Multiplexed shotgun genotyping (MSG) data resolve phylogenetic relationships within and among archipelagos in Macaronesian Tolpis.

PREMISE: Plants endemic to oceanic archipelagos are suitable for studying evolution, being isolated on substrates of different ages. Evolution has been recent, rendering traditionally employed sequences insufficiently variable for resolving relationships. This study includes sampling in the genus Tolpis (Asteraceae) from the Azores, Madeira, and Cape Verde, and expands upon an earlier study demonstrating the efficacy of multiplexed shotgun genotyping (MSG) for resolving relationships in Canarian Tolpis. METHODS: Genomic libraries for 90 accessions of Tolpis and two from the outgroup were generated for genotyping individuals using MSG. Loci were de novo assembled with iPyrad, which clusters MSG loci within and between samples. A maximum likelihood phylogeny was generated with RAxML. Ancestral area reconstruction was inferred using R package BioGeoBEARS. RESULTS: MSG data recovered a highly resolved phylogeny from population to inter-archipelago levels. Ancestral area reconstruction provided biogeographic hypotheses for the radiation of Macaronesian Tolpis. CONCLUSIONS: Four major clades were resolved. The Madeiran endemic T. macrorhiza is sister to other Tolpis. Species from the Canaries, Cape Verdes, and the continent are sister to T. succulenta from Madeira, which has a sister subclade of Azorean populations composed of T. succulenta and T. azorica. Population-level resolution suggests unrecognized taxa on several archipelagos. Ancestral reconstruction suggests initial dispersal from the continent to Madeira, with dispersal to the Azores, then dispersal from Madeira to the Canary Islands, with both subsequent dispersal to the Cape Verdes and back-dispersal to the continent. Single-island radiations and inter-island dispersal are implicated in divergence in Macaronesian Tolpis.

Thoracic Duct Embolization Using Transabdominal Antegrade and Transcervical Retrograde Accesses.

PURPOSE: To evaluate the technical success and clinical outcomes of thoracic duct embolization (TDE) using transabdominal antegrade and transcervical retrograde accesses to treat patients with chyle leak. MATERIALS AND METHODS: This study was a retrospective, nonblinded, single-institution chart review of all patients aged 18 years or older over a 6-year time frame who underwent lymphangiography with attempted TDE for iatrogenic or spontaneous chyle leaks using transabdominal antegrade and/or transcervical retrograde accesses. RESULTS: Ninety-nine patients underwent 113 procedures. Eighty-five patients underwent 1 procedure, and 14 patients required 2 procedures. The technical success rate of TDE was 68% (72/106) with transabdominal antegrade access and 44% (15/34) with transcervical retrograde access. The overall technical success rate of TDE, including both the access methods, was 77% (87/113). The most common reasons for transabdominal access failure were small caliber of the cisterna chyli and thoracic duct (TD) occlusion. Five patients were lost to follow-up. Overall clinical success, defined as resolution of the chyle leak, was achieved in 83% (78/94) of the patients. There were 6 Society of Interventional Radiology (SIR) level 1 adverse events (AEs), 5 SIR level 2 AEs, and 2 SIR level 3 AEs. Nontarget embolization occurred in 2 patients. CONCLUSIONS: Although transcervical retrograde TDE is a challenging procedure, with a lower technical success rate than transabdominal antegrade access, retrograde access improved the technical and clinical success rates of the treatment of chyle leaks in cases of thoracic duct occlusion, small cisterna chyli, and leaks located in the abdomen.

Prey tells, large herbivores fear the human 'super predator'.

Fear of the human 'super predator' has been demonstrated to so alter the feeding behavior of large carnivores as to cause trophic cascades. It has yet to be experimentally tested if fear of humans has comparably large effects on the feeding behavior of large herbivores. We conducted a predator playback experiment exposing white-tailed deer to the vocalizations of humans, extant or locally extirpated non-human predators (coyotes, cougars, dogs, wolves), or non-predator controls (birds), at supplemental food patches to measure the relative impacts on deer feeding behavior. Deer were more than twice as likely to flee upon hearing humans than other predators, and hearing humans was matched only by hearing wolves in reducing overall feeding time gaged by visits to the food patch in the following hour. Combined with previous, site-specific research linking deer fecundity to predator abundance, this study reveals that fear of humans has the potential to induce a larger effect on ungulate reproduction than has ever been reported. By demonstrating that deer most fear the human 'super predator', our results point to the fear humans induce in large ungulates having population- and community-level impacts comparable to those caused by the fear humans induce in large carnivores.

