Monte carlo inference for state-space models of wild animal populations

Summary .  We compare two Monte Carlo (MC) procedures, sequential importance sampling (SIS) and Markov chain Monte Carlo (MCMC), for making Bayesian inferences about the unknown states and parameters of state–space models for animal populations. The procedures were applied to both simulated and real pup count data for the British grey seal metapopulation, as well as to simulated data for a Chinook salmon population. The MCMC implementation was based on tailor-made proposal distributions combined with analytical integration of some of the states and parameters. SIS was implemented in a more generic fashion. For the same computing time MCMC tended to yield posterior distributions with less MC variation across different runs of the algorithm than the SIS implementation with the exception in the seal model of some states and one of the parameters that mixed quite slowly. The efficiency of the SIS sampler greatly increased by analytically integrating out unknown parameters in the observation model. We consider that a careful implementation of MCMC for cases where data are informative relative to the priors sets the gold standard, but that SIS samplers are a viable alternative that can be programmed more quickly. Our SIS implementation is particularly competitive in situations where the data are relatively uninformative; in other cases, SIS may require substantially more computer power than an efficient implementation of MCMC to achieve the same level of MC error.     

Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene

The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.     

Spatial subdivision among assemblages of Spanish mackerel, Scomberomorus commerson (Pisces: Scombridae) across northern Australia: implications for fisheries management

Aim This study investigated the use of stable δ¹³C and δ¹⁸O isotopes in the sagittal otolith carbonate of narrow‐barred Spanish mackerel, Scomberomorus commerson, as indicators of population structure across Australia. Location Samples were collected from 25 locations extending from the lower west coast of Western Australia (30°), across northern Australian waters, and to the east coast of Australia (18°) covering a coastline length of approximately 9500 km, including samples from Indonesia. Methods The stable δ¹³C and δ¹⁸O isotopes in the sagittal otolith carbonate of S. commerson were analysed using standard mass spectrometric techniques. The isotope ratios across northern Australian subregions were subjected to an agglomerative hierarchical cluster analysis to define subregions. Isotope ratios within each of the subregions were compared to assess population structure across Australia. Results Cluster analysis separated samples into four subregions: central Western Australia, north Western Australia, northern Australia and the Gulf of Carpentaria and eastern Australia. Isotope signatures for fish from a number of sampling sites from across Australia and Indonesia were significantly different, indicating population separation. No significant differences were found in otolith isotope ratios between sampling times (no temporal variation). Main conclusions Significant differences in the isotopic signatures of S. commerson demonstrate that there is unlikely to be any substantial movement of fish among these spatially discrete adult assemblages. The lack of temporal variation among otolith isotope ratios indicates that S. commerson populations do not undergo longshore spatial shifts in distribution during their life history. The temporal persistence of spatially explicit stable isotopic signatures indicates that, at these spatial scales, the population units sampled comprise functionally distinct management units or separate ‘stocks’ for many of the purposes of fisheries management. The spatial subdivision evident among populations of S. commerson across northern and western Australia indicates that it may be advantageous to consider S. commerson population dynamics and fisheries management from a metapopulation perspective (at least at the regional level).     

Truncating and missense BMPR2 mutations differentially affect the severity of heritable pulmonary arterial hypertension

Background

Autosomal dominant inheritance of germline mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are a major risk factor for pulmonary arterial hypertension (PAH). While previous studies demonstrated a difference in severity between BMPR2 mutation carriers and noncarriers, it is likely disease severity is not equal among BMPR2 mutations. We hypothesized that patients with missense BMPR2 mutations have more severe disease than those with truncating mutations.

Methods

Testing for BMPR2 mutations was performed in 169 patients with PAH (125 with a family history of PAH and 44 with sporadic disease). Of the 106 patients with a detectable BMPR2 mutation, lymphocytes were available in 96 to functionally assess the nonsense-mediated decay pathway of RNA surveillance. Phenotypic characteristics were compared between BMPR2 mutation carriers and noncarriers, as well as between those carriers with a missense versus truncating mutation.

Results

While there was a statistically significant difference in age at diagnosis between carriers and noncarriers, subgroup analysis revealed this to be the case only for females. Among carriers, there was no difference in age at diagnosis, death, or survival according to exonic location of the BMPR2 mutation. However, patients with missense mutations had statistically significant younger ages at diagnosis and death, as well as shorter survival from diagnosis to death or lung transplantation than those with truncating mutations. Consistent with this data, the majority of missense mutations were penetrant prior to age 36 years, while the majority of truncating mutations were penetrant after age 36 years.

