The impact of combinatorial stress on the growth dynamics and metabolome of Burkholderia mesoacidophila demonstrates the complexity of tolerance mechanisms.

AIMS: The recently sequenced Burkholderia mesoacidophila (previously Pseudomonas mesoacidophila) is a soil organism and as such will be exposed to multiple concurrent stresses in the natural environment. The combinatorial stress potentially experienced by microbes in soil has not been investigated in detail. METHODS AND RESULTS: The impact of combinatorial stress on growth was investigated using tripartite variables-temperature, nutritional environment and either osmotic or oxidative stress. In nutritionally stringent conditions, increasing diamide concentration had no effect on growth while increasing H2 O2 concentration reduced both growth rate and maximum density. Metabolomic studies with oxidative stress revealed specific (unidentified) metabolites associated with diamide tolerance, and an overwhelming dominance of sugars and sugar alcohols in nutritionally stringent conditions with and without the additional stressor. CONCLUSIONS: Combinatorial stress tolerance is complex. Temperature had the greatest independent impact on growth, while the impact of the nutritional environment played a key role in oxidative stress tolerance. In nutritionally stringent conditions, the metabolome suggested different tolerance mechanisms for different types of oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the specificity of the stress response, and the need to consider multiple environmental factors to meaningfully investigate tolerance. Both environmental and clinical settings subject bacteria to combinatorial stress and this should be considered in the design of further studies.

Preoperative sentinel lymph node identification, biopsy and localisation using contrast enhanced ultrasound (CEUS) in patients with breast cancer: a systematic review and meta-analysis.

AIM: To evaluate whether contrast-enhanced ultrasound (CEUS)-guided core biopsy of the sentinel lymph node (SLN) could identify metastatic nodes preoperatively and reduce the number of surgical SLN biopsies in patients with breast cancer and normal axillary B-mode ultrasound; and to establish whether CEUS SLN identification and localisation is a viable alternative to standard lymphatic mapping using isotope and blue dye. MATERIALS AND METHODS: A search of several electronic databases was performed and identified studies were assessed using QUADAS-2 for methodological quality. Pooled estimates of sensitivity and specificity for identification of nodal metastases were calculated. RESULTS: Eleven prospective studies and one retrospective study with 1,520 participants were included. The SLN identification and localisation rate for CEUS-guided skin marking was 70-100%, CEUS guided-wire localisation was 89-97%, and CEUS-guided iodine-125 (125I) seed localisation was 60%. Across the four studies that evaluated preoperative CEUS-guided SLN biopsy, pooled sensitivity for identification of nodal metastases was 54% (95% confidence interval [CI]: 47-61) and pooled specificity 100% (95% CI: 99-100). CONCLUSION: CEUS is a promising technique for preoperative staging of the axilla. CEUS-guided core biopsy has the potential to identify nodal metastases in over half (54%) of patients with normal axillary B-mode ultrasound. CEUS-guided identification and localisation of the SLN may offer a viable alternative to standard lymphatic mapping using isotope and blue dye; however, further prospective studies with larger samples are warranted.

How humans drive speciation as well as extinction.

A central topic for conservation science is evaluating how human activities influence global species diversity. Humanity exacerbates extinction rates. But by what mechanisms does humanity drive the emergence of new species? We review human-mediated speciation, compare speciation and known extinctions, and discuss the challenges of using net species diversity as a conservation objective. Humans drive rapid evolution through relocation, domestication, hunting and novel ecosystem creation-and emerging technologies could eventually provide additional mechanisms. The number of species relocated, domesticated and hunted during the Holocene is of comparable magnitude to the number of observed extinctions. While instances of human-mediated speciation are known, the overall effect these mechanisms have upon speciation rates has not yet been quantified. We also explore the importance of anthropogenic influence upon divergence in microorganisms. Even if human activities resulted in no net loss of species diversity by balancing speciation and extinction rates, this would probably be deemed unacceptable. We discuss why, based upon 'no net loss' conservation literature-considering phylogenetic diversity and other metrics, risk aversion, taboo trade-offs and spatial heterogeneity. We conclude that evaluating speciation alongside extinction could result in more nuanced understanding of biosphere trends, clarifying what it is we actually value about biodiversity.

