Dissecting cell death pathways in fed-batch bioreactors.

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells.

Functional consequences of animal community changes in managed grasslands: An application of the CAFE approach.

In the midst of an ongoing biodiversity crisis, much research has focused on species losses and their impacts on ecosystem functioning. The functional consequences (ecosystem response) of shifts in communities are shaped not only by changes in species richness, but also by compositional shifts that result from species losses and gains. Species differ in their contribution to ecosystem functioning, so species identity underlies the consequences of species losses and gains on ecosystem functions. Such research is critical to better predict the impact of disturbances on communities and ecosystems. We used the "Community Assembly and the Functioning of Ecosystems" (CAFE) approach, a modification of the Price equation to understand the functional consequences and relative effects of richness and composition changes in small nonvolant mammal and dung beetle communities as a result of two common disturbances in North American prairie restorations, prescribed fire and the reintroduction of large grazing mammals. Previous research in this system has shown dung beetles are critically important decomposers, while small mammals modulate much energy in prairie food webs. We found that dung beetle communities were more responsive to bison reintroduction and prescribed fires than small nonvolant mammals. Dung beetle richness increased after bison reintroduction, with higher dung beetle community biomass resulting from changes in remaining species (context-dependent component) rather than species turnover (richness components); prescribed fire caused a minor increase in dung beetle biomass for the same reason. For small mammals, bison reintroduction reduced energy transfer through the loss of species, while prescribed fire had little impact on either small mammal richness or energy transfer. The CAFE approach demonstrates how bison reintroduction controls small nonvolant mammal communities by increasing prairie food web complexity, and increases dung beetle populations with possible benefits for soil health through dung mineralization and soil bioturbation. Prescribed fires, however, have little effect on small mammals and dung beetles, suggesting a resilience to fire. These findings illustrate the key role of re-establishing historical disturbance regimes when restoring endangered prairie ecosystems and their ecological function.

Taxonomic and functional restoration of tallgrass prairie soil microbial communities in comparison to remnant and agricultural soils.

Restoring ecosystems requires the re-establishment of diverse soil microbial communities that drive critical ecosystem functions. In grasslands, restoration and management require the application of disturbances like fire and grazing. Disturbances can shape microbial taxonomic composition and potentially functional composition as well. We characterized taxonomic and functional gene composition of soil communities using whole genome shotgun metagenomic sequencing to determine how restored soil communities differed from pre-restoration agricultural soils and original remnant soils, how management affects soil microbes, and whether restoration and management affect the number of microbial genes associated with carbohydrate degradation. We found distinct differences in both taxonomic and functional diversity and composition among restored, remnant, and agricultural soils. Remnant soils had low taxonomic and functional richness and diversity, as well as distinct composition, indicating that restoration of agricultural soils does not re-create soil microbial communities that match remnants. Prescribed fire management increased functional diversity, which also was higher in more recently planted restorations. Finally, restored and post-fire soils included high abundances of genes encoding cellulose-degrading enzymes, so restorations and their ongoing management can potentially support functions important in carbon cycling.

Restoration age and reintroduced bison may shape soil bacterial communities in restored tallgrass prairies.

Knowledge of how habitat restoration shapes soil microbial communities often is limited despite their critical roles in ecosystem function. Soil community diversity and composition change after restoration, but the trajectory of these successional changes may be influenced by disturbances imposed for habitat management. We studied soil bacterial communities in a restored tallgrass prairie chronosequence for >6 years to document how diversity and composition changed with age, management through fire, and grazing by reintroduced bison, and in comparison to pre-restoration agricultural fields and remnant prairies. Soil C:N increased with restoration age and bison, and soil pH first increased and then declined with age, although bison weakened this pattern. Bacterial richness and diversity followed a similar hump-shaped pattern as soil pH, such that the oldest restorations approached the low diversity of remnant prairies. ß-diversity patterns indicated that composition in older restorations with bison resembled bison-free sites, but over time they became more distinct. In contrast, younger restorations with bison maintained unique compositions throughout the study, suggesting bison disturbances may cause a different successional trajectory. We used a novel random forest approach to identify taxa that indicate these differences, finding that they were frequently associated with bacteria that respond to grazing in other grasslands.

