Drivers of microbial metabolism, nutrient cycling and greenhouse gas production in agricultural streambed sediments

Drivers of carbon and nitrogen cycling, and associated greenhouse gas (GHG) production, were investigated in streambed sediments. Research was conducted to address a lack of availability of adequate porewater sampling technologies and standard protocols, and to determine the effect of temperature, sediment properties and season as primary drivers of nutrient cycling and GHG production in the streambed. A high-resolution sampler of nitrate isotope and concentration data was developed by confirming diffusive equilibrium in thin-film (DET) gel samplers did not cause fractionation of nitrate isotopes. An investigation of commonly used sampling techniques provided information on the most appropriate samplers to use and illustrated that ammonium concentrations vary significantly between sampling techniques. Thermal sensitivity of CO$_2$ and CH$_4$ emissions was dependent on sediment type, organic matter and geology, with these factors having a major effect. CO$_2$ and CH$_4$ concentrations were higher in sand than gravel sediments, but season had a minor influence. Nitrogen cycling was highest in sand than gravel sediments, resulting in high rates of denitrification and low N$_2$O concentrations in the sand sediments. Nitrogen cycling and associated N$_2$O concentrations varied greatly with season in gravel sediments. This indicates that different greenhouse gases may be produced in different areas of the streambed

Antibodies as clinical tools for tuberculosis

Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Global research efforts to improve TB control are hindered by insufficient understanding of the role that antibodies play in protective immunity and pathogenesis. This impacts knowledge of rational and optimal vaccine design, appropriate diagnostic biomarkers, and development of therapeutics. Traditional approaches for the prevention and diagnosis of TB may be less efficacious in high prevalence, remote, and resource-poor settings. An improved understanding of the immune response to the causative agent of TB, Mycobacterium tuberculosis (Mtb), will be crucial for developing better vaccines, therapeutics, and diagnostics. While memory CD4+ T cells and cells and cytokine interferon gamma (IFN-g) have been the main identified correlates of protection in TB, mounting evidence suggests that other types of immunity may also have important roles. TB serology has identified antibodies and functional characteristics that may help diagnose Mtb infection and distinguish between different TB disease states. To date, no serological tests meet the World Health Organization (WHO) requirements for TB diagnosis, but multiplex assays show promise for improving the sensitivity and specificity of TB serodiagnosis. Monoclonal antibody (mAb) therapies and serum passive infusion studies in murine models of TB have also demonstrated some protective outcomes. However, animal models that better reflect the human immune response to Mtb are necessary to fully assess the clinical utility of antibody-based TB prophylactics and therapeutics. Candidate TB vaccines are not designed to elicit an Mtb-specific antibody response, but evidence suggests BCG and novel TB vaccines may induce protective Mtb antibodies. The potential of the humoral immune response in TB monitoring and control is being investigated and these studies provide important insight into the functional role of antibody-mediated immunity against TB. In this review, we describe the current state of development of antibody-based clinical tools for TB, with a focus on diagnostic, therapeutic, and vaccine-based applications

The genetics and epidemiology of neuropathic pain-like symptoms in people with osteoarthritis and post-total joint replacement

