorafenib and an ERBB1/2/4 inhibitor should be explored in a new phase I trial in solid tumor patients

Rapid Evolution of Enormous, Multichromosomal Genomes in Flowering Plant Mitochondria with Exceptionally High Mutation Rates

A pair of species within the genus Silene have evolved the largest known mitochondrial genomes, coinciding with extreme changes in mutation rate, recombination activity, and genome structure

<i>Silene</i> mitochondrial genome sizes relative to all sequenced mitochondrial and eubacterial genomes from the National Center for Biotechnology Information (NCBI) Genome database.

<p><i>Silene</i> mitochondrial genome sizes relative to all sequenced mitochondrial and eubacterial genomes from the National Center for Biotechnology Information (NCBI) Genome database.</p

Repeat-mediated recombinational activity in the low mutation rate <i>S. latifolia</i> and <i>S. vulgaris</i> mitochondrial genomes (A) and the fast-evolving <i>S. noctiflora</i> and <i>S. conica</i> mitochondrial genomes (B).

<p>Each point represents a pair of repeats, and its position on the <i>y</i>-axis denotes the proportion of recombinant genome conformations detected with paired-end 454 reads. The dashed lines indicate the level at which equal frequencies of read pairs support recombinant and nonrecombinant conformations. The <i>S. latifolia</i> mitochondrial genome was not sequenced with 454 paired-end reads, but Southern blot hybridizations indicated that alternative genome conformations associated with its six-copy 1.4-kb repeat exist at roughly equivalent frequencies <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio.1001241-Sloan3" target="_blank">[38]</a>, as indicated by the large X.</p

Summary of four <i>Silene</i> mitochondrial genomes.

a<p>Duplicate genes/introns are included in length and coverage statistics but excluded from reported counts.</p>b<p>Two of the <i>S. vulgaris</i> plastid-derived tRNA genes may not be functional (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio-1001241-g004" target="_blank">Figure 4</a>).</p>c<p>Intron lengths only include <i>cis</i>-spliced introns.</p>d<p>Excludes regions of plastid-origin.</p>e<p>Excludes regions of plastid-origin and regions conserved in other plant mitochondrial genomes.</p>f<p>Predicted.</p

Protein and RNA gene content in sequenced seed plant mitochondrial genomes.

<p>Dark shading indicates the presence of an intact reading frame or folding structure, whereas light shading indicates the presence of only a putative pseudogene. The numbers at the bottom of each group indicate the total number of intact genes for that species. Note that the <i>ccmFc</i> gene, which is universally present in all other seed plants surveyed to date <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio.1001241-Adams1" target="_blank">[104]</a>, is classified as a pseudogene in <i>S. conica</i>. It has experienced numerous structural mutations in this lineage, including multiple frame shifts in the second exon that introduce premature stop codons. However, cDNA sequencing confirmed that this gene is transcribed, spliced, and RNA edited in <i>S. conica</i> (unpublished data), so it is possible that the gene is still functional in its truncated form. In some cases, the presence of an intact gene may not indicate functionality. This is particularly true for tRNA genes embedded within recently transferred regions of plastid DNA <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio.1001241-Alverson1" target="_blank">[20]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio.1001241-Leon1" target="_blank">[105]</a>. For example, the <i>trnN(guu)</i> and <i>trnR(acg)</i> genes in <i>S. vulgaris</i> may not be functional, as they are within a 2.6-kb region that appears to have been recently transferred from the plastid genome (on the basis of its perfect sequence identity with the exception of a single 18-bp deletion). These two tRNA genes are not orthologous to the plastid-derived copies of <i>trnN(guu)</i> and <i>trnR(acg)</i> in other seed plant mitochondria. Intron-containing plastid-derived tRNA genes such as <i>trnA(ugc)</i> in <i>Bambusa</i>, <i>trnV(uac)</i> in <i>Cycas</i>, <i>trnK(uuu)</i> in <i>Vitis</i>, and <i>trnI(gau)</i> in <i>Zea</i> are also unlikely to be functional. In <i>Cycas</i>, the <i>trnL(uaa)</i>, <i>trnP(ugg)</i>, <i>trnQ(uug)</i>, <i>trnR(ucu)</i>, and <i>trnV(uac)-</i> Ψ genes are classified on the basis of sequence homology to other land plant tRNAs even though their genomically encoded anticodons differ (CAA, CGG, CUG, CCU, and CAC, respectively). It is possible that these anticodons undergo C-to-U RNA editing to restore the ancestral codon as has been observed in other vascular plants <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio.1001241-Grewe1" target="_blank">[106]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001241#pbio.1001241-Grewe2" target="_blank">[107]</a>. Plastid-derived tRNAs with substitutions in their anticodons, such as <i>Citrullus trnT(ugu)</i> and <i>Silene latifolia trnP(ugg)</i>, are also classified (as pseudogenes) on the basis of homology.</p

Sequence divergence, genome size, and gene content in seed plant mitochondria.

<p>Branch lengths are scaled to the number of synonymous nucleotide substitution per site (<i>d</i>_S) on the basis of an analysis of all shared protein genes. Genome size ranges are reported for species with multiple sequences available. Gene counts exclude duplicates and putative pseudogenes.</p