Concerted cutting by Spo11 illuminates meiotic DNA break mechanics.

Genetic recombination arises during meiosis through the repair of DNA double-strand breaks (DSBs) that are created by Spo11, a topoisomerase-like protein1,2. Spo11 DSBs form preferentially in nucleosome-depleted regions termed hotspots3,4, yet how Spo11 engages with its DNA substrate to catalyse DNA cleavage is poorly understood. Although most recombination events are initiated by a single Spo11 cut, here we show in Saccharomyces cerevisiae that hyperlocalized, concerted Spo11 DSBs separated by 33 to more than 100 base pairs also form, which we term 'double cuts'. Notably, the lengths of double cuts vary with a periodicity of 10.5 base pairs, which is conserved in yeast and mice. This finding suggests a model in which the orientation of adjacent Spo11 molecules is fixed relative to the DNA helix-a proposal supported by the in vitro DNA-binding properties of the Spo11 core complex. Deep sequencing of meiotic progeny identifies recombination scars that are consistent with repair initiated from gaps generated by adjacent Spo11 DSBs. Collectively, these results revise our present understanding of the mechanics of Spo11-DSB formation and expand on the original concepts of gap repair during meiosis to include DNA gaps that are generated by Spo11 itself.

Costs of antibiotic resistance genes depend on host strain and environment and can influence community composition.

Antibiotic resistance genes (ARGs) benefit host bacteria in environments containing corresponding antibiotics, but it is less clear how they are maintained in environments where antibiotic selection is weak or sporadic. In particular, few studies have measured if the direct effect of ARGs on host fitness is fixed or if it depends on the host strain, perhaps marking some ARG-host combinations as selective refuges that can maintain ARGs in the absence of antibiotic selection. We quantified the fitness effects of six ARGs in 11 diverse Escherichia spp. strains. Three ARGs (blaTEM-116, cat and dfrA5, encoding resistance to ß-lactams, chloramphenicol, and trimethoprim, respectively) imposed an overall cost, but all ARGs had an effect in at least one host strain, reflecting a significant strain interaction effect. A simulation predicts these interactions can cause the success of ARGs to depend on available host strains, and, to a lesser extent, can cause host strain success to depend on the ARGs present in a community. These results indicate the importance of considering ARG effects across different host strains, and especially the potential of refuge strains to allow resistance to persist in the absence of direct selection, in efforts to understand resistance dynamics.

Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon.

Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.

Fitness of evolving bacterial populations is contingent on deep and shallow history but only shallow history creates predictable patterns.

Long-term evolution experiments have tested the importance of genetic and environmental factors in influencing evolutionary outcomes. Differences in phylogenetic history, recent adaptation to distinct environments and chance events, all influence the fitness of a population. However, the interplay of these factors on a population's evolutionary potential remains relatively unexplored. We tracked the outcome of 2000 generations of evolution of four natural isolates of Escherichia coli bacteria that were engineered to also create differences in shallow history by adding previously identified mutations selected in a separate long-term experiment. Replicate populations started from each progenitor evolved in four environments. We found that deep and shallow phylogenetic histories both contributed significantly to differences in evolved fitness, though by different amounts in different selection environments. With one exception, chance effects were not significant. Whereas the effect of deep history did not follow any detectable pattern, effects of shallow history followed a pattern of diminishing returns whereby fitter ancestors had smaller fitness increases. These results are consistent with adaptive evolution being contingent on the interaction of several evolutionary forces but demonstrate that the nature of these interactions is not fixed and may not be predictable even when the role of chance is small.

Predicting microbial growth in a mixed culture from growth curve data.

Determining the fitness of specific microbial genotypes has extensive application in microbial genetics, evolution, and biotechnology. While estimates from growth curves are simple and allow high throughput, they are inaccurate and do not account for interactions between costs and benefits accruing over different parts of a growth cycle. For this reason, pairwise competition experiments are the current "gold standard" for accurate estimation of fitness. However, competition experiments require distinct markers, making them difficult to perform between isolates derived from a common ancestor or between isolates of nonmodel organisms. In addition, competition experiments require that competing strains be grown in the same environment, so they cannot be used to infer the fitness consequence of different environmental perturbations on the same genotype. Finally, competition experiments typically consider only the end-points of a period of competition so that they do not readily provide information on the growth differences that underlie competitive ability. Here, we describe a computational approach for predicting density-dependent microbial growth in a mixed culture utilizing data from monoculture and mixed-culture growth curves. We validate this approach using 2 different experiments with Escherichia coli and demonstrate its application for estimating relative fitness. Our approach provides an effective way to predict growth and infer relative fitness in mixed cultures.