Aggregation dynamics explain vegetation patch-size distributions.

Vegetation patch-size distributions have been an intense area of study for theoreticians and applied ecologists alike in recent years. Of particular interest is the seemingly ubiquitous nature of power-law patch-size distributions emerging in a number of diverse ecosystems. The leading explanation of the emergence of these power-laws is due to local facilitative mechanisms. There is also a common transition from power law to exponential distribution when a system is under global pressure, such as grazing or lack of rainfall. These phenomena require a simple mechanistic explanation. Here, we study vegetation patches from a spatially implicit, patch dynamic viewpoint. We show that under minimal assumptions a power-law patch-size distribution appears as a natural consequence of aggregation. A linear death term also leads to an exponential term in the distribution for any non-zero death rate. This work shows the origin of the breakdown of the power-law under increasing pressure and shows that in general, we expect to observe a power law with an exponential cutoff (rather than pure power laws). The estimated parameters of this distribution also provide insight into the underlying ecological mechanisms of aggregation and death.

Studies on the synthesis, stability and conformation of 2-sulfonyl-oxetane fragments.

2-(Arylsulfonyl)oxetanes have been prepared as new structural motifs of interest for medicinal chemistry. These are designed to fit within fragment space and be suitable for screening in fragment based drug discovery, as well as being suitable for further elaboration or incorporation into drug-like compounds. The oxetane ring is constructed through an efficient C-C bond forming cyclisation which allows the incorporation of a wide range of aryl-sulfonyl groups. Furthermore, biaryl-containing compounds can be accessed through Suzuki-Miyaura coupling from halogenated derivatives. With a number of oxetane containing fragment compounds available, their pH stability was assessed, indicating good half-life values for mono-substituted aryl sulfonyl oxetanes across the pH range (1 to 10). Solubility and metabolic stability data is also reported. Finally, the conformation of the fragments is assessed computationally, providing an indication of possible binding orientations.

Importance of baseline specification in evaluating conservation interventions and achieving no net loss of biodiversity.

There is an urgent need to improve the evaluation of conservation interventions. This requires specifying an objective and a frame of reference from which to measure performance. Reference frames can be baselines (i.e., known biodiversity at a fixed point in history) or counterfactuals (i.e., a scenario that would have occurred without the intervention). Biodiversity offsets are interventions with the objective of no net loss of biodiversity (NNL). We used biodiversity offsets to analyze the effects of the choice of reference frame on whether interventions met stated objectives. We developed 2 models to investigate the implications of setting different frames of reference in regions subject to various biodiversity trends and anthropogenic impacts. First, a general analytic model evaluated offsets against a range of baseline and counterfactual specifications. Second, a simulation model then replicated these results with a complex real world case study: native grassland offsets in Melbourne, Australia. Both models showed that achieving NNL depended upon the interaction between reference frame and background biodiversity trends. With a baseline, offsets were less likely to achieve NNL where biodiversity was decreasing than where biodiversity was stable or increasing. With a no-development counterfactual, however, NNL was achievable only where biodiversity was declining. Otherwise, preventing development was better for biodiversity. Uncertainty about compliance was a stronger determinant of success than uncertainty in underlying biodiversity trends. When only development and offset locations were considered, offsets sometimes resulted in NNL, but not across an entire region. Choice of reference frame determined feasibility and effort required to attain objectives when designing and evaluating biodiversity offset schemes. We argue the choice is thus of fundamental importance for conservation policy. Our results shed light on situations in which biodiversity offsets may be an inappropriate policy instrument.

Slow fitness recovery in a codon-modified viral genome.

Extensive synonymous codon modification of viral genomes appears to be an effective way of attenuating strains for use as live vaccines. An assumption of this method is that codon changes have individually small effects, such that codon-attenuated viruses will be slow to evolve back to high fitness (and thus to high virulence). The major capsid gene of the bacterial virus T7 was modified to have varying levels of suboptimal synonymous codons in different constructs, and fitnesses declined linearly with the number of changes. Adaptation of the most extreme design, with 182 codon changes, resulted in a slow fitness recovery by standards of previous experimental evolution with this virus, although fitness effects of substitutions were higher than expected from the average effect of an engineered codon modification. Molecular evolution during recovery was modest, and changes evolved both within the modified gene and outside it. Some changes within the modified gene evolved in parallel across replicates, but with no obvious explanation. Overall, the study supports the premise that codon-modified viruses recover fitness slowly, although the evolution is substantially more rapid than expected from the design principle.

