Accelerated Evolution by Diversity-Generating Retroelements.

Diversity-generating retroelements (DGRs) create vast amounts of targeted, functional diversity by facilitating the rapid evolution of ligand-binding protein domains. Thousands of DGRs have been identified in bacteria, archaea, and their respective viruses. They are broadly distributed throughout the microbial world, with enrichment observed in certain taxa and environments. The diversification machinery works through a novel mechanism termed mutagenic retrohoming, whereby nucleotide sequence information is copied from an invariant DNA template repeat (TR) into an RNA intermediate, selectively mutagenized at TR adenines during cDNA synthesis by a DGR-encoded reverse transcriptase, and transferred to a variable repeat (VR) region within a variable-protein gene (54). This unidirectional flow of information leaves TR-DNA sequences unmodified, allowing for repeated rounds of mutagenic retrohoming to optimize variable-protein function. DGR target genes are often modular and can encode one or more of a wide variety of discrete functional domains appended to a diversifiable ligand-binding motif. Bacterial variable proteins often localize to cellsurfaces, although a subset appear to be cytoplasmic, while phage-encoded DGRs commonly diversify tail fiber-associated receptor-binding proteins. Here, we provide a comprehensive review of the mechanism and consequences of accelerated protein evolution by these unique and beneficial genetic elements.

Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor.

Chromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and ß-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the ß-globin promoter in GATA1 null erythroblasts, in which the ß-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the ß-globin promoter substantially activated ß-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.

Action of a minimal contractile bactericidal nanomachine.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1-4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

The temporal dynamics of task processing and choice in a novel multitasking paradigm.

This study investigated the temporal dynamics of task performance and voluntary task choice within a multitasking paradigm in which the task-related processing outcomes themselves determined the to-be-performed task. In the novel forced-no-go trials, the stimulus for one task required an overt response, but the stimulus for the other task was associated with a no-go response. Task performance results showed that participants often processed the no-go task's stimulus before switching to the go-task. Dual-task interference effects and switch costs indicated various forms of multitasking interference, with their underlying causes appearing to overlap, as engagement in parallel processing seemed to be limited by switch-related reconfiguration processes. Intermixing free-choice trials, where both stimuli were associated with overt responses, revealed costs associated with switching between processing modes, providing new evidence that the distinctions between free and forced task goals stem from differences in their internal representations rather than alterations in processing due to different presentations in the environment. Task choice results align with this perspective, demonstrating a preference for repeating a free- over a forced-choice task. Furthermore, these free-choice results illuminate the interplay of cognitive (task-repetition bias) and environmental constraints (first-task bias) in shaping task choices: It appears that task-specific information increases goal activations for both task goals concurrently, with participants favoring central processing of the second- over the first-presented task to optimize their behavior when shorter central processing is required (task repetition). Overall, this study offers new insights into the dynamics of task processing and choice in environments requiring the balance of multiple tasks.

How Many Participants? How Many Trials? Maximizing the Power of Reaction Time Studies.

Due to limitations in the resources available for carrying out reaction time (RT) experiments, researchers often have to choose between testing relatively few participants with relatively many trials each or testing relatively many participants with relatively few trials each. To compare the experimental power that would be obtained under each of these options, I simulated virtual experiments using subsets of participants and trials from eight large real RT datasets examining 19 experimental effects. The simulations compared designs using the first N T trials from N P randomly selected participants, holding constant the total number of trials across all participants, N P × N T . The [ N P , N T ] combination maximizing the power to detect each effect depended on how the mean and variability of that effect changed with practice. For most effects, power was greater in designs having many participants with few trials each rather than the reverse, suggesting that researchers should usually try to recruit large numbers of participants for short experimental sessions. In some cases, power for a fixed total number of trials across all participants was maximized by having as few as two trials per participant in each condition. Where researchers can make plausible predictions about how their effects will change over the course of a session, they can use those predictions to increase their experimental power.