Dynamics of bacterial adaptation.

Determining pattern in the dynamics of population evolution is a long-standing focus of evolutionary biology. Complementing the study of natural populations, microbial laboratory evolution experiments have become an important tool for addressing these dynamics because they allow detailed and replicated analysis of evolution in response to controlled environmental and genetic conditions. Key findings include a tendency for smoothly declining rates of adaptation during selection in constant environments, at least in part a reflection of antagonism between accumulating beneficial mutations, and a large number of beneficial mutations available to replicate populations leading to significant, but relatively low genetic parallelism, even as phenotypic characteristics show high similarity. Together, there is a picture of adaptation as a process with a varied and largely unpredictable genetic basis leading to much more similar phenotypic outcomes. Increasing sophistication of sequencing and genetic tools will allow insight into mechanisms behind these and other patterns.

Tel1(ATM)-mediated interference suppresses clustered meiotic double-strand-break formation.

Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)--a process that is mediated by the conserved DNA damage response kinase, Tel1(ATM). The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1(ATM) prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.

The impact and recovery of asteroid 2018 LA.

The June 2, 2018, impact of asteroid 2018 LA over Botswana is only the second asteroid detected in space prior to impacting over land. Here, we report on the successful recovery of meteorites. Additional astrometric data refine the approach orbit and define the spin period and shape of the asteroid. Video observations of the fireball constrain the asteroid's position in its orbit and were used to triangulate the location of the fireball's main flare over the Central Kalahari Game Reserve. 23 meteorites were recovered. A consortium study of eight of these classifies Motopi Pan as a HED polymict breccia derived from howardite, cumulate and basaltic eucrite, and diogenite lithologies. Before impact, 2018 LA was a solid rock of ~156 cm diameter with high bulk density ~2.85 g/cm3, a relatively low albedo pv ~ 0.25, no significant opposition effect on the asteroid brightness, and an impact kinetic energy of ~0.2 kt. The orbit of 2018 LA is consistent with an origin at Vesta (or its Vestoids) and delivery into an Earth-impacting orbit via the v6 resonance. The impact that ejected 2018 LA in an orbit towards Earth occurred 22.8 ± 3.8 Ma ago. Zircons record a concordant U-Pb age of 4563 ± 11 Ma and a consistent 207Pb/206Pb age of 4563 ± 6 Ma. A much younger Pb-Pb phosphate resetting age of 4234 ± 41 Ma was found. From this impact chronology, we discuss what is the possible source crater of Motopi Pan and the age of Vesta's Veneneia impact basin.

Effects of Beneficial Mutations in pykF Gene Vary over Time and across Replicate Populations in a Long-Term Experiment with Bacteria.

The fitness effects of mutations can depend on the genetic backgrounds in which they occur and thereby influence future opportunities for evolving populations. In particular, mutations that fix in a population might change the selective benefit of subsequent mutations, giving rise to historical contingency. We examine these effects by focusing on mutations in a key metabolic gene, pykF, that arose independently early in the history of 12 Escherichia coli populations during a long-term evolution experiment. Eight different evolved nonsynonymous mutations conferred similar fitness benefits of ∼10% when transferred into the ancestor, and these benefits were greater than the one conferred by a deletion mutation. In contrast, the same mutations had highly variable fitness effects, ranging from ∼0% to 25%, in evolved clones isolated from the populations at 20,000 generations. Two mutations that were moved into these evolved clones conferred similar fitness effects in a given clone, but different effects between the clones, indicating epistatic interactions between the evolved pykF alleles and the other mutations that had accumulated in each evolved clone. We also measured the fitness effects of six evolved pykF alleles in the same populations in which they had fixed, but at seven time points between 0 and 50,000 generations. Variation in fitness effects was high at intermediate time points, and declined to a low level at 50,000 generations, when the mean fitness effect was lowest. Our results demonstrate the importance of genetic context in determining the fitness effects of different beneficial mutations even within the same gene.

The fitness challenge of studying molecular adaptation.

Advances in bioinformatics and high-throughput genetic analysis increasingly allow us to predict the genetic basis of adaptive traits. These predictions can be tested and confirmed, but the molecular-level changes - i.e. the molecular adaptation - that link genetic differences to organism fitness remain generally unknown. In recent years, a series of studies have started to unpick the mechanisms of adaptation at the molecular level. In particular, this work has examined how changes in protein function, activity, and regulation cause improved organismal fitness. Key to addressing molecular adaptations is identifying systems and designing experiments that integrate changes in the genome, protein chemistry (molecular phenotype), and fitness. Knowledge of the molecular changes underpinning adaptations allow new insight into the constraints on, and repeatability of adaptations, and of the basis of non-additive interactions between adaptive mutations. Here we critically discuss a series of studies that examine the molecular-level adaptations that connect genetic changes and fitness.