Termite sensitivity to temperature affects global wood decay rates.

Deadwood is a large global carbon store with its store size partially determined by biotic decay. Microbial wood decay rates are known to respond to changing temperature and precipitation. Termites are also important decomposers in the tropics but are less well studied. An understanding of their climate sensitivities is needed to estimate climate change effects on wood carbon pools. Using data from 133 sites spanning six continents, we found that termite wood discovery and consumption were highly sensitive to temperature (with decay increasing >6.8 times per 10°C increase in temperature)-even more so than microbes. Termite decay effects were greatest in tropical seasonal forests, tropical savannas, and subtropical deserts. With tropicalization (i.e., warming shifts to tropical climates), termite wood decay will likely increase as termites access more of Earth's surface.

The effect of natural disturbances on forest biodiversity: an ecological synthesis.

Disturbances alter biodiversity via their specific characteristics, including severity and extent in the landscape, which act at different temporal and spatial scales. Biodiversity response to disturbance also depends on the community characteristics and habitat requirements of species. Untangling the mechanistic interplay of these factors has guided disturbance ecology for decades, generating mixed scientific evidence of biodiversity responses to disturbance. Understanding the impact of natural disturbances on biodiversity is increasingly important due to human-induced changes in natural disturbance regimes. In many areas, major natural forest disturbances, such as wildfires, windstorms, and insect outbreaks, are becoming more frequent, intense, severe, and widespread due to climate change and land-use change. Conversely, the suppression of natural disturbances threatens disturbance-dependent biota. Using a meta-analytic approach, we analysed a global data set (with most sampling concentrated in temperate and boreal secondary forests) of species assemblages of 26 taxonomic groups, including plants, animals, and fungi collected from forests affected by wildfires, windstorms, and insect outbreaks. The overall effect of natural disturbances on α-diversity did not differ significantly from zero, but some taxonomic groups responded positively to disturbance, while others tended to respond negatively. Disturbance was beneficial for taxonomic groups preferring conditions associated with open canopies (e.g. hymenopterans and hoverflies), whereas ground-dwelling groups and/or groups typically associated with shady conditions (e.g. epigeic lichens and mycorrhizal fungi) were more likely to be negatively impacted by disturbance. Across all taxonomic groups, the highest α-diversity in disturbed forest patches occurred under moderate disturbance severity, i.e. with approximately 55% of trees killed by disturbance. We further extended our meta-analysis by applying a unified diversity concept based on Hill numbers to estimate α-diversity changes in different taxonomic groups across a gradient of disturbance severity measured at the stand scale and incorporating other disturbance features. We found that disturbance severity negatively affected diversity for Hill number q = 0 but not for q = 1 and q = 2, indicating that diversity-disturbance relationships are shaped by species relative abundances. Our synthesis of α-diversity was extended by a synthesis of disturbance-induced change in species assemblages, and revealed that disturbance changes the ß-diversity of multiple taxonomic groups, including some groups that were not affected at the α-diversity level (birds and woody plants). Finally, we used mixed rarefaction/extrapolation to estimate biodiversity change as a function of the proportion of forests that were disturbed, i.e. the disturbance extent measured at the landscape scale. The comparison of intact and naturally disturbed forests revealed that both types of forests provide habitat for unique species assemblages, whereas species diversity in the mixture of disturbed and undisturbed forests peaked at intermediate values of disturbance extent in the simulated landscape. Hence, the relationship between α-diversity and disturbance severity in disturbed forest stands was strikingly similar to the relationship between species richness and disturbance extent in a landscape consisting of both disturbed and undisturbed forest habitats. This result suggests that both moderate disturbance severity and moderate disturbance extent support the highest levels of biodiversity in contemporary forest landscapes.

Coupling picodroplet microfluidics with plate imaging for the rapid creation of biomanufacturing suitable cell lines with high probability and improved multi-step assurance of monoclonality.