Conclusion

In this cohort, BMPR2 mutation carriers have more severe PAH disease than noncarriers, but this is only the case for females. Among carriers, patients with missense mutations that escape nonsense-mediated decay have more severe disease than those with truncating mutations. These findings suggest that treatment and prevention strategies directed specifically at BMPR2 pathway defects may need to vary according to the type of mutation.     

Endogenous Phenazine Antibiotics Promote Anaerobic Survival of Pseudomonas aeruginosa via Extracellular Electron Transfer

Antibiotics are increasingly recognized as having other, important physiological functions for the cells that produce them. An example of this is the effect that phenazines have on signaling and community development for Pseudomonas aeruginosa (L. E. Dietrich, T. K. Teal, A. Price-Whelan, and D. K. Newman, Science 321:1203-1206, 2008). Here we show that phenazine-facilitated electron transfer to poised-potential electrodes promotes anaerobic survival but not growth of Pseudomonas aeruginosa PA14 under conditions of oxidant limitation. Other electron shuttles that are reduced but not made by PA14 do not facilitate survival, suggesting that the survival effect is specific to endogenous phenazines.Phenazines have long been recognized for their redox properties. While most attention concerning their redox activity has focused on their role in generating reactive oxygen species in the context of infection (13, 14, 19, 22), as early as 1931, Friedheim hypothesized that phenazine reduction might benefit producer cells as an alternative respiratory pigment (12). Several years ago, our group suggested that the context in which this might be most important would be in biofilms, where cell densities are high and access to oxidants is limited (16, 31); consistent with this, we recently showed that mutants unable to produce phenazines are defective in community development (8). While this phenotype is likely due to many factors, including a signaling function for phenazines in later stages of growth (9), given the 1931 hypothesis by Friedheim (12) and our related recent work demonstrating that phenazines control redox homeostasis in Pseudomonas aeruginosa (30), we reasoned that phenazines might contribute to the survival of cells experiencing oxidant limitation.As a first step toward testing this, we investigated the effect of redox-active small molecules on anaerobic survival of P. aeruginosa PA14 in stationary-phase planktonic cultures. We justified beginning with planktonic cultures rather than biofilms based on previous studies which have suggested that cells in stationary-phase planktonic culture physiologically resemble cells in established biofilms (15, 35, 38). Moreover, by working with cells in planktonic cultures, we could build on voltammetric methods that had previously been used to determine how metabolism changes in Escherichia coli in the presence of the synthetic redox-shuttle ferricyanide (36). Similar voltammetric approaches have also been used to study how phenazines (32, 33) and structurally related flavins (23) mediate power generation by microbial fuel cells.We assembled bulk electrolysis-based glass bioreactors housed within an O₂- and H₂-free glove box (MBraun) and controlled by a multichannel potentiostat (series G 300; Gamry) outside the glove box. Each bioreactor held a graphite rod working electrode (Alfa Aesar) with an operating surface area of 6 cm², a Ag/AgCl reference electrode (RE-5B; BASi) with a constant potential of +207 mV versus that of the normal hydrogen electrode (NHE), and about 100 ml MOPS (morpholinepropanesulfonic acid) culture medium (100 mM MOPS at pH 7.2, 93 mM NH₄Cl, 43 mM NaCl, 2.2 mM KH₂PO₄, 1 mM MgSO₄, 5.0 μM FeCl₃) (modified from reference 29). The bioreactor was joined by a fritted glass junction to a small side chamber, in which a Pt counter electrode made from Pt mesh (Alfa Aesar) soldered to a copper wire completed the circuit. In order to selectively examine different redox-active small molecules, we used the phenazine-null mutant of PA14 (Δphz) which is deleted in its two phenazine biosynthetic operons (9).We began by focusing on three endogenous phenazines—pyocyanin (PYO), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine (1-OHPHZ)—that are known to be excreted by PA14 during stationary-phase growth cycle in laboratory cultures (9, 17). We harvested cells from cultures grown aerobically on Luria-Bertani (LB; Fisher Scientific) medium at 37°C and concentrated and resuspended them in anaerobic MOPS medium at 10⁹ CFU/ml. These resuspended cells were incubated in bioreactors over a period of 7 days at 30°C. To perform the survival experiments, we incubated dense suspensions (10⁹ cells/ml) in the MOPS medium containing 20 mM d-glucose to ensure excess electron donor, added ∼90 μM phenazine (PYO, PCA, or 1-OHPHZ), and poised the working electrode at +200 mV versus that of the NHE to make certain it was just high enough to efficiently oxidize bacterially reduced phenazine but not other medium components (e.g., d-glucose). To confirm the poised potential was appropriate to ensure that phenazine was the sole reversible redox-active component, we compared the cyclic voltammetries (CV) of Δphz cultures with or without phenazine at the time point immediately prior to the start of the survival test. Using the CV method