Benefit of transferred mutations is better predicted by the fitness of recipients than by their ecological or genetic relatedness.

The effect of a mutation depends on its interaction with the genetic background in which it is assessed. Studies in experimental systems have demonstrated that such interactions are common among beneficial mutations and often follow a pattern consistent with declining evolvability of more fit genotypes. However, these studies generally examine the consequences of interactions between a small number of focal mutations. It is not clear, therefore, that findings can be extrapolated to natural populations, where new mutations may be transferred between genetically divergent backgrounds. We build on work that examined interactions between four beneficial mutations selected in a laboratory-evolved population of Escherichia coli to test how they interact with the genomes of diverse natural isolates of the same species. We find that the fitness effect of transferred mutations depends weakly on the genetic and ecological similarity of recipient strains relative to the donor strain in which the mutations were selected. By contrast, mutation effects were strongly inversely correlated to the initial fitness of the recipient strain. That is, there was a pattern of diminishing returns whereby fit strains benefited proportionally less from an added mutation. Our results strengthen the view that the fitness of a strain can be a major determinant of its ability to adapt. They also support a role for barriers of transmission, rather than differential selection of transferred DNA, as an explanation of observed phylogenetically determined patterns of restricted recombination among E. coli strains.

Cellular Growth Arrest and Persistence from Enzyme Saturation.

Metabolic efficiency depends on the balance between supply and demand of metabolites, which is sensitive to environmental and physiological fluctuations, or noise, causing shortages or surpluses in the metabolic pipeline. How cells can reliably optimize biomass production in the presence of metabolic fluctuations is a fundamental question that has not been fully answered. Here we use mathematical models to predict that enzyme saturation creates distinct regimes of cellular growth, including a phase of growth arrest resulting from toxicity of the metabolic process. Noise can drive entry of single cells into growth arrest while a fast-growing majority sustains the population. We confirmed these predictions by measuring the growth dynamics of Escherichia coli utilizing lactose as a sole carbon source. The predicted heterogeneous growth emerged at high lactose concentrations, and was associated with cell death and production of antibiotic-tolerant persister cells. These results suggest how metabolic networks may balance costs and benefits, with important implications for drug tolerance.

The environment affects epistatic interactions to alter the topology of an empirical fitness landscape.

The fitness effect of mutations can be influenced by their interactions with the environment, other mutations, or both. Previously, we constructed 32 ( = 25) genotypes that comprise all possible combinations of the first five beneficial mutations to fix in a laboratory-evolved population of Escherichia coli. We found that (i) all five mutations were beneficial for the background on which they occurred; (ii) interactions between mutations drove a diminishing returns type epistasis, whereby epistasis became increasingly antagonistic as the expected fitness of a genotype increased; and (iii) the adaptive landscape revealed by the mutation combinations was smooth, having a single global fitness peak. Here we examine how the environment influences epistasis by determining the interactions between the same mutations in two alternative environments, selected from among 1,920 screened environments, that produced the largest increase or decrease in fitness of the most derived genotype. Some general features of the interactions were consistent: mutations tended to remain beneficial and the overall pattern of epistasis was of diminishing returns. Other features depended on the environment; in particular, several mutations were deleterious when added to specific genotypes, indicating the presence of antagonistic interactions that were absent in the original selection environment. Antagonism was not caused by consistent pleiotropic effects of individual mutations but rather by changing interactions between mutations. Our results demonstrate that understanding adaptation in changing environments will require consideration of the combined effect of epistasis and pleiotropy across environments.

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming.

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors.

Homeostatic regulation of meiotic DSB formation by ATM/ATR.

Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.

Adaptive evolution of the lactose utilization network in experimentally evolved populations of Escherichia coli.

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network.

Diagnosis and management of dementia in primary care: exploratory study.