BACKGROUND: There is an expectation from regulatory agencies that cell lines used in the commercial production of biopharmaceuticals are derived from a single cell progenitor. Traditional methods of single cell cloning include the use of the limiting dilution cloning method which often requires multiple rounds of low cell density cell plating and either microscopic evaluation that wells contain single cells and/or the calculation of a statistically derived probability of monoclonality. METHODS AND RESULTS: We have combined the single cell screening, deposition and picodroplet imaging ability of Sphere Fluidics' Cyto-Mine technology with the plate imaging capability of the Solentim Cell Metric to create a novel workflow for the generation of high producing clonal cell lines with both high probability and assurance of monoclonality. The efficiency of three key stages of the process (single cell picodroplet encapsulation, single picodroplet dispensation and single cell settling in the focal plane of the plate imager) was determined and a probability calculation was derived using the Wilson Score Interval method. The combined probability that a single cell is encapsulated into a picodroplet, is deposited into the correct well of a 96-well plate and that a cell settles into the focal plane of the plate imager yields a combined > 99% probability of monoclonality. Furthermore, visual verification of a single cell progenitor is obtained at multiple steps throughout the cloning workflow. CONCLUSION: This novel methodology for the rapid creation of high quality clonal cell lines for biomanufacturing purposes has many advantages over more traditional approaches including improved assurance of single cell derivation, integrated imaging capability, assay flexibility, equipment utilization time and in-process cell line segregation.

Disturbance-Induced Trophic Niche Shifts In Ground Beetles (Coleoptera: Carabidae) In Restored Grasslands.

Ecosystem restoration is a critical component of land management, countering the loss of native biodiversity. Restoration efforts are enhanced by reintroducing naturally occurring ecosystem processes, including disturbances that may impact species characteristics such as niche position or niche size. In grasslands, grazing and fire affect plant diversity and habitat complexity, which potentially influence insect dietary behaviors and thus their contributions to functions like seed and arthropod predation. Using carbon and nitrogen stable isotopes, we characterized variation in the dietary niche of six ground beetle species (Coleoptera: Carabidae) in response to grazing by reintroduced bison and prescribed fire disturbances in twenty tallgrass prairies. Management disturbances did not affect activity density for most beetle species and mean trophic position was mostly unaffected. However, five of six species exhibited increased trophic niche area and breadth with disturbances, indicating a switch to a more generalist diet that incorporated a wider range of food items. The combination of bison and fire impacts may increase vegetation patchiness and heterogeneity, driving these diet changes. Morphological traits and microhabitat preferences might mediate response to disturbances and the resulting heterogeneity. Combining prescribed fire and grazing, which increases plant diversity and vegetation structural diversity, may help beetle communities establish over time and support the ecological functions to which these insects contribute.

Introduced ecological engineers drive behavioral changes of grasshoppers, consequently linking to its abundance in two grassland plant communities.

Introduced ecosystem engineers are expected to have extensive ecological impacts on a broad range of resident biota by altering the physical-chemical structure of ecosystems. Livestock that are potentially important introduced ecosystem engineers in grassland systems could create and/or modify habitats for native plant-dwelling insects. Yet, there is little knowledge of how insects respond to engineering effects of introduced livestock. To bridge this gap, we tested how domestic sheep affects the behavior and abundance of a native grasshopper Euchorthippus unicolor at both low (11.8 ± 0.4 plant species per plot) and high (19.8 ± 0.5 plant species per plot) diversity sites. Results found grasshoppers shifted their resting and feeding locations from the upper to the intermediate or low layers of vegetation, and fed on more plants species following livestock engineering effects. In the low plant diversity habitats, grazing caused grasshoppers to increase switching frequency, spend more time searching for host plants, and reduce time spent feeding, but had opposite effects on all the three behaviors in the high-diversity habitats. Moreover, grazing engineering effects on behavioral changes of grasshoppers were potentially related to their abundance. Overall, this study highlights native insect species' behavior and abundance in responses to introduced ecological engineers, and suggests that ecosystem engineers of non-native species have strong and important impacts extending beyond their often most obvious and frequently documented direct ecological effects.

