Polymers and biopolymers at interfaces.

This review updates recent progress in the understanding of the behaviour of polymers at surfaces and interfaces, highlighting examples in the areas of wetting, dewetting, crystallization, and 'smart' materials. Recent developments in analysis tools have yielded a large increase in the study of biological systems, and some of these will also be discussed, focussing on areas where surfaces are important. These areas include molecular binding events and protein adsorption as well as the mapping of the surfaces of cells. Important techniques commonly used for the analysis of surfaces and interfaces are discussed separately to aid the understanding of their application.

Effects of prior exposure to antibiotics on bacterial adaptation to phages.

Understanding adaptation to complex environments requires information about how exposure to one selection pressure affects adaptation to others. For bacteria, antibiotics and viral parasites (phages) are two of the most common selection pressures and are both relevant for treatment of bacterial infections: increasing antibiotic resistance is generating significant interest in using phages in addition or as an alternative to antibiotics. However, we lack knowledge of how exposure to antibiotics affects bacterial responses to phages. Specifically, it is unclear how the negative effects of antibiotics on bacterial population growth combine with any possible mutagenic effects or physiological responses to influence adaptation to other stressors such as phages, and how this net effect varies with antibiotic concentration. Here, we experimentally addressed the effect of pre-exposure to a wide range of antibiotic concentrations on bacterial responses to phages. Across 10 antibiotics, we found a strong association between their effects on bacterial population size and subsequent population growth in the presence of phages (which in these conditions indicates phage-resistance evolution). We detected some evidence of mutagenesis among populations treated with fluoroquinolones and ß-lactams at sublethal doses, but these effects were small and not consistent across phage treatments. These results show that, although stressors such as antibiotics can boost adaptation to other stressors at low concentrations, these effects are weak compared to the effect of reduced population growth at inhibitory concentrations, which in our experiments strongly reduced the likelihood of subsequent phage-resistance evolution.

Spatial heterogeneity lowers rather than increases host-parasite specialization.

Abiotic environmental heterogeneity can promote the evolution of diverse resource specialists, which in turn may increase the degree of host-parasite specialization. We coevolved Pseudomonas fluorescens and lytic phage ϕ2 in spatially structured populations, each consisting of two interconnected subpopulations evolving in the same or different nutrient media (homogeneous and heterogeneous environments, respectively). Counter to the normal expectation, host-parasite specialization was significantly lower in heterogeneous compared with homogeneous environments. This result could not be explained by dispersal homogenizing populations, as this would have resulted in the heterogeneous treatments having levels of specialization equal to or greater than that of the homogeneous environments. We argue that selection for costly generalists is greatest when the coevolving species are exposed to diverse environmental conditions and that this can provide an explanation for our results. A simple coevolutionary model of this process suggests that this can be a general mechanism by which environmental heterogeneity can reduce rather than increase host-parasite specialization.

Parasite host range and the evolution of host resistance.

Parasite host range plays a pivotal role in the evolution and ecology of hosts and the emergence of infectious disease. Although the factors that promote host range and the epidemiological consequences of variation in host range are relatively well characterized, the effect of parasite host range on host resistance evolution is less well understood. In this study, we tested the impact of parasite host range on host resistance evolution. To do so, we used the host bacterium Pseudomonas fluorescens SBW25 and a diverse suite of coevolved viral parasites (lytic bacteriophage Φ2) with variable host ranges (defined here as the number of host genotypes that can be infected) as our experimental model organisms. Our results show that resistance evolution to coevolved phages occurred at a much lower rate than to ancestral phage (approximately 50% vs. 100%), but the host range of coevolved phages did not influence the likelihood of resistance evolution. We also show that the host range of both single parasites and populations of parasites does not affect the breadth of the resulting resistance range in a naïve host but that hosts that evolve resistance to single parasites are more likely to resist other (genetically) more closely related parasites as a correlated response. These findings have important implications for our understanding of resistance evolution in natural populations of bacteria and viruses and other host-parasite combinations with similar underlying infection genetics, as well as the development of phage therapy.

Genotype-by-environment interactions due to antibiotic resistance and adaptation in Escherichia coli.

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype-by-environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic-free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic-free conditions (LB medium), and did the same for the antibiotic-sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant-evolved genotypes were more phenotypically different from the ancestor and from each other than resistant-nonevolved and sensitive-evolved genotypes. This suggests genotype-by-environment interactions generated by antibiotic resistance mutations, observed previously in short-term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long-term selection on resistance mutations from fitness effects in a single environment.