described in detail previously (39), we found that Δphz cultures supplemented with phenazine exhibited single anodic (oxidation) and cathodic (reduction) peaks; these peaks were absent when phenazine was not present. This is illustrated in Fig. ​Fig.11 for PYO. For Δphz cultures supplemented with phenazine, we collected supernatants at the beginning and the end of each survival test for high-performance liquid chromatography (HPLC) analyses using a previously developed method (9). In both cases, HPLC samples yielded the same single phenazine peak with characteristic peak size, implying that degradation did not occur in these experiments. Throughout the incubation period, we continuously recorded the anodic (oxidation) current as well as the charge transferred due to the oxidation of an electrochemically active component(s) at the working electrode. Periodically, we sampled to measure viability by means of counting the number of CFU on LB agar (11).Open in a separate windowFIG. 1.Representative cyclic voltammetry (CV) of Δphz mutant cultures of P. aeruginosa PA14 incubated anaerobically in 100 ml MOPS medium containing 20 mM glucose, supplemented with 90 μM PYO (dark trace) or no PYO (light trace). PYO is the only electrochemically active component with single anodic (oxidation) and cathodic (reduction) peaks characteristic of itself. CV experiments were performed at 100 mV/s, with electrodes consisting of a stationary gold disk working electrode (BASi), an Ag/AgCl reference electrode, and a Pt counter electrode.We observed that the Δphz mutant maintained a constant viable cell number at the original 10⁹ CFU/ml over 7 days, characteristic of survival but not growth (Fig. ​(Fig.2).2). In contrast, when we incubated the Δphz mutant in the bioreactors without adding phenazine or applying a potential or both, the cells sustained their viability up to day 3 and then dropped logarithmically down to 0.1 to 1% of the original 10⁹ CFU/ml by day 7 (Fig. ​(Fig.2).2). Our electrochemical observations were in agreement with the number of CFU results. Without phenazine supplementation, we observed a constant anodic current in the range of 5 to 10 μA with the poised potential, most likely reflecting a background current due to slow oxidation of medium components, which was not able to help Δphz survive over 7 days. In the presence of phenazine, however, the anodic current increased from the background level to 80 ± 10 μA for PYO and PCA and 45 ± 10 μA for 1-OHPHZ within 2 h and stayed at the high current levels with slow decay (less than 20%) throughout the incubation period. The slow current decay was likely due to electrode fouling (23) and/or the accumulation of toxic metabolic by-products in the batch reactors over time. The facile reversibility of redox-active phenazines, which are reduced within the bacterial cell and oxidized outside the cell by the working electrode, led to the high current readings and was key to Δphz survival.Open in a separate windowFIG. 2.PYO (a), PCA (b), and 1-OHPHZ (c) function as electron shuttles (▪) to promote anaerobic survival of the Δphz mutant of P. aeruginosa PA14 when cells are incubated anaerobically in MOPS-buffered medium containing 20 mM d-glucose and ∼90 μM phenazine (PYO, PCA, or 1-OHPHZ) and with the graphite rod working electrode poised at +0.2 V versus that of the NHE. Survival was determined by measuring the number of CFU on LB agar plates. The number of CFU of Δphz anaerobic incubations without phenazine (▿), poised potential (▵), or both (⋄) served as a control. Error bars represent standard deviations from at least triplicate samples in each experimental set. Plots represent results from at least three independent experiments.We then estimated the average number of redox cycles (defined as “a”) for each phenazine molecule, which is known to undergo two-electron oxidation-reduction (39), throughout the 7-day incubation based on the equation adapted from Faraday''s law for bulk electrolysis (1), Q = 2FN = 2F(acv), with c as the phenazine concentration (in M), v as reaction volume (in liters), F as Faraday''s constant (96,485 C/mole), N (which equals acv) as the amount of phenazine (in moles) involved in the electrolysis, and Q (in coulombs) as the net charge quantity associated with the electrochemical oxidation of reduced phenazine during electrolysis (by subtracting the background charge without phenazine from the total charge passed with phenazine). By recording Q, and knowing c and v, we calculated the number of redox cycles over 7 days for PYO, PCA, and 1-OHPHZ to be 31, 22, and 14, respectively. Moreover, each of the three phenazines showed the color characteristic of its oxidized form during redox cycling rather than that of its reduced form, which was apparent when cycling was prevented by not applying the poised potential (39); this indicated that intracellular phenazine reduction was the rate-limiting step of each redox cycle. In addition, we observed the following correlation between the reaction kinetics and phenazine thermodynamic properties: both the phenazine reduction potential (Table ​(Table1)1) and the intracellular reduction rate decreased in the order PYO > PCA > 1-OHPHZ. In summary, despite different electron-shuttling efficiencies, all three phenazines supported Δphz survival equally well within the testing period by acting as electron acceptors (Fig. ​(Fig.22).