Effects of management outweigh effects of plant diversity on restored animal communities in tallgrass prairies.

A primary goal of ecological restoration is to increase biodiversity in degraded ecosystems. However, the success of restoration ecology is often assessed by measuring the response of a single functional group or trophic level to restoration, without considering how restoration affects multitrophic interactions that shape biodiversity. An ecosystem-wide approach to restoration is therefore necessary to understand whether animal responses to restoration, such as changes in biodiversity, are facilitated by changes in plant communities (plant-driven effects) or disturbance and succession resulting from restoration activities (management-driven effects). Furthermore, most restoration ecology studies focus on how restoration alters taxonomic diversity, while less attention is paid to the response of functional and phylogenetic diversity in restored ecosystems. Here, we compared the strength of plant-driven and management-driven effects of restoration on four animal communities (ground beetles, dung beetles, snakes, and small mammals) in a chronosequence of restored tallgrass prairie, where sites varied in management history (prescribed fire and bison reintroduction). Our analyses indicate that management-driven effects on animal communities were six-times stronger than effects mediated through changes in plant biodiversity. Additionally, we demonstrate that restoration can simultaneously have positive and negative effects on biodiversity through different pathways, which may help reconcile variation in restoration outcomes. Furthermore, animal taxonomic and phylogenetic diversity responded differently to restoration, suggesting that restoration plans might benefit from considering multiple dimensions of animal biodiversity. We conclude that metrics of plant diversity alone may not be adequate to assess the success of restoration in reassembling functional ecosystems.

Reintroduced grazers and prescribed fire effects on beetle assemblage structure and function in restored grasslands.

Ecological restoration seeks to reestablish functioning ecosystems, but planning and evaluation often focus on taxonomic community structure and neglect consumers and their functional roles. The functional trait composition of insect assemblages, which make up the majority of animal diversity in many systems, can reveal how they are affected by restoration management and the consequences for ecosystem function. We sampled ground beetle (Coleoptera: Carabidae) assemblages in restored tallgrass prairies varying in management with prescribed fire and reintroduced American bison (Bison bison) to describe their taxonomic and functional trait structure. We also measured seed and arthropod predation to relate management, beetle assemblage characteristics, and function, and to test if function is maximized by trait diversity, dominant trait values, or beetle abundance. Beetle assemblages primarily varied with restoration age, declining over time in richness and both taxonomic and functional diversity, but bison presence also influenced taxonomic composition. Prescribed fire reduced seed predation in summer and arthropod predation in fall. Although seed predation was unrelated to beetle assemblages, arthropod predation was greater in sites with higher abundances of carnivorous ground beetles. The relatively weak impacts of fire and bison on functional assemblage structure is a promising sign that these management disturbances, aimed at supporting a diverse native plant community, are not detrimental to beetle assemblages. The significance of reduced predator function following prescribed fire will depend on the restoration context and whether seed or arthropod predation relates to management goals.

Flowering plant composition shapes pathogen infection intensity and reproduction in bumble bee colonies.

Pathogens pose significant threats to pollinator health and food security. Pollinators can transmit diseases during foraging, but the consequences of plant species composition for infection is unknown. In agroecosystems, flowering strips or hedgerows are often used to augment pollinator habitat. We used canola as a focal crop in tents and manipulated flowering strip composition using plant species we had previously shown to result in higher or lower bee infection in short-term trials. We also manipulated initial colony infection to assess impacts on foraging behavior. Flowering strips using high-infection plant species nearly doubled bumble bee colony infection intensity compared to low-infection plant species, with intermediate infection in canola-only tents. Both infection treatment and flowering strips reduced visits to canola, but we saw no evidence that infection treatment shifted foraging preferences. Although high-infection flowering strips increased colony infection intensity, colony reproduction was improved with any flowering strips compared to canola alone. Effects of flowering strips on colony reproduction were explained by nectar availability, but effects of flowering strips on infection intensity were not. Thus, flowering strips benefited colony reproduction by adding floral resources, but certain plant species also come with a risk of increased pathogen infection intensity.