No effect of host-parasite co-evolution on host range expansion.

Antagonistic co-evolution between hosts and parasites (reciprocal selection for resistance and infectivity) is hypothesized to play an important role in host range expansion by selecting for novel infectivity alleles, but tests are lacking. Here, we determine whether experimental co-evolution between a bacterium (Pseudomonas fluorescens SBW25) and a phage (SBW25Φ2) affects interstrain host range: the ability to infect different strains of P. fluorescens other than SBW25. We identified and tested a genetically and phenotypically diverse suite of co-evolved phage variants of SBW25Φ2 against both sympatric and allopatric co-evolving hosts (P. fluorescens SBW25) and a large set of other P. fluorescens strains. Although all co-evolved phage had a greater host range than the ancestral phage and could differentially infect co-evolved variants of P. fluorescens SBW25, none could infect any of the alternative P. fluorescens strains. Thus, parasite generalism at one genetic scale does not appear to affect generalism at other scales, suggesting fundamental genetic constraints on parasite adaptation for this virus.

Torsional electromechanical systems based on carbon nanotubes.

Carbon nanotubes (CNTs) are among the most highly studied nanomaterials due to their unique (and intertwined) mechanical and electrical properties. Recent advances in fabrication have allowed devices to be fabricated that are capable of applying a twisting force to individual CNTs while measuring mechanical and electrical response. Here, we review major results from this emerging field of study, revealing new properties of the material itself and opening possibilities for advances in future devices.

Multiplicity of infection does not accelerate infectivity evolution of viral parasites in laboratory microcosms.

Coinfection with multiple parasite genotypes [multiplicity of infection (MOI)] creates within-host competition and opportunities for parasite recombination and is therefore predicted to be important for both parasite and host evolution. We tested for a difference in the infectivity of viral parasites (lytic phage Φ2) and resistance of their bacterial hosts (Pseudomonas fluorescens SBW25) under both high and low MOI during coevolution in laboratory microcosms. Results show that MOI has no effect on infectivity and resistance evolution during coevolution over ∼80 generations of host growth, and this is true when the experiment is initiated with wild-type viruses and hosts, or with viruses and hosts that have already been coevolving for ∼330 generations. This suggests that MOI does not have a net effect of accelerating parasite adaptation to hosts through recombination, or slowing adaptation to hosts through between-parasite conflict in this system.

p53-dependent cell death/apoptosis is required for a productive adenovirus infection.

The p53 tumor suppressor protein binds to both cellular and viral proteins, which influence its biological activity. One such protein is the large E1b tumor antigen (E1b58kDa) from adenoviruses (Ads), which abrogates the ability of p53 to transactivate various promoters. This inactivation of p53 function is believed to be the mechanism by which E1b58kDa contributes to the cell transformation process. Although the p53-E1b58kDa complex occurs during infection and is conserved among different serotypes, there are limited data demonstrating that it has a role in virus replication. However, loss of p53 expression occurs after adenovirus infection of human cells and an E1b58kDa deletion mutant (Onyx-015, also called dl 1520) selectively replicates in p53-defective cells. These (and other) data indicate a plausible hypothesis is that loss of p53 function may be conducive to efficient adenovirus replication. However, wild-type (wt) Ad5 grows more efficiently in cells expressing a wt p53 protein. These studies indicate that the hypothesis may be an oversimplification. Here, we show that cells expressing wt p53, as well as p53-defective cells, allow adenovirus replication, but only cells expressing wt p53 show evidence of virus-induced cytopathic effect. This correlates with the ability of adenovirus to induce cell death. Our data indicate that p53 plays a necessary part in mediating cellular destruction to allow a productive adenovirus infection. In contrast, p53-deficient cells are less sensitive to the cytolytic effects of adenovirus and as such raise questions about the use of E1b58kDa-deficient adenoviruses in tumor therapy.

Translocation of RecA-coated double-stranded DNA through solid-state nanopores.

We report translocation of double-stranded DNA (dsDNA) molecules that are coated with RecA protein through solid-state nanopores. Translocation measurements show current-blockade events with a wide variety in time duration (10-4-10-1 s) and conductance blockade values (3-14 nS). Large blockades (11.4+/-0.7 nS) are identified as being caused by translocations of RecA-dsDNA filaments. We confirm these results through a variety of methods, including changing molecular length and using an optical tweezer system to deliver bead-functionalized molecules to the nanopore. We further distinguish two different regimes of translocation: a low-voltage regime (<150 mV) in which the event rate increases exponentially with voltage, and a high-voltage regime in which it remains constant. Our results open possibilities for a variety of future experiments with (partly) protein-coated DNA molecules, which is interesting for both fundamental science and genomic screening applications.