TABLE 1.

Properties and results summarized from experiments for testing the roles of endogenous phenazines and other type redox-active compounds in promoting anaerobic survival of P. aeruginosa

Protective Efficacy of Intermittent Preventive Treatment of Malaria in Infants (IPTi) Using Sulfadoxine-Pyrimethamine and Parasite Resistance

Background

Intermittent Preventive Treatment of malaria in infants using sulfadoxine-pyrimethamine (SP-IPTi) is recommended by WHO for implementation in settings where resistance to SP is not high. Here we examine the relationship between the protective efficacy of SP-IPTi and measures of SP resistance.

Methods and Results

We analysed the relationship between protective efficacy reported in the 7 SP-IPTi trials and contemporaneous data from 6 in vivo efficacy studies using SP and 7 molecular studies reporting frequency of dhfr triple and dhps double mutations within 50km of the trial sites. We found a borderline significant association between frequency of the dhfr triple mutation and protective efficacy to 12 months of age of SP-IPTi. This association is significantly biased due to differences between studies, namely number of doses of SP given and follow up times. However, fitting a simple probabilistic model to determine the relationship between the frequency of the dhfr triple, dhps double and dhfr/dhps quintuple mutations associated with resistance to SP and protective efficacy, we found a significant inverse relationship between the dhfr triple mutation frequency alone and the dhfr/dhps quintuple mutations and efficacy at 35 days post the 9 month dose and up to 12 months of age respectively.

Conclusions

A significant relationship was found between the frequency of the dhfr triple mutation and SP-IPTi protective efficacy at 35 days post the 9 month dose. An association between the protective efficacy to 12 months of age and dhfr triple and dhfr/dhps quintuple mutations was found but should be viewed with caution due to bias. It was not possible to define a more definite relationship based on the data available from these trials.     

The Cost-Effectiveness of Intermittent Preventive Treatment for Malaria in Infants in Sub-Saharan Africa

Background

Intermittent preventive treatment in infants (IPTi) has been shown to decrease clinical malaria by approximately 30% in the first year of life and is a promising malaria control strategy for Sub-Saharan Africa which can be delivered alongside the Expanded Programme on Immunisation (EPI). To date, there have been limited data on the cost-effectiveness of this strategy using sulfadoxine pyrimethamine (SP) and no published data on cost-effectiveness using other antimalarials.

Methods

We analysed data from 5 countries in sub-Saharan Africa using a total of 5 different IPTi drug regimens; SP, mefloquine (MQ), 3 days of chlorproguanil-dapsone (CD), SP plus 3 days of artesunate (SP-AS3) and 3 days of amodiaquine-artesunate (AQ3-AS3).The cost per malaria episode averted and cost per Disability-Adjusted Life-Year (DALY) averted were modeled using both trial specific protective efficacy (PE) for all IPTi drugs and a pooled PE for IPTi with SP, malaria incidence, an estimated malaria case fatality rate of 1.57%, IPTi delivery costs and country specific provider and household malaria treatment costs.

Findings

In sites where IPTi had a significant effect on reducing malaria, the cost per episode averted for IPTi-SP was very low, USD 1.36–4.03 based on trial specific data and USD 0.68–2.27 based on the pooled analysis. For IPTi using alternative antimalarials, the lowest cost per case averted was for AQ3-AS3 in western Kenya (USD 4.62) and the highest was for MQ in Korowge, Tanzania (USD 18.56). Where efficacious, based only on intervention costs, IPTi was shown to be cost effective in all the sites and highly cost-effective in all but one of the sites, ranging from USD 2.90 (Ifakara, Tanzania with SP) to USD 39.63 (Korogwe, Tanzania with MQ) per DALY averted. In addition, IPTi reduced health system costs and showed significant savings to households from malaria cases averted. A threshold analysis showed that there is room for the IPTi-efficacy to fall and still remain highly cost effective in all sites where IPTi had a statistically significant effect on clinical malaria.

Conclusions

IPTi delivered alongside the EPI is a highly cost effective intervention against clinical malaria with a range of drugs in a range of malaria transmission settings. Where IPTi did not have a statistically significant impact on malaria, generally in low transmission sites, it was not cost effective.