A platform for context-specific genetic engineering of recombinant protein production by CHO cells.

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.

From plant fungi to bee parasites: mycorrhizae and soil nutrients shape floral chemistry and bee pathogens.

Bee populations have experienced declines in recent years, due in part to increased disease incidence. Multiple factors influence bee-pathogen interactions, including nectar and pollen quality and secondary metabolites. However, we lack an understanding of how plant interactions with their environment shape bee diet quality. We examined how plant interactions with the belowground environment alter floral rewards and, in turn, bee-pathogen interactions. Soil-dwelling mycorrhizal fungi are considered plant mutualists, although the outcome of the relationship depends on environmental conditions such as nutrients. In a 2 × 2 factorial design, we asked whether mycorrhizal fungi and nutrients affect concentrations of nectar and pollen alkaloids (anabasine and nicotine) previously shown to reduce infection by the gut pathogen Crithidia in the native bumble bee Bombus impatiens. To ask how plant interactions affect this common bee pathogen, we fed pollen and nectar from our treatment plants, and from a wildflower pollen control with artificial nectar, to bees infected with Crithidia. Mycorrhizal fungi and fertilizer both influenced flowering phenology and floral chemistry. While we found no anabasine or nicotine in nectar, high fertilizer increased anabasine and nicotine in pollen. Arbuscular mycorrhizal fungi (AMF) decreased nicotine concentrations, but the reduction due to AMF was stronger in high than low-nutrient conditions. AMF and nutrients also had interactive effects on bee pathogens via changes in nectar and pollen. High fertilizer reduced Crithidia cell counts relative to low fertilizer in AMF plants, but increased Crithidia in non-AMF plants. These results did not correspond with effects of fertilizer and AMF on pollen alkaloid concentrations, suggesting that other components of pollen or nectar were affected by treatments and shaped pathogen counts. Our results indicate that soil biotic and abiotic environment can alter bee-pathogen interactions via changes in floral rewards, and underscore the importance of integrative studies to predict disease dynamics and ecological outcomes.

Preinfection Effects of Nectar Secondary Compounds on a Bumble Bee Gut Pathogen.

Bumble bee pollinators can be exposed to pathogens when foraging on flowers previously visited by infected individuals. Infectious cells may be deposited in floral nectar, providing a site for pathogens to interact with nectar secondary compounds prior to infecting bees. Some nectar secondary compounds can reduce pathogen counts in infected bumble bees, but we know less about how exposure to these compounds directly affects pathogens prior to being ingested by their host. We exposed the trypanosomatid gut pathogen, Crithidia bombi (Lipa & Triggiani 1988) (Trypanosomatida: Trypanosomatidae), to six different compounds found in nectar (aucubin, catalpol, nicotine, thymol, anabasine, and citric acid) for 1-h prior to ingestion by Bombus impatiens (Cresson 1863) (Hymenoptera: Apidae) workers that were then reared for 1 wk on a control diet. All of these compounds except citric acid reduce pathogen levels when consumed in hosts after infection, and citric acid is a common preservative found in citrus fruits and some honeys. We found that both citric acid and aucubin reduced Crithidia cell counts compared with controls. However, catalpol, nicotine, thymol, and anabasine did not have significant effects on Crithidia levels. These results suggest that Crithidia exposure in some floral nectars may reduce cell viability, resulting in a lower risk to visiting pollinators, but this effect may not be widespread across all flowering species.

Soil microbial community composition in tallgrass prairie restorations converge with remnants across a 27-year chronosequence.

Restoration and management of natural ecosystems is a critical strategy in mitigating global biodiversity loss. This is exemplified in the American Midwest by efforts aimed at reclaiming historical grasslands lost to high-yield agriculture. While restorations traditionally take the form of plant reintroduction and management, advances in microbial analyses suggest that soil communities could be indicators restoration success. However, current understanding of key microbial taxa and functional activities in both natural and restored ecosystems is limited. Here, we investigated the impact of nearly 30 years of carefully managed restoration on soil microbial communities at the Nachusa Grasslands in northern Illinois, USA. We characterized bacterial and archaeal communities in a chronosequence of restored tallgrass prairies ranging from 1 to 27 years old across a growing season and compared them to communities in pre-restoration agricultural fields and remnant prairies. Results indicate that older restorations harboured communities statistically distinct from newer restorations. These communities converged toward those in local prairie remnants, suggesting that plant-focussed restoration has yielded soil bacterial communities reflective of a successful restoration. Recovery of microbial clades within the Verrucomicrobia and Acidobacteria are an important feature of this convergence, and these groups could be targeted for future soil-focussed, bottom-up restoration studies.