ELX-02 Generates Protein via Premature Stop Codon Read-Through without Inducing Native Stop Codon Read-Through Proteins.

ELX-02 is a clinical stage, small-molecule eukaryotic ribosomal selective glycoside acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5 mg/kg). These samples were processed to collect white blood cells and then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100 µg/ml) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense-mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified by using proteomic analysis was lower than estimated, and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles. SIGNIFICANCE STATEMENT: ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here by using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases.

Dose reduction using digital fluoroscopy versus digital subtraction angiography in endovascular aneurysm repair: A prospective randomized trial.

OBJECTIVE: Endovascular aneurysm repair (EVAR) can result in high radiation dose to patients and operators. This prospective randomized study aimed to assess whether patient radiation dose sustained during EVAR could be decreased by predominantly using digital fluoroscopy (DF) vs the standard technique using digital subtraction angiography (DSA). METHODS: Between February 2011 and June 2017, patients with EVAR of infrarenal abdominal aortic aneurysms were prospectively enrolled and randomly assigned to a standard treatment DSA cohort or a DF cohort in which two or fewer DSA acquisitions were allowed for confirmatory imaging. Primary end points included dose-area product (DAP) and cumulative air kerma. Secondary end points included technical success and conversion to DSA standard treatment (if DF was inadequate for visualization). RESULTS: For all 43 patients enrolled (26 in the DF cohort, 17 in the DSA cohort), technical success was 100%. Of the 26 DF patients, 5 (19%) required conversion to the DSA cohort. In an intention-to-treat analysis, mean DAP was significantly lower in the DF cohort than in the DSA cohort (132 vs 174 Gy·cm2; P = .04). When patients were separated by number of DSA acquisitions (two or fewer vs three or more), mean DAP decreased 41% (109 vs 185 Gy·cm2; P = .005) and cumulative air kerma decreased 40% (578 vs 964 mGy; P = .004). CONCLUSIONS: In most patients (81%), DF or limited DSA was adequate for visualization during EVAR. In both intention-to-treat DF and limited-DSA cohorts, mean DAP was significantly decreased. If image quality allows, a DF-only or limited-DSA approach to EVAR decreases radiation dose.

Intravitreal administration of small molecule read-through agents demonstrate functional activity in a nonsense mutation mouse model.

The prevalence of nonsense mutations as a class within genetic diseases such as inherited retinal disorders (IRDs) presents an opportunity to develop a singular, common therapeutic agent for patients whose treatment options are otherwise limited. We propose a novel approach to addressing IRDs utilizing Eukaryotic Ribosome Selective Glycosides, ELX-01 and ELX-06, delivered to the eye by intravitreal (IVT) injection. We assessed read-through activity in vitro using a plasmid-based dual luciferase assay and in vivo in a mouse model of oculocutaneous albinism type 2. These models interrogate a naturally occurring R262X nonsense mutation in the OCA2 gene. ELX-01 and ELX-06 both produced a concentration-dependent increase in read-through of the OCA2 R262X mutation in the dual luciferase assay, with an effect at the top concentration which is superior to both gentamicin and G418. When testing both compounds in vivo, a single IVT injection produced a dose-dependent increase in melanin, consistent with compound read-through activity and functional restoration of the Oca2 protein. These results establish that ELX-01 and ELX-06 produce read-through of a premature stop codon in the OCA2 gene both in vitro and in vivo. The in vivo results suggest that these compounds can be dosed IVT to achieve read-through at the back of the eye. These data also suggest that ELX-01 or ELX-06 could serve as a common therapeutic agent across nonsense mutation-mediated IRDs and help to establish a target exposure range for development of a sustained release IVT formulation.

Genome-wide genotyping estimates mating system parameters and paternity in the island species Tolpis succulenta.