Background: Osteoarthritis (OA) is the most common form of arthritis and one of the leading causes of pain and disability worldwide. The only long-term treatment for severe, end-stage OA is removal of the damaged tissues by a total joint replacement (TJR). However for some individuals, pain continues to be a concern post-TJR. As well as this, a proportion of individuals with OA and post-TJR suffer with neuropathic pain-like symptoms (NP). NP is caused by changes or damage to the nervous system, which can occur as a result of nerve injury or chronic pain. NP differs from nociceptive pain in that symptoms can occur in response to a non-harmful stimulus, or in the absence of a stimulus. It is also hard to treat NP effectively. The molecular mechanisms underlying pain and NP in OA are still not well understood and therefore require further study. The high prevalence of OA means research in this field has the potential to impact many individuals. Objectives: The aims of this thesis were a) to describe the effects of NP on post-TJR satisfaction, b) to investigate the genetic contribution to NP in people post-TJR using a genome-wide approach and c) to analyse two candidate genes for an association with pain in people with symptomatic OA or asymptomatic OA. Methods: Individuals from three independent Nottinghamshire-based cohorts were analysed: 1) the Genetics of OA and Lifestyle (GOAL) study, 2) the Nottingham Genetics of OA Study (NGOAS) and 3) individuals recruited from Nottinghamshire who formed part of the Arthritis Research Council OA Genetics (arcOGEN) Consortium. Participants from all three studies were X-rayed at the time of recruitment and the Kellgren-Lawrence (K/L) grading system was used to classify radiographic severity of OA at the joints of interest. All participants answered questionnaires containing validated assessment tools. NP was assessed using the painDETECT questionnaire. Logistic regression analysis was used to identify factors associated with NP in people with OA and post-TJR. A modified LASSO variable reduction method was used to identify the contribution of these factors to post-TJR satisfaction. A genome-wide association scan (GWAS) was used to assess the genetic contribution to NP post-TJR. Candidate gene analysis allowed variants in two genes to be investigated for an association with pain and NP in people with OA and post-TJR. Results: NP was found to be a very strong predictor of post-TJR satisfaction: AUC=0.79 (0.75-0.82). Other contributing factors were the site of the TJR (knee or hip replacement), history of revision surgery and other measures of pain. The use of opioid medications was also significant associated with a lower likelihood of satisfaction post-TJR: OR=0.54 95% CI 0.36-0.80, p=0.002 after adjustment for covariates and pain intensity. Overall, possible NP affected 17.3% of post-TJR patients (10.2% of those satisfied, 41.9% of those partially or not satisfied). A reproducible genetic effect was found between a single nucleotide polymorphism (SNP) in the protein kinase C alpha (PRKCA) gene and the risk of possible NP in people with knee pain, knee OA, hip OA and post-TJR: OR= OR=2.41 95% CI 1.74-3.34, p=1.29x10-7 after meta-analysis in three cohorts. Candidate gene analysis found a consistent genetic effect in the substance P receptor gene (TACR1). The G allele at rs11688000 was associated with lower risk of symptomatic OA (OR=0.84 95% CI 0.70-1.00, p=0.047). This variant was also associated in four independent cohorts, yielding an overall OR=0.80 95% 0.70-0.91, p=8.66x10-4 after meta-analysis in five cohorts (total symptomatic OA n=1,566, total asymptomatic OA n=894). A SNP in the interleukin-15 receptor alpha (IL15RA) gene was found to be significantly associated with the risk of symptomatic OA versus asymptomatic OA: OR=1.56 95% 1.18-2.07, p=0.002. This remained significant (OR=1.43 95% CI 1.04-1.99, p=0.029) after adjustment for covariates (age, sex and body mass index) and tibiofemoral K/L knee OA radiographic grade. Another variant in the IL15RA gene was significantly associated with NP post-TJR: OR=0.76 95% 0.63-0.92, p=0.005 after meta-analysis in two cohorts. Conclusion: The significant contribution of NP to post-TJR satisfaction highlights the importance of identifying cases of NP in people with OA. The PDQ used here to measure NP is a validated tool which could easily be introduced more widely into clinical practice post-TJR. These results also highlight the prevalence of NP symptoms post-TJR. My work also has identified a biologically relevant genetic effect for the risk of NP post-TJR. The SNPs identified in this GWAS provide the first results from a GWAS on NP in OA and could inform future replication studies in additional cohorts. The association of variants in the TACR1 and IL15RA genes with pain in people with OA and, the case of IL15RA, with NP pain post-TJR suggests potential treatment targets for future studies into therapies to treat pain and NP in people with OA

Streambed organic matter controls on carbon dioxide and methane emissions from streams

Greenhouse gas (GHG) emissions of carbon dioxide (CO2) and methane (CH4) from streambeds are currently understudied. There is a paucity of research exploring organic matter (OM) controls on GHG production by microbial metabolic activity in streambeds, which is a major knowledge gap given the increased inputs of allochthonous carbon to streams, especially in agricultural catchments. This study aims to contribute to closing this knowledge gap by quantifying how contrasting OM contents in different sediments affect streambed GHG production and associated microbial metabolic activity. We demonstrate, by means of an incubation experiment, that streambed sediments have the potential to produce substantial amounts of GHG, controlled by sediment OM quantity and quality. We observed streambed CO2 production rates that can account for 35% of total stream evasion estimated in previous studies, ranging between 1.4 and 86% under optimal conditions. Methane production varied stronger than CO2 between different geologic backgrounds, suggesting OM quality controls between streambed sediments. Moreover, our results indicate that streambed sediments may produce much more CO2 than quantified to date, depending on the quantity and quality of the organic matter, which has direct implications for global estimates of C fluxes in stream ecosystems

Opening opportunities for high-resolution isotope analysis - Quantification of δ15NNO3 and δ18ONO3 in diffusive equilibrium in thin–film passive samplers