Estimating the proportions and latencies of reaction time outliers: A pooling method and case study of lexical decision tasks.

A methodological problem in most reaction time (RT) studies is that some measured RTs may be outliers-that is, they may be very fast or very slow for reasons unconnected to the task-related processing of interest. Numerous ad hoc methods have been suggested to discriminate between such outliers and the valid RTs of interest, but it is extremely difficult to determine how well these methods work in practice because virtually nothing is known about the actual characteristics of outliers in real RT datasets. This article proposes a new method of pooling cumulative distribution function values for examining empirical RT distributions to assess both the proportions of outliers and their latencies relative to those of the valid RTs. As the method is developed, its strengths and weaknesses are examined using simulations based on previously suggested ad hoc models for RT outliers with particular assumed proportions and distributions of valid RTs and outliers. The method is then applied to several large RT datasets from lexical decision tasks, and the results provide the first empirically based description of outlier RTs. For these datasets, fewer than 1% of the RTs seem to be outliers, and the median outlier latency appears to be approximately 4-6 standard deviations of RT above the mean of the valid RT distribution.

Phylogeographic relationships and morphological evolution between cave and surface Astyanax mexicanus populations (De Filippi 1853) (Actinopterygii, Characidae).

The Astyanax mexicanus complex includes two different morphs, a surface- and a cave-adapted ecotype, found at three mountain ranges in Northeastern Mexico: Sierra de El Abra, Sierra de Guatemala and Sierra de la Colmena (Micos). Since their discovery, multiple studies have attempted to characterize the timing and the number of events that gave rise to the evolution of these cave-adapted ecotypes. Here, using RADseq and genome-wide sequencing, we assessed the phylogenetic relationships, genetic structure and gene flow events between the cave and surface Astyanax mexicanus populations, to estimate the tempo and mode of evolution of the cave-adapted ecotypes. We also evaluated the body shape evolution across different cave lineages using geometric morphometrics to examine the role of phylogenetic signal versus environmental pressures. We found strong evidence of parallel evolution of cave-adapted ecotypes derived from two separate lineages of surface fish and hypothesize that there may be up to four independent invasions of caves from surface fish. Moreover, a strong congruence between the genetic structure and geographic distribution was observed across the cave populations, with the Sierra de Guatemala the region exhibiting most genetic drift among the cave populations analysed. Interestingly, we found no evidence of phylogenetic signal in body shape evolution, but we found support for parallel evolution in body shape across independent cave lineages, with cavefish from the Sierra de El Abra reflecting the most divergent morphology relative to surface and other cavefish populations.

Optimizing Research Output: How Can Psychological Research Methods Be Improved?

Recent evidence suggests that research practices in psychology and many other disciplines are far less effective than previously assumed, which has led to what has been called a "crisis of confidence" in psychological research (e.g., Pashler & Wagenmakers 2012). In response to the perceived crisis, standard research practices have come under intense scrutiny, and various changes have been suggested to improve them. The burgeoning field of metascience seeks to use standard quantitative data-gathering and modeling techniques to understand the reasons for inefficiency, to assess the likely effects of suggested changes, and ultimately to tell psychologists how to do better science. We review the pros and cons of suggested changes, highlighting the many complex research trade-offs that must be addressed to identify better methods.

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell-cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

Percentile rank pooling: A simple nonparametric method for comparing group reaction time distributions with few trials.

Although many studies of reaction time (RT) focus on a single measure of central tendency such as the mean RT, a more detailed picture of the underlying processes can be gained by looking at full distributions of RTs. Unfortunately, for practical reasons it is sometimes difficult to obtain enough trials per participant in a condition of interest to construct such a distribution with existing methods. The purpose of this article is to propose a method of forming group RT distributions that can be used to compare the full distributions of RTs even in an infrequent condition with only a few trials per participant. In brief, the percentile ranks of each participant's infrequent-condition RTs are scored relative to a larger pool including that participant's RTs in other conditions, and a histogram of the infrequent-condition's percentile ranks is then formed by pooling across participants. The resulting histogram of infrequent-condition RT ranks shows where the RTs in that condition tend to fall relative to the other conditions, and this histogram can reveal systematic patterns in the infrequent-condition's RT distribution. To illustrate the method, I present histograms of the ranks of infrequent error RTs (~ 5% of trials), ranked relative to correct responses, in real data sets from Simon and lexical decision tasks.