OBJECTIVE: To assess the current identification and management of patients with dementia in a primary care setting; to determine the accuracy of identification of dementia by primary care physicians; to examine reasons (triggers) for referral of patients with suspected dementia to the geriatric assessment team (GAT) from the primary care setting; and to compare indices of identification and management of dementia between the GAT and primary care network (PCN) physicians and between the GAT and community care (CC). DESIGN: Retrospective chart review and comparisons, based on quality indicators of dementia care as specified in the Third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia, were conducted from matching charts obtained from 3 groups of health care providers. SETTING: Semirural region in the province of Alberta involving a PCN, CC, and a GAT. PARTICIPANTS: One hundred patients who had been assessed by the GAT randomly selected from among those diagnosed with dementia or mild cognitive impairment by the GAT. MAIN OUTCOME MEASURES: Diagnosis of dementia and indications of high-quality dementia care listed in PCN, CC, and GAT charts. RESULTS: Only 59% of the patients diagnosed with dementia by the GAT had a documented diagnosis of dementia in their PCN charts. None of the 12 patients diagnosed with mild cognitive impairment by the GAT had been diagnosed by the PCN. Memory decline was the most common reason for referral to the GAT. There were statistically significant differences between the PCN and the GAT on all quality indicators of dementia, with underuse of diagnostic and functional assessment tools and lack of attention to wandering, driving, medicolegal, and caregiver issues, and underuse of community supports in the PCN. There was higher congruence between CC and the GAT on assessment and care indices. CONCLUSION: Dementia care remains a challenge in primary care. Within our primary care setting, there are opportunities for synergistic collaboration among the health care professionals from the PCN, CC, and the GAT. Currently they exist as individual entities in the system. An integrated model of care is required in order to build capacity to meet the needs of an aging population.

Interaction with a phage gene underlie costs of a ß-lactamase.

The fitness cost of an antibiotic resistance gene (ARG) can differ across host strains, creating refuges that allow the maintenance of an ARG in the absence of direct selection for its resistance phenotype. Despite the importance of such ARG-host interactions for predicting ARG dynamics, the basis of ARG fitness costs and their variability between hosts are not well understood. We determined the genetic basis of a host-dependent cost of a ß-lactamase, blaTEM-116*, that conferred a significant cost in one Escherichia coli strain but was close to neutral in 11 other Escherichia spp. strains. Selection of a blaTEM-116*-encoding plasmid in the strain in which it initially had a high cost resulted in rapid and parallel compensation for that cost through mutations in a P1-like phage gene, relAP1. When the wild-type relAP1 gene was added to a strain in which it was not present and in which blaTEM-116* was neutral, it caused the ARG to become costly. Thus, relAP1 is both necessary and sufficient to explain blaTEM-116* costs in at least some host backgrounds. To our knowledge, these findings represent the first demonstrated case of the cost of an ARG being influenced by a genetic interaction with a phage gene. The interaction between a phage gene and a plasmid-borne ARG highlights the complexity of selective forces determining the maintenance and spread of ARGs and, by extension, encoding phage and plasmids in natural bacterial communities.IMPORTANCEAntibiotic resistance genes (ARGs) play a major role in the increasing problem of antibiotic resistance in clinically relevant bacteria. Selection of these genes occurs in the presence of antibiotics, but their eventual success also depends on the sometimes substantial costs they impose on host bacteria in antibiotic-free environments. We evolved an ARG that confers resistance to penicillin-type antibiotics in one host in which it did confer a cost and in one host in which it did not. We found that costs were rapidly and consistently reduced through parallel genetic changes in a gene encoded by a phage that was infecting the costly host. The unmutated version of this gene was sufficient to cause the ARG to confer a cost in a host in which it was originally neutral, demonstrating an antagonism between the two genetic elements and underlining the range and complexity of pressures determining ARG dynamics in natural populations.

Interferon-stimulated neutrophils as a predictor of immunotherapy response.

Despite the remarkable success of anti-cancer immunotherapy, its effectiveness remains confined to a subset of patients-emphasizing the importance of predictive biomarkers in clinical decision-making and further mechanistic understanding of treatment response. Current biomarkers, however, lack the power required to accurately stratify patients. Here, we identify interferon-stimulated, Ly6Ehi neutrophils as a blood-borne biomarker of anti-PD1 response in mice at baseline. Ly6Ehi neutrophils are induced by tumor-intrinsic activation of the STING (stimulator of interferon genes) signaling pathway and possess the ability to directly sensitize otherwise non-responsive tumors to anti-PD1 therapy, in part through IL12b-dependent activation of cytotoxic T cells. By translating our pre-clinical findings to a cohort of patients with non-small cell lung cancer and melanoma (n = 109), and to public data (n = 1440), we demonstrate the ability of Ly6Ehi neutrophils to predict immunotherapy response in humans with high accuracy (average AUC ≈ 0.9). Overall, our study identifies a functionally active biomarker for use in both mice and humans.