Optimizing the Application Timing and Dosage of Metarhizium brunneum (Hypocreales: Clavicipitaceae) as a Biological Control Agent of Aedes aegypti (Diptera: Culicidae) Larvae.

Aedes aegypti (Diptera: Culicidae) is the principal vector of dengue and other viruses that cause disease among 100 to 400 million people each year. The recent development of widespread insecticidal resistance has led to the rapid development of biological control solutions aimed at larval control. While the efficacy of Metarhizium brunneum has been shown against Aedes larvae, the impact of larval population dynamics will need to be determined to formulate effective control strategies. In this study, larvae were subjected to four concentrations of M. brunneum (105, 106, 107, 108 conidia ml-1). Larvae were found to be susceptible to M. brunneum with dose-dependent efficacy. When constant larval immigration was added as a parameter, peak mortality was consistently found to occur on the fourth day, before a significant reduction in control efficacy linked to a decline in conidial availability within the water column. This suggests that M. brunneum treatments should be applied at a concentration 1 × 107 conidia ml-1 every four days to effectively control mosquito larvae in the field, regardless of the fungal formulation, water volume, or larval density. Understanding fungal-mosquito dynamics is critical in developing appropriate control programs as it helps optimize the fungal control agent's dose and frequency of application.

Nature-positive goals for an organization's food consumption.

Organizations are increasingly committing to biodiversity protection targets with focus on 'nature-positive' outcomes, yet examples of how to feasibly achieve these targets are needed. Here we propose an approach to achieve nature-positive targets with respect to the embodied biodiversity impacts of an organization's food consumption. We quantify these impacts using a comprehensive database of life-cycle environmental impacts from food, and map exploratory strategies to meet defined targets structured according to a mitigation and conservation hierarchy. By considering the varying needs and values across the organization's internal community, we identify a range of targeted approaches towards mitigating impacts, which balance top-down and bottom-up actions to different degrees. Delivering ambitious nature-positive targets within current constraints will be challenging, particularly given the need to mitigate cumulative impacts. Our results evidence that however committed an organization is to being nature positive in its food provision, this is unachievable in the absence of systems change.

In vivo growth rates are poorly correlated with phage therapy success in a mouse infection model.

Two classes of phages yield profoundly different levels of recovery in mice experimentally infected with an Escherichia coli O18:K1:H7 strain. Phages requiring the K1 capsule for infection (K1-dep) rescue virtually all infected mice, whereas phages not requiring the capsule (K1-ind) rescue modest numbers (∼30%). To rescue infected mice, K1-ind phages require at least a 10(6)-fold-higher inoculum than K1-dep phages. Yet their in vivo growth dynamics are only modestly inferior to those of K1-dep phages, and competition between the two phage types in the same mouse reveals only a slight growth advantage for the K1-dep phage. The in vivo growth rate seems unlikely to be the primary determinant of phage therapy success. An alternative explanation is that the success of K1-dep phages is due substantially to their proteomic composition. They encode an enzyme that degrades the K1 capsule, which has been shown in other work to be sufficient to cure infection in the complete absence of phages.

Lessons from the use of telemedicine in the austere military environment and the implications for deployed surgical teams.

Over the last 20 years, there have been significant changes in UK surgical training. Civilian surgical training may no longer prepare military surgeons for the range of skills they require on operations. One method to address gaps in knowledge or experience is to use telemedicine to facilitate specialist consultations from UK-based specialists to deployed medical teams. In the UK Defence Medical Services (DMS), this capability is called real-time clinical support (RTCS). RTCS provides a direct audio-visual link from a deployed location anywhere in the world to a supporting medical specialist in the UK. RTCS is currently delivered via a combination of off-the-shelf hardware and commercially available software. This article will outline the current use of RTCS, with emphasis on deployed surgical teams in austere environments, and discuss the advantages and limitations of this capability. However, it must be emphasised that no technology can be a substitute for clinical training and experience. Although several limitations remain, the authors believe that RTCS offers potential benefits for the DMS and could be an important tool aiding deployed clinicians. It can also be argued that by engaging with the concept now, the DMS can shape future developments in this sphere.

