Modeling methanogenesis with a genome-scale metabolic reconstruction of Methanosarcina barkeri

We present a genome-scale metabolic model for the archaeal methanogen Methanosarcina barkeri. We characterize the metabolic network and compare it to reconstructions from the prokaryotic, eukaryotic and archaeal domains. Using the model in conjunction with constraint-based methods, we simulate the metabolic fluxes and resulting phenotypes induced by different environmental and genetic conditions. This represents the first large-scale simulation of either a methanogen or an archaeal species. Model predictions are validated by comparison to experimental growth measurements and phenotypes of M. barkeri on different substrates. The predicted growth phenotypes for wild type and mutants of the methanogenic pathway have a high level of agreement with experimental findings. We further examine the efficiency of the energy-conserving reactions in the methanogenic pathway, specifically the Ech hydrogenase reaction, and determine a stoichiometry for the nitrogenase reaction. This work demonstrates that a reconstructed metabolic network can serve as an analysis platform to predict cellular phenotypes, characterize methanogenic growth, improve the genome annotation and further uncover the metabolic characteristics of methanogenesis

Designing a physical activity parenting course : parental views on recruitment, content and delivery

Background Many children do not engage in sufficient levels of physical activity (PA) and spend too much time screen-viewing (SV). High levels of SV (e.g. watching TV, playing video games and surfing the internet) and low levels of PA have been associated with adverse health outcomes. Parenting courses may hold promise as an intervention medium to change children’s PA and SV. The current study was formative work conducted to design a new parenting programme to increase children’s PA and reduce their SV. Specifically, we focussed on interest in a course, desired content and delivery style, barriers and facilitators to participation and opinions on control group provision. Methods In-depth telephone interviews were conducted with thirty two parents (29 female) of 6–8 year olds. Data were analysed thematically. An anonymous online survey was also completed by 750 parents of 6–8 year old children and descriptive statistics calculated. Results Interview participants were interested in a parenting course because they wanted general parenting advice and ideas to help their children be physically active. Parents indicated that they would benefit from knowing how to quantify their child’s PA and SV levels. Parents wanted practical ideas of alternatives to SV. Most parents would be unable to attend unless childcare was provided. Schools were perceived to be a trusted source of information about parenting courses and the optimal recruitment location. In terms of delivery style, the majority of parents stated they would prefer a group-based approach that provided opportunities for peer learning and support with professional input. Survey participants reported the timing of classes and the provision of childcare were essential factors that would affect participation. In terms of designing an intervention, the most preferred control group option was the opportunity to attend the same course at a later date. Conclusions Parents are interested in PA/SV parenting courses but the provision of child care is essential for attendance. Recruitment is likely to be facilitated via trusted sources. Parents want practical advice on how to overcome barriers and suggest advice is provided in a mutually supportive group experience with expert input

Some considerations in the design and interpretation of antimalarial drug trials in uncomplicated falciparum malaria

BACKGROUND: Treatments for uncomplicated falciparum malaria should have high cure rates. The World Health Organization has recently set a target cure rate of 95% assessed at 28 days. The use of more effective drugs, with longer periods of patient follow-up, and parasite genotyping to distinguish recrudescence from reinfection raise issues related to the design and interpretation of antimalarial treatment trials in uncomplicated falciparum malaria which are discussed here. METHODS: The importance of adequate follow-up is presented and the advantages and disadvantages of non-inferiority trials are discussed. The different methods of interpreting trial results are described, and the difficulties created by loss to follow-up and missing or indeterminate genotyping results are reviewed. CONCLUSION: To characterize cure rates adequately assessment of antimalarial drug efficacy in uncomplicated malaria requires a minimum of 28 days and as much as 63 days follow-up after starting treatment. The longer the duration of follow-up in community-based assessments, the greater is the risk that this will be incomplete, and in endemic areas, the greater is the probability of reinfection. Recrudescence can be distinguished from reinfection using PCR genotyping but there are commonly missing or indeterminate results. There is no consensus on how these data should be analysed, and so a variety of approaches have been employed. It is argued that the correct approach to analysing antimalarial drug efficacy assessments is survival analysis, and patients with missing or indeterminate PCR results should either be censored from the analysis, or if there are sufficient data, results should be adjusted based on the identified ratio of new infections to recrudescences at the time of recurrent parasitaemia. Where the estimated cure rates with currently recommended treatments exceed 95%, individual comparisons with new regimens should generally be designed as non-inferiority trials with sample sizes sufficient to determine adequate precision of cure rate estimates (such that the lower 95% confidence interval bound exceeds 90%)