Role of diversity-generating retroelements for regulatory pathway tuning in cyanobacteria.

BACKGROUND: Cyanobacteria maintain extensive repertoires of regulatory genes that are vital for adaptation to environmental stress. Some cyanobacterial genomes have been noted to encode diversity-generating retroelements (DGRs), which promote protein hypervariation through localized retrohoming and codon rewriting in target genes. Past research has shown DGRs to mainly diversify proteins involved in cell-cell attachment or viral-host attachment within viral, bacterial, and archaeal lineages. However, these elements may be critical in driving variation for proteins involved in other core cellular processes. RESULTS: Members of 31 cyanobacterial genera encode at least one DGR, and together, their retroelements form a monophyletic clade of closely-related reverse transcriptases. This class of retroelements diversifies target proteins with unique domain architectures: modular ligand-binding domains often paired with a second domain that is linked to signal response or regulation. Comparative analysis indicates recent intragenomic duplication of DGR targets as paralogs, but also apparent intergenomic exchange of DGR components. The prevalence of DGRs and the paralogs of their targets is disproportionately high among colonial and filamentous strains of cyanobacteria. CONCLUSION: We find that colonial and filamentous cyanobacteria have recruited DGRs to optimize a ligand-binding module for apparent function in signal response or regulation. These represent a unique class of hypervariable proteins, which might offer cyanobacteria a form of plasticity to adapt to environmental stress. This analysis supports the hypothesis that DGR-driven mutation modulates signaling and regulatory networks in cyanobacteria, suggestive of a new framework for the utility of localized genetic hypervariation.

Follistatin and Soluble Endoglin Predict 1-Year Nonrelapse Mortality after Allogeneic Hematopoietic Cell Transplantation.

Damage-associated angiogenic factors (AFs), including follistatin (FS) and soluble endoglin (sEng), are elevated in circulation at the onset of acute graft-versus-host disease (GVHD). We hypothesized that regimen-related tissue injury also might be associated with aberrant AF levels and sought to determine the relevance of these AF on nonrelapse mortality (NRM) in patients with acute GVHD and those without acute GVHD. To test our hypothesis, we analyzed circulating levels of FS, sEng, angiopoietin-2 (Ang2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) A and B, placental growth factor (PlGF), and soluble VEGF receptor (sVEGFR)-1 and -2, in plasma samples from patients enrolled on Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0402 (n = 221), which tested GVHD prophylaxis after myeloablative hematopoietic stem cell transplantation (HCT). We found that the interaction between FS and sEng had an additive effect in their association with 1-year NRM. In multivariate analysis, patients with the highest levels of day +28 FS and sEng had a 14.9-fold greater hazard ratio (HR) of NRM (95% confidence interval, 3.2 to 69.4; P < .01) when compared with low levels of FS and sEng. We validated these findings using an external cohort of patients (n = 106). Pre-HCT measurements of FS and sEng were not associated with NRM, suggesting that elevations in these factors early post-HCT may be consequences of early regimen-related toxicity. Determining the mechanisms responsible for patient-specific vulnerability to treatment toxicities and endothelial damage associated with specific AF elevation may guide interventions to reduce NRM post-HCT.

Beyond mean reaction times: Combining distributional analyses with processing stage manipulations in the Simon task.