The fate of nitrate transported across groundwater-surface water interfaces has been intensively studied in recent decades. The interfaces between aquifers and rivers or lakes have been identified as biogeochemical hotspots with steep redox gradients. However, a detailed understanding of the spatial heterogeneity and potential temporal variability of these hotspots, and the consequences for nitrogen processing, is still hindered by a paucity of adequate measurement techniques. A novel methodology is presented here, using Diffusive Equilibrium in Thin-film (DET) gels as high-spatial-resolution passive-samplers of δ15NNO3 and δ18ONO3 to investigate nitrogen cycling. Fractionation of δ15NNO3 and δ18ONO3 during diffusion of nitrate through the DET gel was determined using varying equilibrium times and nitrate concentrations. This demonstrated that nitrate isotopes of δ15NNO3 and δ18ONO3 do not fractionate when sampled with a DET gel. δ15NNO3 values from the DET gels ranged between 2.3 ± 0.2 and 2.7 ± 0.3‰ for a NO3– stock solution value of 2.7 ± 0.4‰, and δ18ONO3 values ranged between 18.3 ± 1.0 and 21.5 ± 0.8‰ for a NO3– stock solution of 19.7 ± 0.9‰. Nitrate recovery and isotope values were independent of equilibrium time and nitrate concentration. Additionally, an in situ study showed that nitrate concentration and isotopes provide unique, high-resolution data that enable improved understanding of nitrogen cycling in freshwater sediments

Towards a comprehensive understanding of the structural dynamics of a bacterial diterpene synthase during catalysis

Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C–C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins’ structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2’s catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis

Seasonal variability of sediment controls of nitrogen cycling in an agricultural stream

Agricultural streams receive large inputs of nutrients, such as nitrate (NO3−) and ammonium (NH4+), which impact water quality and stream health. Streambed sediments are hotspots of biogeochemical reactivity, characterised by high rates of nutrient attenuation and denitrification. High concentrations of nitrous oxide (N2O) previously observed in stream sediments point to incomplete denitrification, with sediments acting as a potentially significant source of global N2O. We investigated the effect of sediment type and seasonal variation on denitrification and N2O production in the streambed of an agricultural UK stream. Denitrification was strongly controlled by sediment type, with sand-dominated sediments exhibiting potential rates of denitrification almost 10 times higher than those observed in gravel-dominated sediments (0.026 ± 0.004 N2O–N μg g−1 h−1 for sand-dominated and 0.003 ± 0.003 N2O–N μg g−1 h−1 for gravel-dominated). In-situ measurements supported this finding, with higher concentrations of NO3−, nitrite (NO2−) and N2O observed in the porewaters of gravel-dominated sediments. Denitrification varied substantially between seasons, with denitrification increasing from winter to autumn. Our results indicate highest NO3− reduction occurred in sand-dominated sediments whilst highest N2O concentrations occurred in gravel-dominated sediments. This suggests that finer-grained streambeds could play an important role in removing excess nitrogen from agricultural catchments without producing excess N2O

Reply to ‘Pseudoreplication and greenhouse-gas emissions from rivers'

Tiegs et al.1 highlight the significance and relevance of the findings of Comer-Warner et al.2 on greenhouse-gas emissions from streambed sediments but raise questions about some aspects of the experimental design. We support their call for more detailed field and laboratory-based studies on this subject. However, we believe that their concerns relate to uncertainties and limitations in the experimental design that were discussed explicitly in the original paper (and accompanying transparent peer review process—available online), or represent criticisms related to highly improbable minor anomalies that may unnecessarily dismiss experimental results as discussed below

Bidirectional association between disturbed sleep and neuropathic pain symptoms : a prospective cohort study in post-total joint replacement participants

Background Disturbed sleep is strongly correlated with chronic pain. The aim of this study was to examine the association between sleep disturbance and incident joint pain focusing on neuropathic-like pain symptoms. Methods A total of 423 individuals who had undergone total joint replacement (TJR) for osteoarthritis were assessed at the mean time of 3.6 years post-surgery and again at 5.9 years post-TJR, using the Medical Outcomes Survey sleep subscale, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and painDETECT questionnaire instruments. Cox hazard ratios (HRs) and 95% confidence intervals (CIs) were computed adjusting for age, body mass index, sex, and use of hypnotic and analgesic medication. Results The presence of neuropathic pain symptoms predicted incidence of disturbed sleep after adjustment for covariates and pain severity (adjusted HR [aHR] 2.01, 95% CI: 1.00–4.10; p<0.05). There was no association between joint pain and incidence of disturbed sleep when individuals with neuropathic pain symptoms at the baseline visit were excluded (aHR 1.11, 95% CI: 0.47–2.67). Disturbed sleep at baseline predicted incident neuropathic joint pain symptoms (aHR 2.75, 95% CI: 1.21–6.26; p<0.016) but had no effect on incidence of joint pain when all types of pain were considered together (aHR 0.63, 95% CI: 0.30–1.39). Conclusion These data suggest a causal bidirectional link between sleep disturbance and joint pain with neuropathic features but not with other types of joint pain