The timings of host diapause and epidemic progression mediate host genetic diversity and future epidemic size in Daphnia-parasite populations.

Epidemics commonly exert parasite-mediated selection and cause declines in host population genetic diversity. This can lead to evolution of resistance in the long term and smaller subsequent epidemics. Alternatively, the loss of genetic diversity can increase host vulnerability to future disease spread and larger future epidemics. Matters are made more complex by the fact that a great many host organisms produce diapausing life stages in response to environmental change (often as a result of sexual reproduction; e.g. plant seeds and invertebrate resting eggs). These diapausing stages can disrupt the relationship between past epidemics, host genetic diversity and future epidemics because they allow host dispersal through time. Specifically, temporally dispersing hosts avoid infection and thus selection from contemporary parasites, and also archive genetic variation for the future. We studied 80 epidemics in 20 semi-natural populations of the temporally dispersing crustacean Daphnia magna and its sterilizing bacterial parasite Pasteuria ramosa, and half of these populations experienced a simulated environmental disturbance treatment. We found that early initiation of diapause relative to the timing of the epidemic led to greater host genetic diversity and reduced epidemic size in the subsequent year, but this was unaffected by environmental disturbance.

Radiation-mediated supply of genetic variation outweighs the effects of selection and drift in Chernobyl Daphnia populations.

Populations experiencing varying levels of ionizing radiation provide an excellent opportunity to study the fundamental drivers of evolution. Radiation can cause mutations and thus supply genetic variation; it can also selectively remove individuals that are unable to cope with the physiological stresses associated with radiation exposure, or non-selectively cull swathes of the population, reducing genetic variation. Since the nuclear power plant explosion in 1986, the Chernobyl area has experienced a spatially heterogeneous exposure to varying levels of ionizing radiation. We sampled Daphnia pulex (a freshwater crustacean) from lakes across the Chernobyl area, genotyped them at ten microsatellite loci and also calculated the current radiation dose rates. We then investigated whether the pattern of genetic diversity was positively associated with radiation dose rates, consistent with radiation-mediated supply of de novo mutations, or negatively associated with radiation dose rates, as would be expected with strong radiation-mediated selection. We found that measures of genetic diversity, including expected heterozygosity and mean allelic richness (an unbiased indicator of diversity), were significantly higher in lakes that experienced the highest radiation dose rates. This suggests that mutation outweighs selection as the key evolutionary force in populations exposed to high radiation dose rates. We also found significant but weak population structure, indicative of low genetic drift and clear evidence for isolation-by-distance between populations. This further suggests that gene flow between nearby populations is eroding population structure and that mutational input in high radiation lakes could, ultimately, supply genetic variation to lower radiation sites.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis.

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.

Simulated climate change, epidemic size, and host evolution across host-parasite populations.

Climate change is causing warmer and more variable temperatures as well as physical flux in natural populations, which will affect the ecology and evolution of infectious disease epidemics. Using replicate seminatural populations of a coevolving freshwater invertebrate-parasite system (host: Daphnia magna, parasite: Pasteuria ramosa), we quantified the effects of ambient temperature and population mixing (physical flux within populations) on epidemic size and population health. Each population was seeded with an identical suite of host genotypes and dose of parasite transmission spores. Biologically reasonable increases in environmental temperature caused larger epidemics, and population mixing reduced overall epidemic size. Mixing also had a detrimental effect on host populations independent of disease. Epidemics drove parasite-mediated selection, leading to a loss of host genetic diversity, and mixed populations experienced greater evolution due to genetic drift over the season. These findings further our understanding of how diversity loss will reduce the host populations' capacity to respond to changes in selection, therefore stymying adaptation to further environmental change.

Ecology directs host-parasite coevolutionary trajectories across Daphnia-microparasite populations.

Host-parasite interactions often fuel coevolutionary change. However, parasitism is one of a myriad of possible ecological interactions in nature. Biotic (for example, predation) and abiotic (for example, temperature) variation can amplify or dilute parasitism as a selective force on hosts and parasites, driving population variation in (co)evolutionary trajectories. We dissected the relationships between wider ecology and coevolutionary trajectory using 16 ecologically complex Daphnia magna-Pasteuria ramosa ponds seeded with an identical starting host (Daphnia) and parasite (Pasteuria) population. We show, using a time-shift experiment and outdoor population data, how multivariate biotic and abiotic ecological differences between ponds caused coevolutionary divergence. Wider ecology drove variation in host evolution of resistance, but not parasite infectivity; parasites subsequently coevolved in response to the changing complement of host genotypes, such that parasites adapted to historically resistant host genotypes. Parasitism was a stronger interaction for the parasite than for its host, probably because the host is the principal environment and selective force, whereas for hosts, parasite-mediated selection is one of many sources of selection. Our findings reveal the mechanisms through which wider ecology creates coevolutionary hotspots and coldspots in biologically realistic arenas of host-parasite interaction, and sheds light on how the ecological theatre can affect the (co)evolutionary play.

Environmental variation causes different (co) evolutionary routes to the same adaptive destination across parasite populations.

Epidemics are engines for host-parasite coevolution, where parasite adaptation to hosts drives reciprocal adaptation in host populations. A key challenge is to understand whether parasite adaptation and any underlying evolution and coevolution is repeatable across ecologically realistic populations that experience different environmental conditions, or if each population follows a completely unique evolutionary path. We established twenty replicate pond populations comprising an identical suite of genotypes of crustacean host, Daphnia magna, and inoculum of their parasite, Pasteuria ramosa. Using a time-shift experiment, we compared parasite infection traits before and after epidemics and linked patterns of parasite evolution with shifts in host genotype frequencies. Parasite adaptation to the sympatric suite of host genotypes came at a cost of poorer performance on foreign genotypes across populations and environments. However, this consistent pattern of parasite adaptation was driven by different types of frequency-dependent selection that was contingent on an ecologically relevant environmental treatment (whether or not there was physical mixing of water within ponds). In unmixed ponds, large epidemics drove rapid and strong host-parasite coevolution. In mixed ponds, epidemics were smaller and host evolution was driven mainly by the mixing treatment itself; here, host evolution and parasite evolution were clear, but coevolution was absent. Population mixing breaks an otherwise robust coevolutionary cycle. These findings advance our understanding of the repeatability of (co)evolution across noisy, ecologically realistic populations.

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.

BACKGROUND: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. METHODS: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0.9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. FINDINGS: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3.7%, 95% CI 0.1-7.3; p=0.046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. INTERPRETATION: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials. FUNDING: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.

Quantification of mRNA using real-time PCR and Western blot analysis of MAPK events in chondrocyte/agarose constructs.

In vitro models of chondrocyte mechanobiology have been used to compare the intracellular signalling pathways altered in normal and osteoarthritis-affected cartilage. However, differences in the model system and type of loading configuration have led to complicated pathways. This chapter is a follow-on of previous studies from our group utilising 3D agarose as a physiological model to study mechanotransduction pathways. Experimental methods are described to assess targets at the protein and gene expression level by Western blot analysis and real-time PCR, respectively. This chapter provides a quantitative gene expression approach to explore the intracellular pathways activated by both mechanical loading and inflammatory mediators and examine upstream phosphorylation events. Ultimately, development of methods used to analyse mechano-sensitive pathways will provide important information for the identification of appropriate pharmacological and physiotherapeutic agents for the treatment of osteoarthritis.