PREMISE: The mating system has profound consequences, not only for ecology and evolution, but also for the conservation of threatened or endangered species. Unfortunately, small populations are difficult to study owing to limits on sample size and genetic marker diversity. Here, we estimated mating system parameters in three small populations of an island plant using genomic genotyping. Although self-incompatible (SI) species are known to often set some self-seed, little is known about how "leaky SI" affects selfing rates in nature or the role that multiple paternity plays in small populations. METHODS: We generalized the BORICE mating system program to determine the siring pattern within maternal families. We applied this algorithm to maternal families from three populations of Tolpis succulenta from Madeira Island and genotyped the progeny using RADseq. We applied BORICE to estimate each individual offspring as outcrossed or selfed, the paternity of each outcrossed offspring, and the level of inbreeding of each maternal plant. RESULTS: Despite a functional self-incompatibility system, these data establish T. succulenta as a pseudo-self-compatible (PSC) species. Two of 75 offspring were strongly indicated as products of self-fertilization. Despite selfing, all adult maternal plants were fully outbred. There was high differentiation among and low variation within populations, consistent with a history of genetic isolation of these small populations. There were generally multiple sires per maternal family. Twenty-two percent of sib contrasts (between outcrossed offspring within maternal families) shared the same sire. CONCLUSIONS: Genome-wide genotyping, combined with appropriate analytical methods, enables estimation of mating system and multiple paternity in small populations. These data address questions about the evolution of reproductive traits and the conservation of threatened populations.

Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine.

Ten-eleven translocation (TET) enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) to yield 5-hydroxymethylcytosine (hmC) and the rarer bases 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Stepwise oxidation obscures how each individual base forms and functions in epigenetic regulation, and prompts the question of whether TET enzymes primarily serve to generate hmC or are adapted to produce fC and caC as well. By mutating a single, conserved active site residue in human TET2, Thr1372, we uncovered enzyme variants that permit oxidation to hmC but largely eliminate fC and caC. Biochemical analyses, combined with molecular dynamics simulations, elucidated an active site scaffold that is required for wild-type (WT) stepwise oxidation and that, when perturbed, explains the mutants' hmC-stalling phenotype. Our results suggest that the TET2 active site is shaped to enable higher-order oxidation and provide the first TET variants that could be used to probe the biological functions of hmC separately from fC and caC.

Correction to: Factors driving adaptive radiation in plants of oceanic islands: a case study from the Juan Fernández Archipelago.

The article Factors driving adaptive radiation in plants of oceanic islands: a case study from the Juan Fernández Archipelago, written by Koji Takayama, Daniel J. Crawford, Patricio López­Sepúlveda, Josef Greimler, Tod F. Stuessy was originally published electronically on the publisher's internet portal (currently SpringerLink) on 13 March 2018 without open access.

Factors driving adaptive radiation in plants of oceanic islands: a case study from the Juan Fernández Archipelago.

Adaptive radiation is a common evolutionary phenomenon in oceanic islands. From one successful immigrant population, dispersal into different island environments and directional selection can rapidly yield a series of morphologically distinct species, each adapted to its own particular environment. Not all island immigrants, however, follow this evolutionary pathway. Others successfully arrive and establish viable populations, but they remain in the same ecological zone and only slowly diverge over millions of years. This transformational speciation, or anagenesis, is also common in oceanic archipelagos. The critical question is why do some groups radiate adaptively and others not? The Juan Fernández Islands contain 105 endemic taxa of angiosperms, 49% of which have originated by adaptive radiation (cladogenesis) and 51% by anagenesis, hence providing an opportunity to examine characteristics of taxa that have undergone both types of speciation in the same general island environment. Life form, dispersal mode, and total number of species in progenitors (genera) of endemic angiosperms in the archipelago were investigated from literature sources and compared with modes of speciation (cladogenesis vs. anagenesis). It is suggested that immigrants tending to undergo adaptive radiation are herbaceous perennial herbs, with leaky self-incompatible breeding systems, good intra-island dispersal capabilities, and flexible structural and physiological systems. Perhaps more importantly, the progenitors of adaptively radiated groups in islands are those that have already been successful in adaptations to different environments in source areas, and which have also undergone eco-geographic speciation. Evolutionary success via adaptive radiation in oceanic islands, therefore, is less a novel feature of island lineages but rather a continuation of tendency for successful adaptive speciation in lineages of continental source regions.

The genetic breakdown of sporophytic self-incompatibility in Tolpis coronopifolia (Asteraceae).

Angiosperm diversity has been shaped by mating system evolution, with the most common transition from outcrossing to self-fertilizing. To investigate the genetic basis of this transition, we performed crosses between two species endemic to the Canary Islands, the self-compatible (SC) species Tolpis coronopifolia and its self-incompatible (SI) relative Tolpis santosii. We scored self-compatibility as self-seed set of recombinant plants within two F2 populations. To map and genetically characterize the breakdown of SI, we built a draft genome sequence of T. coronopifolia, genotyped F2 plants using multiplexed shotgun genotyping (MSG), and located MSG markers to the genome sequence. We identified a single quantitative trait locus (QTL) that explains nearly all variation in self-seed set in both F2 populations. To identify putative causal genetic variants within the QTL, we performed transcriptome sequencing on mature floral tissue from both SI and SC species, constructed a transcriptome for each species, and then located each predicted transcript to the T. coronopifolia genome sequence. We annotated each predicted gene within the QTL and found two strong candidates for SI breakdown. Each gene has a coding sequence insertion/deletion mutation within the SC species that produces a truncated protein. Homologs of each gene have been implicated in pollen development, pollen germination, and pollen tube growth in other species.

Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism.

Modification of cytosine-guanine dinucleotides (CpGs) is a key part of mammalian epigenetic regulation and helps shape cellular identity. Tet enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) in CpGs to 5-hydroxymethylcytosine (hmC), or onward to 5-formylcytosine (fC) or 5-carboxylcytosine (caC). The multiple mC oxidation products, while intricately linked, are postulated to play independent epigenetic roles, making it critical to understand how the products of stepwise oxidation are established and maintained. Using highly sensitive isotope-based studies, we newly show that Tet2 can yield fC and caC by iteratively acting in a single encounter with mC-containing DNA, without release of the hmC intermediate, and that the modification state of the complementary CpG has little impact on Tet2 activity. By revealing Tet2 as an iterative, de novo mC oxygenase, our study provides insight into how features intrinsic to Tet2 shape the epigenetic landscape.

Different modes of retrovirus restriction by human APOBEC3A and APOBEC3G in vivo.

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.

High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.

Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9-11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function.

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site.

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.

Relationships and genetic consequences of contrasting modes of speciation among endemic species of Robinsonia (Asteraceae, Senecioneae) of the Juan Fernández Archipelago, Chile, based on AFLPs and SSRs.

This study analyses and compares the genetic signatures of anagenetic and cladogenetic speciation in six species of the genus Robinsonia (Asteraceae, Senecioneae), endemic to the Juan Fernández Islands, Chile. Population genetic structure was analyzed by amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeat, SSR) markers from 286 and 320 individuals, respectively, in 28 populations. Each species is genetically distinct. Previous hypotheses of classification among these species into subgenera and sections, via morphological, phytochemical, isozymic and internal transcribed spacer (ITS) data, have been confirmed, except that R. saxatilis appears to be related to R. gayana rather than R. evenia. Analysis of phylogenetic results and biogeographic context suggests that five of these species have originated by cladogenesis and adaptive radiation on the older Robinson Crusoe Island. The sixth species, R. masafuerae, restricted to the younger Alejandro Selkirk Island, is closely related to and an anagenetic derivative of R. evenia from Robinson Crusoe. Microsatellite and AFLP data reveal considerable genetic variation among the cladogenetically derived species of Robinsonia, but within each the genetic variation is lower, highlighting presumptive genetic isolation and rapid radiation. The anagenetically derived R. masafuerae harbors a level of genetic variation similar to that of its progenitor, R. evenia. This is the first direct comparison of the genetic consequences of anagenetic and cladogenetic speciation in plants of an oceanic archipelago.

Multiplexed-shotgun-genotyping data resolve phylogeny within a very recently derived insular lineage.

PREMISE OF THE STUDY: Endemic plants on oceanic islands have long served as model systems for studying patterns and processes of evolution. However, phylogenetic studies of island plants frequently illustrate a decoupling of molecular divergence and ecological/morphological diversity, resulting in phylogenies lacking the resolution required to interpret patterns of evolution in a phylogenetic context. The current study uses the primarily Macaronesian flowering plant genus Tolpis to illustrate the utility of multiplexed shotgun genotyping (MSG) for resolving relationships at relatively deep (among archipelagos) and very shallow (within archipelagos) nodes in this small, yet diverse insular plant lineage that had not been resolved with other molecular markers. METHODS: Genomic libraries for 27 accessions of Macaronesian Tolpis were generated for genotyping individuals using MSG, a form of reduced-representation sequencing, similar to restriction-site-associated DNA markers (RADseq). The resulting data files were processed using the program pyRAD, which clusters MSG loci within and between samples. Phylogenetic analyses of the aligned data matrix were conducted using RAxML. KEY RESULTS: Analysis of MSG data recovered a highly resolved phylogeny with generally strong support, including the first robust inference of relationships within the highly diverse Canary Island clade of Tolpis. CONCLUSIONS: The current study illustrates the utility of MSG data for resolving relationships in lineages that have undergone recent, rapid diversification resulting in extensive ecological and morphological diversity. We suggest that a similar approach may prove generally useful for other rapid plant radiations where resolving phylogeny has been difficult.