Optimality models in the age of experimental evolution and genomics.

Optimality models have been used to predict evolution of many properties of organisms. They typically neglect genetic details, whether by necessity or design. This omission is a common source of criticism, and although this limitation of optimality is widely acknowledged, it has mostly been defended rather than evaluated for its impact. Experimental adaptation of model organisms provides a new arena for testing optimality models and for simultaneously integrating genetics. First, an experimental context with a well-researched organism allows dissection of the evolutionary process to identify causes of model failure--whether the model is wrong about genetics or selection. Second, optimality models provide a meaningful context for the process and mechanics of evolution, and thus may be used to elicit realistic genetic bases of adaptation--an especially useful augmentation to well-researched genetic systems. A few studies of microbes have begun to pioneer this new direction. Incompatibility between the assumed and actual genetics has been demonstrated to be the cause of model failure in some cases. More interestingly, evolution at the phenotypic level has sometimes matched prediction even though the adaptive mutations defy mechanisms established by decades of classic genetic studies. Integration of experimental evolutionary tests with genetics heralds a new wave for optimality models and their extensions that does not merely emphasize the forces driving evolution.

Compensatory evolution for a gene deletion is not limited to its immediate functional network.

BACKGROUND: Genetic disruption of an important phenotype should favor compensatory mutations that restore the phenotype. If the genetic basis of the phenotype is modular, with a network of interacting genes whose functions are specific to that phenotype, compensatory mutations are expected among the genes of the affected network. This perspective was tested in the bacteriophage T3 using a genome deleted of its DNA ligase gene, disrupting DNA metabolism. RESULTS: In two replicate, long-term adaptations, phage compensatory evolution accommodated the low ligase level provided by the host without reinventing its own ligase. In both lines, fitness increased substantially but remained well below that of the intact genome. Each line accumulated over a dozen compensating mutations during long-term adaptation, and as expected, many of the compensatory changes were within the DNA metabolism network. However, several compensatory changes were outside the network and defy any role in DNA metabolism or biochemical connection to the disruption. In one line, these extra-network changes were essential to the recovery. The genes experiencing compensatory changes were moderately conserved between T3 and its relative T7 (25% diverged), but the involvement of extra-network changes was greater in T3. CONCLUSION: Compensatory evolution was only partly limited to the known functionally interacting partners of the deleted gene. Thus gene interactions contributing to fitness were more extensive than suggested by the functional properties currently ascribed to the genes. Compensatory evolution offers an easy method of discovering genome interactions among specific elements that does not rest on an a priori knowledge of those elements or their interactions.

Temperature-dependent sex determination in turtles.

The sex of hatchling map turtles is determined by incubation temperature of eggs in the laboratory as well as in nature. Temperature controls sex differentiation rather than causing a differential mortality of sexes. Temperature has no effect on sex determination in a soft-shelled turtle.

Mammalian ZFY sequences exist in reptiles regardless of sex-determining mechanism.

In some reptiles, egg incubation temperature determines whether the embryo hatches as male or female; in others, sex chromosomes determine sex. A cloned gene (ZFY) representing the putative testis-determining factor in mammals was hybridized to genomic DNA of reptiles with sex chromosomes and to DNA of reptiles with temperature-dependent sex determination. No sex differences in hybridization patterns were observed. Hybridization of ZFY to polyadenylated RNA indicates that reptilian versions of this gene are expressed in embryos of both sexes during the temperature-sensitive period. If these highly conserved sequences are important in reptilian sex determination, then temperature-dependent and genotypic sex determination may have a similar molecular basis. For reptiles with XX/XY or ZZ/ZW systems, the absence of sex differences in hybridization patterns raises the question of whether the ZFY sequences reside on their sex chromosomes.

Experimental phylogenetics: generation of a known phylogeny.