Decay of unused characters by selection and drift.

The reduction and loss of redundant phenotypic characters is a common feature of evolution. However, the mechanisms that drive deterioration of unused characters remain unclear. Here, we outline a simple framework where the relative importance of selective and neutral processes varies with environmental factors, because of variation in the fitness costs associated with unused traits. We tested our hypotheses using experimental evolution of the bacterium Pseudomonas fluorescens in spatially uniform environments. Results show that an unused character, swimming motility, decayed over evolutionary time and the rate of this decay varied among selection environments with different levels of resource availability. This is explained in the context of an environment-specific genetic correlation between motility and fitness, which is negative when resources are limited but neutral at higher resource levels. Thus, selection against an unused character was most effective in environments where the fitness cost was the greatest. This suggests that the same character can decay by different mechanisms depending upon environmental factors and supports previous evidence to show that resource availability can critically affect the outcomes of evolution.

Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.

Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks, leading to knockout of both these proteins in 8-10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca(2+)-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca(2+) and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.

Specific p53-DNA complexes contain an mdm2-related protein.

The mdm2 gene encodes a family of proteins, a subset of which bind p53 and negatively regulate its function as a transcription factor. We now show that an anti-mdm-2 monoclonal antibody, 2A10, recognises a protein present in rabbit reticulocyte lysate which binds murine p53 translated in vitro. Deletion of p53 residues 10-35, which encompass the mdm-2 binding site, abolished binding of this 2A10-reactive protein. Binding was also dependent upon p53 protein conformation and may require nascent p53 polypeptide since binding was lost following conformational shifting of the temperature-sensitive mutant A135V. Previous studies have shown that mdm-2-p53 complexes fail to exhibit detectable sequence-specific DNA binding. However, our present results demonstrate that p53 in complex with an mdm-2-related protein in vitro retained sequence-specific DNA binding capacity. Non-transformed (but not transformed) 3T3 cells were also found to express a similar 2A10-reactive protein, detectable by gel shift analysis of cellular p53 in complex with a specific DNA target. Mdm-2 in rabbit reticulocyte lysate and in normal, non-transformed 3T3 cells may represent constitutively expressed protein. Our results raise the possibility that constitutive mdm-2 may enhance and/or suppress functions of p53 as yet unidentified.

Structural and kinetic analysis of p53-DNA complexes and comparison of human and murine p53.

Sequence-specific DNA binding by p53 is dependent upon protein conformation. The 1620+ form correlates with wild type p53 suppressor function and is a prerequisite for binding to the DNA consensus p53-CON in vitro. It has been reported that murine p53 changes conformation on interaction with high affinity DNA target sequences and in the present study we have analysed p53-DNA complexes using conformation-specific monoclonal antibodies against p53. For murine p53 (mp53) we show (i) the 1620+ form is retained and stabilised in complex with DNA, and (ii) the complexes are dissociated by the PAb1620 monoclonal antibody. In contrast, PAb1620 did not detect nor dissociate human p53-DNA complexes nor did it interfere with complex formation. In competition experiments murine p53 replaced human p53 (hp53) in p53-DNA complexes and this correlated with the greater lability observed for hp53-DNA complexes at a given temperature. Mixed human-murine p53 oligomers were competent for DNA binding with an estimated affinity around 5 x 10(-10) M, similar to that observed for either human or murine p53 alone. The potential significance of these observations is discussed in relation p53 function in vivo.

Mitochondrial fusion and fission proteins: novel therapeutic targets for combating cardiovascular disease.

Mitochondria are no longer considered to be solely the static powerhouses of the cell. While they are undoubtedly essential to sustaining life and meeting the energy requirements of the cell through oxidative phosphorylation, they are now regarded as highly dynamic organelles with multiple functions, playing key roles in cell survival and death. In this review, we discuss the emerging role of mitochondrial fusion and fission proteins, as novel therapeutic targets for treating a wide range of cardiovascular diseases.

DJ-1 protects against cell death following acute cardiac ischemia-reperfusion injury.

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia-reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia-reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1(L166P) and DJ-1(Cys106A) mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.