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-κB activation

The potential for inhibitors of nuclear factor-κB (NF-κB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-κB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-κB inhibitor SN50 (18 μM) attenuated the expression of 205 proteasome α-subunits, two subunits of the 195 regulator MSSI and p42, and the ubiquitin-conjugating enzyme, E214k, as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-κB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 μM) and resveratrol (30 μM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg-1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg-1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-κB DNA-binding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-κB may prove useful for the treatment of muscle wasting in cancer cachexia

Population pharmacokinetics of artesunate and amodiaquine in African children

<p>Abstract</p> <p>Background</p> <p>Pharmacokinetic (PK) data on amodiaquine (AQ) and artesunate (AS) are limited in children, an important risk group for malaria. The aim of this study was to evaluate the PK properties of a newly developed and registered fixed dose combination (FDC) of artesunate and amodiaquine.</p> <p>Methods</p> <p>A prospective population pharmacokinetic study of AS and AQ was conducted in children aged six months to five years. Participants were randomized to receive the new artesunate and amodiaquine FDC or the same drugs given in separate tablets. Children were divided into two groups of 70 (35 in each treatment arm) to evaluate the pharmacokinetic properties of AS and AQ, respectively. Population pharmacokinetic models for dihydroartemisinin (DHA) and desethylamodiaquine (DeAq), the principal pharmacologically active metabolites of AS and AQ, respectively, and total artemisinin anti-malarial activity, defined as the sum of the molar equivalent plasma concentrations of DHA and artesunate, were constructed using the non-linear mixed effects approach. Relative bioavailability between products was compared by estimating the ratios (and 95% CI) between the areas under the plasma concentration-time curves (AUC).</p> <p>Results</p> <p>The two regimens had similar PK properties in young children with acute malaria. The ratio of loose formulation to fixed co-formulation AUCs, was estimated as 1.043 (95% CI: 0.956 to 1.138) for DeAq. For DHA and total anti-malarial activity AUCs were estimated to be the same. Artesunate was rapidly absorbed, hydrolysed to DHA, and eliminated. Plasma concentrations were significantly higher following the first dose, when patients were acutely ill, than after subsequent doses when patients were usually afebrile and clinically improved. Amodiaquine was converted rapidly to DeAq, which was then eliminated with an estimated median (range) elimination half-life of 9 (7 to 12) days. Efficacy was similar in the two treatments groups, with cure rates of 0.946 (95% CI: 0.840–0.982) in the AS+AQ group and 0.892 (95% CI: 0.787 – 0.947) in the AS/AQ group. Four out of five patients with PCR confirmed recrudescences received AQ doses < 10 mg/kg. Both regimens were well tolerated. No child developed severe, post treatment neutropaenia (<1,000/μL). There was no evidence of AQ dose related hepatotoxicity, but one patient developed an asymptomatic rise in liver enzymes that was resolving by Day-28.</p> <p>Conclusion</p> <p>The bioavailability of the co-formulated AS-AQ FDC was similar to that of the separate tablets for desethylamodiaquine, DHA and the total anti-malarial activity. These data support the use this new AS-AQ FDC in children with acute uncomplicated falciparum malaria.</p

Human cytomegalovirus gene UL76 induces IL-8 expression through activation of the DNA damage response.