Although methods of phylogenetic estimation are used routinely in comparative biology, direct tests of these methods are hampered by the lack of known phylogenies. Here a system based on serial propagation of bacteriophage T7 in the presence of a mutagen was used to create the first completely known phylogeny. Restriction-site maps of the terminal lineages were used to infer the evolutionary history of the experimental lines for comparison to the known history and actual ancestors. The five methods used to reconstruct branching pattern all predicted the correct topology but varied in their predictions of branch lengths; one method also predicts ancestral restriction maps and was found to be greater than 98 percent accurate.

Different trajectories of parallel evolution during viral adaptation.

The molecular basis of adaptation is a major focus of evolutionary biology, yet the dynamic process of adaptation has been explored only piecemeal. Experimental evolution of two bacteriophage lines under strong selection led to over a dozen nucleotide changes genomewide in each replicate. At least 96 percent of the amino acid substitutions appeared to be adaptive, and half the changes in one line also occurred in the other. However, the order of these changes differed between replicates, and parallel substitutions did not reflect the changes with the largest beneficial effects or indicate a common trajectory of adaptation.

Predicting evolution from genomics: experimental evolution of bacteriophage T7.

A wealth of molecular biology has been exploited in designing and interpreting experimental evolution studies with bacteriophage T7. The modest size of its genome (40 kb dsDNA) and the ease of making genetic constructs, combined with the many genetic resources for its host (Escherichia coli), have enabled comprehensive and detailed studies of experimental adaptations. In several studies, the genome was specifically altered (gene knockouts, gene replacements, reordering of genetic elements) such that a priori knowledge of genetics and biochemistry of the phage could be used to predict the pathways of compensatory evolution when the modified phage is adapted to recover fitness. In other work, the phage has been adapted to specific environmental conditions chosen to select phenotypic outcomes with a quantitative basis, and the molecular bases of that evolution have been explored. Predicting the outcomes of these adaptations has been challenging. In hindsight, one-third to one-half of the compensatory nucleotide changes observed during the adaptation can be rationalized based on T7 biology. This rationalization usually only applies at the genetic level-a gene product may be known to be involved in the affected pathway, but it usually remains unknown how the observed change affects activity. The progress is encouraging, but the prediction of experimental evolution pathways remains far from complete, and is still sometimes confounded by observation when an adaptation yields a completely unexpected outcome.

The optimal burst of mutation to create a phenotype.

Mutagenesis is commonly applied to genes and genomes to create novel variants with desired properties. This paper calculates the level of mutagenesis that maximizes the appearance of favorable mutants, assuming that the mutagenesis is applied in a single episode. The downside of mutagenesis is that a substantial fraction of mutations will destroy gene/genome function. The upside of mutagenesis is the production of beneficial mutations, but the desired phenotype may require that 1, 2 or more beneficial mutations be present simultaneously (the phenotype dimensionality). The optimum level of mutagenesis is sensitive to both properties. In the simplest model, the mutation optimum occurs when number of lethal equivalents per genome equals the phenotype dimensionality, a result first derived by Mundry and Gierer [1958. Production of mutations in tobacco mosaic virus by chemical treatment of its nucleic acid in vitro. Z. Vererbungsl. 89 (4), 614-630]. This level of mutation is shown to be an upper bound for the optimum in various extensions of the model, and the recovery of mutants is also reasonably tolerant to deviations from the optimum.

From bad to good: Fitness reversals and the ascent of deleterious mutations.

Deleterious mutations are considered a major impediment to adaptation, and there are straightforward expectations for the rate at which they accumulate as a function of population size and mutation rate. In a simulation model of an evolving population of asexually replicating RNA molecules, initially deleterious mutations accumulated at rates nearly equal to that of initially beneficial mutations, without impeding evolutionary progress. As the mutation rate was increased within a moderate range, deleterious mutation accumulation and mean fitness improvement both increased. The fixation rates were higher than predicted by many population-genetic models. This seemingly paradoxical result was resolved in part by the observation that, during the time to fixation, the selection coefficient (s) of initially deleterious mutations reversed to confer a selective advantage. Significantly, more than half of the fixations of initially deleterious mutations involved fitness reversals. These fitness reversals had a substantial effect on the total fitness of the genome and thus contributed to its success in the population. Despite the relative importance of fitness reversals, however, the probabilities of fixation for both initially beneficial and initially deleterious mutations were exceedingly small (on the order of 10(-5) of all mutations).