Simulating high frequency water quality monitoring data using a catchment runoff attenuation flux tool (CRAFT).

High resolution water quality data has recently become widely available from numerous catchment based monitoring schemes. However, the models that can reproduce time series of concentrations or fluxes have not kept pace with the advances in monitoring data. Model performance at predicting phosphorus (P) and sediment concentrations has frequently been poor with models not fit for purpose except for predicting annual losses. Here, the data from the Eden Demonstration Test Catchments (DTC) project have been used to calibrate the Catchment Runoff Attenuation Flux Tool (CRAFT), a new, parsimonious model developed with the aim of modelling both the generation and attenuation of nutrients and sediments in small to medium sized catchments. The CRAFT has the ability to run on an hourly timestep and can calculate the mass of sediments and nutrients transported by three flow pathways representing rapid surface runoff, fast subsurface drainage and slow groundwater flow (baseflow). The attenuation feature of the model is introduced here; this enables surface runoff and contaminants transported via this pathway to be delayed in reaching the catchment outlet. It was used to investigate some hypotheses of nutrient and sediment transport in the Newby Beck Catchment (NBC) Model performance was assessed using a suite of metrics including visual best fit and the Nash-Sutcliffe efficiency. It was found that this approach for water quality models may be the best assessment method as opposed to using a single metric. Furthermore, it was found that, when the aim of the simulations was to reproduce the time series of total P (TP) or total reactive P (TRP) to get the best visual fit, that attenuation was required. The model will be used in the future to explore the impacts on water quality of different mitigation options in the catchment; these will include attenuation of surface runoff.

The Medicines Advice Service Evaluation (MASE): a randomised controlled trial of a pharmacist-led telephone based intervention designed to improve medication adherence.

AIM: To test the effectiveness of a tailored, pharmacist-led centralised advice service to improve adherence to patients on established medications. METHODS: A parallel group randomised controlled trial was conducted. Patients prescribed at least one oral medication for type 2 diabetes and/or lipid regulation were eligible to participate. 677 patients of a mail-order pharmacy were recruited and randomised (340 intervention, 337 control). The intervention comprised two tailored telephone consultations with a pharmacist, 4-6 weeks apart, plus a written summary of the discussion and a medicines reminder chart. The primary outcome was self-reported adherence to medication at 6-month follow-up, collected via a postal questionnaire, analysed using generalised estimating equations. Secondary outcomes included prescription refill adherence, lipid and glycaemic control and patient satisfaction. RESULTS: In intention-to-treat analysis 36/340 (10.6%) of the intervention group were non-adherent (<90% of medication taken in the past 7 days) at 6 months compared with 66/337 (19.6%) in the control group, yielding an unadjusted OR of 1.54 (95% CI 1.11 to 2.15, p=0.01). Analyses of dispensing data also showed that the odds of being classified as adherent (≥90%) were 60% greater for the intervention group compared with the control group (OR 1.60, 95% CI 1.14 to 2.24, p<0.01). In a subsample of patients who provided blood samples, glycaemic and lipid control did not differ significantly between groups (p=0.06 and p=0.24, respectively) but positive trends were observed. Ninety-two per cent of intervention group patients reported that they were satisfied with the service overall. CONCLUSIONS: A telephone intervention, led by a pharmacist and tailored to the individuals' needs, can significantly improve medication adherence in patients with long-term conditions, using a mail-order pharmacy. Further work is needed to confirm a trend towards improved clinical outcome. TRIAL REGISTRATION NUMBER: NCT01864239.