Human cytomegalovirus (HCMV), a β-herpesvirus, has evolved many strategies to subvert both innate and adaptive host immunity in order to ensure its survival and propagation within the host. Induction of IL-8 is particularly important during HCMV infection as neutrophils, primarily attracted by IL-8, play a key role in virus dissemination. Moreover, IL-8 has a positive effect in the replication of HCMV. This work has identified an HCMV gene (UL76), with the relevant property of inducing IL-8 expression at both transcriptional and protein levels. Up-regulation of IL-8 by UL76 results from activation of the NF-kB pathway as inhibition of both IKK-β activity or degradation of Ikβα abolishes the IL-8 induction and, concomitantly, expression of UL76 is associated with the translocation of p65 to the nucleus where it binds to the IL-8 promoter. Furthermore, the UL76-mediated induction of IL-8 requires ATM and is correlated with the phosphorylation of NEMO on serine 85, indicating that UL76 activates NF-kB pathway by the DNA Damage response, similar to the impact of genotoxic drugs. More importantly, a UL76 deletion mutant virus was significantly less efficient in stimulating IL-8 production than the wild type virus. In addition, there was a significant reduction of IL-8 secretion when ATM -/- cells were infected with wild type HCMV, thus, indicating that ATM is also involved in the induction of IL-8 by HCMV. In conclusion, we demonstrate that expression of UL76 gene induces IL-8 expression as a result of the DNA damage response and that both UL76 and ATM have a role in the mechanism of IL-8 induction during HCMV infection. Hence, this work characterizes a new role of the activation of DNA Damage response in the context of host-pathogen interactions

NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin–proteasome system in skeletal muscle

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-κB (NF-κB) in regulating muscle protein degradation and expression of the ubiquitin–proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C2C12 myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-κBα, an NF-κB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-κBα, nuclear accumulation of NF-κB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-κBα proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-κB. Proteolysis-inducing factor also induced increased expression of the ubiquitin–proteasome pathway, as determined by ‘chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the β-subunits of the proteasome, protein expression of 20S α-subunits and the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme, E214k, in cells containing wild-type, but not mutant, I-κBα. The ability of mutant I-κBα to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin–proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-κB

Ethological principles predict the neuropeptides co-opted to influence parenting

Ethologists predicted that parental care evolves by modifying behavioural precursors in the asocial ancestor. As a corollary, we predict that the evolved mechanistic changes reside in genetic pathways underlying these traits. Here we test our hypothesis in female burying beetles, Nicrophorus vespilloides, an insect where caring adults regurgitate food to begging, dependent offspring. We quantify neuropeptide abundance in brains collected from three behavioural states: solitary virgins, individuals actively parenting or post-parenting solitary adults and quantify 133 peptides belonging to 18 neuropeptides. Eight neuropeptides differ in abundance in one or more states, with increased abundance during parenting in seven. None of these eight neuropeptides have been associated with parental care previously, but all have roles in predicted behavioural precursors for parenting. Our study supports the hypothesis that predictable traits and pathways are targets of selection during the evolution of parenting and suggests additional candidate neuropeptides to study in the context of parenting

Early Predictors of Objectively Measured Physical Activity and Sedentary Behaviour in 8–10 Year Old Children: The Gateshead Millennium Study

With a number of studies suggesting associations between early life influences and later chronic disease risk, it is suggested that associations between early growth and later physical activity (PA) may be a mediator. However, conflicting evidence exists for association between birth weight and childhood PA. In addition, it is important to know what other, potentially modifiable, factors may influence PA in children given its' association with childhood and later adiposity. We used the Gateshead Millennium Study (GMS) to identify predictors of childhood PA levels. The GMS is a cohort of 1029 infants born in 1999–2000 in Gateshead in northern England. Throughout infancy and early childhood, detailed information was collected. Assessments at age 9 years included body composition, objective measures of habitual PA and a range of lifestyle factors. Mean total volumes of PA (accelerometer count per minute, cpm) and moderate-vigorous intensity PA (MVPA), and the percentage of time spent in sedentary behaviour (%SB) were quantified and related to potential predictors using linear regression and path analysis. Children aged 8–10 years were included. Significant differences were seen in all three outcome variables between sexes and season of measurement (p<0.001). Restricting children’s access to television was associated with decreased MVPA. Increased paternal age was associated with significant increases in %SB (p = 0.02), but not MVPA or total PA. Increased time spent in out of school sports clubs was significantly associated with decreased %SB (p = 0.02). No significant associations were seen with birth weight. A range of factors, directly or indirectly, influenced PA and sedentary behaviour. However, associations differed between the different constructs of PA and %SB. Exploring further the sex differences in PA would appear to be useful, as would encouraging children to join out of school sports clubs