We combined analyses of reaction time (RT) distributions with experimental manipulations of different processing stages (perception, decision, motor execution) in a Simon task to investigate which changes in Simon effects could be explained entirely by fading irrelevant response activation. Consistent with fading activation accounts, the Simon effect on mean RT was usually smaller for conditions with slower responses (Expts. 1-3 but not Expt. 4), and delta plot analyses revealed that it was always smaller for the slower responses within each condition. Critically, however, these analyses also revealed that some experimental manipulations produced upward or downward shifts in the RT delta plots, thus altering the Simon effect on mean RT in ways that could not be explained by fading activation. The results demonstrate the power of combining RT distributional analyses with experimental manipulations to reveal mechanisms contributing to the Simon effect that would not be revealed using only mean RT. We consider alternatives to fading activation accounts of decreasing delta plots and discuss the contribution of different cognitive stages in modulating Simon effects.

Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey.

Diversity-generating retroelements (DGRs) are novel genetic elements that use reverse transcription to generate vast numbers of sequence variants in specific target genes. Here, we present a detailed comparative bioinformatic analysis that depicts the landscape of DGR sequences in nature as represented by data in GenBank. Over 350 unique DGRs are identified, which together form a curated reference set of putatively functional DGRs. We classify target genes, variable repeats and DGR cassette architectures, and identify two new accessory genes. The great variability of target genes implies roles of DGRs in many undiscovered biological processes. There is much evidence for horizontal transfers of DGRs, and we identify lineages of DGRs that appear to have specialized properties. Because GenBank contains data from only 10% of described species, the compilation may not be wholly representative of DGRs present in nature. Indeed, many DGR subtypes are present only once in the set and DGRs of the candidate phylum radiation bacteria, and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea archaea, are exceptionally diverse in sequence, with little information available about functions of their target genes. Nonetheless, this study provides a detailed framework for classifying and studying DGRs as they are uncovered and studied in the future.

Are model parameters linked to processing stages? An empirical investigation for the ex-Gaussian, ex-Wald, and EZ diffusion models.

In previous research, the parameters of the ex-Gaussian distribution have been subject to a wide variety of interpretations. The present study investigated whether the ex-Gaussian model is capable of distinguishing effects on separate processing stages (i.e., pre-motor vs. motor). In order to do so, we used datasets where the locus of effect was quite clear. Specifically, we analyzed data from experiments comparing hand vs. foot responses-presumably differing in the motor stage-and from experiments in which the lateralized readiness potential was used to localize experimental effects into premotor vs. motor processes. Moreover, we broadened the scope to two other descriptive RT models: the ex-Wald and EZ diffusion models. To the extent possible with each of these models, we reanalyzed the RT data of 19 clearly localized experimental effects from 12 separate experiments reported in seven previously published articles. Unfortunately, we did not find a clear pattern of results for any of the models, with no clear link between effects on one of the model's parameters and effects on different processing stages. The present results suggest that one should resist the temptation to associate specific processing stages with individual parameters of the ex-Gaussian, ex-Wald, and EZ diffusion models.

DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat (TR) to a variable repeat (VR) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the Bordetella phage DGR is primed by an adenine residue in TR RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is VR-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of TR RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation.

Incorporation of a chiral gem-disubstituted nitrogen heterocycle yields an oxazolidinone antibiotic with reduced mitochondrial toxicity.

gem-Disubstituted N-heterocycles are rarely found in drugs, despite their potential to improve the drug-like properties of small molecule pharmaceuticals. Linezolid, a morpholine heterocycle-containing oxazolidinone antibiotic, exhibits significant side effects associated with human mitochondrial protein synthesis inhibition. We synthesized a gem-disubstituted linezolid analogue that when compared to linezolid, maintains comparable (albeit slightly diminished) activity against bacteria, comparable in vitro physicochemical properties, and a decrease in undesired mitochondrial protein synthesis (MPS) inhibition. This research contributes to the structure-activity-relationship data surrounding oxazolidinone MPS inhibition, and may inspire investigations into the utility of gem-disubstituted N-heterocycles in medicinal chemistry.

Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-σ factor.

The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

BACKGROUND: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. RESULTS: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. CONCLUSIONS: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.