Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial sub populations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 μM after 30 min of incubation and the orange nucleic acid probe at 1 μM after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 μM concentrations and higher in both probes exhibited a type of non-specific staining, whereby 'live' cells produced a target fluorescence, leading to an over representation of 'non-viable' cell numbers. The positive controls (heat killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively

Cyanobacterial Abundance and Microcystin Profiles in Two Southern British Lakes: The Importance of Abiotic and Biotic Interactions.

Freshwater cyanobacteria blooms represent a risk to ecological and human health through induction of anoxia and release of potent toxins; both conditions require water management to mitigate risks. Many cyanobacteria taxa may produce microcystins, a group of toxic cyclic heptapeptides. Understanding the relationships between the abiotic drivers of microcystins and their occurrence would assist in the implementation of targeted, cost-effective solutions to maintain safe drinking and recreational waters. Cyanobacteria and microcystins were measured by flow cytometry and liquid chromatography coupled to tandem mass spectrometry in two interconnected reservoirs varying in age and management regimes, in southern Britain over a 12-month period. Microcystins were detected in both reservoirs, with significantly higher concentrations in the southern lake (maximum concentration >7 µg L-1). Elevated microcystin concentrations were not positively correlated with numbers of cyanobacterial cells, but multiple linear regression analysis suggested temperature and dissolved oxygen explained a significant amount of the variability in microcystin across both reservoirs. The presence of a managed fishery in one lake was associated with decreased microcystin levels, suggestive of top down control on cyanobacterial populations. This study supports the need to develop inclusive, multifactor holistic water management strategies to control cyanobacterial risks in freshwater bodies

Exploiting eco-physiological niche to facilitate the separation of the freshwater cyanobacteria Microcystis sp. and Synechococcus sp.

In a novel approach to separate the co-occurring freshwater cyanobacteria Microcystis and Synechoccous, published ecological characteristics are used to manipulate temperature and nutrient concentrations to successfully establish a unialgal Microcystis strain. The simple protocol has implications for future cyanobacterial culturing approaches and the establishment of new cyanobacteria strains

A pragmatic randomized waitlist-controlled effectiveness and cost-effectiveness trial of digital interventions for depression and anxiety

Utilization of internet-delivered cognitive behavioural therapy (iCBT) for treating depression and anxiety disorders in stepped-care models, such as the UK’s Improving Access to Psychological Therapies (IAPT), is a potential solution for addressing the treatment gap in mental health. We investigated the effectiveness and cost-effectiveness of iCBT when fully integrated within IAPT stepped-care settings. We conducted an 8-week pragmatic randomized controlled trial with a 2:1 (iCBT intervention: waiting-list) allocation, for participants referred to an IAPT Step 2 service with depression and anxiety symptoms (Trial registration: ISRCTN91967124). The primary outcomes measures were PHQ-9 (depressive symptoms) and GAD-7 (anxiety symptoms) and WSAS (functional impairment) as a secondary outcome. The cost-effectiveness analysis was based on EQ-5D-5L (preference-based health status) to elicit the quality-adjust life year (QALY) and a modified-Client Service Receipt Inventory (care resource-use). Diagnostic interviews were administered at baseline and 3 months. Three-hundred and sixty-one participants were randomized (iCBT, 241; waiting-list, 120). Intention-to-treat analyses showed significant interaction effects for the PHQ-9 (b = −2.75, 95% CI −4.00, −1.50) and GAD-7 (b = −2.79, 95% CI −4.00, −1.58) in favour of iCBT at 8-week and further improvements observed up to 12-months. Over 8-weeks the probability of cost-effectiveness was 46.6% if decision makers are willing to pay £30,000 per QALY, increasing to 91.2% when the control-arm’s outcomes and costs were extrapolated over 12-months. Results indicate that iCBT for depression and anxiety is effective and potentially cost-effective in the long-term within IAPT. Upscaling the use of iCBT as part of stepped care could help to enhance IAPT outcomes. The pragmatic trial design supports the ecological validity of the findings

Predator-prey interactions between the ciliate Blepharisma americanum and toxic (Microcystis spp.) and non-toxic (Chlorella vulgaris, Microcystis sp.) photosynthetic microbes.

Despite free-living protozoa being a major factor in modifying aquatic autotrophic biomass ciliate cyanobacteria interactions and their functional ecological roles have been poorly described, especially with toxic cyanobacteria. Trophic relationships have been neglected and grazing experiments give contradictory evidence when toxic taxa such as Microcystis are involved. Here two toxic Microcystis strains (containing microcystins), one non-toxic Microcystis strain and a non-toxic green alga, Chlorella vulgaris, were used to investigate predator-prey interactions with a phagotrophic ciliate, Blepharisma americanum. Flow cytometric analysis for microalgal measurements and a rapid UHPLC-MS/MS protocol to quantify microcystins showed non-toxic photosynthetic microbes were significantly grazed by B. americanum, which sustained ciliate populations. In contrast, despite constant ingestion of toxic Microcystis rapid egestion of cells occurred. The lack of digestion resulted in no significant control of toxic cyanobacteria densities, a complete reduction in ciliate numbers, and no observable encystment or cannibalistic behaviour (gigantism). Individual B. americanum morphological responses (biovolume and cell width) showed a significant decrease over time when sustained on non-toxic Microcystis compared 55 to grazed C. vulgaris populations, supporting previous studies that cyanobacteria may be a relatively poor source of nutrition. Results here provide an insight into the ecological interactions of ciliates and cyanobacteria, and for the first time B. americanum is shown to have the capacity to suppress potentially bloom-forming cyanobacteria. However, grazing can be significantly altered by the presence of microcystins, which could have an impact on bloom dynamics and overall community structure

Effects of H2O2 on growth, metabolic activity and membrane integrity in three strains of Microcystis aeruginosa.

The application of hydrogen peroxide (H2O2) as a management tool to control Microcystis blooms has become increasingly popular due to its short lifetime and targeted action. H2O2 increases intracellular reactive oxygen species resulting in oxidative stress and subsequently cell death. H2O2 is naturally produced in freshwater bodies as a result of photocatalytic reactions between dissolved organic carbon and sunlight. Previously, some studies have suggested that this environmental source of H2O2 selectively targets for toxigenic cyanobacteria strains in the genus Microcystis. Also, past studies only focused on the morphological and biochemical changes of H2O2-induced cell death in Microcystis with little information available on the effects of different H2O2 concentrations on growth, esterase activity and membrane integrity. Therefore, this study investigated the effects of non-lethal (40-4000 nM) concentrations on percentage cell death; with a focus on sub-lethal (50 μM) and lethal (275 μM; 500 μM) doses of H2O2 on growth, cells showing esterase activity and membrane integrity. The non-lethal dose experiment was part of a preliminary study. Results showed a dose- and time-dependent relationship in all three Microcystis strains post H2O2 treatment. H2O2 resulted in a significant increase in intracellular reactive oxygen species, decreased chlorophyll a content, decreased growth rate and esterase activity. Interestingly, at sub-lethal (50 μM H2O2 treatment), percentage of dead cells in microcystin-producing strains was significantly higher (p < 0.05) than that in non-microcystin-producing strains at 72 h. These findings further cement our understanding of the influence of H2O2 on different strains of Microcystis and its impact on membrane integrity and metabolic physiology: important to future toxic bloom control programmes

A multiscale analysis of gene flow for the New England cottontail, an imperiled habitat specialist in a fragmented landscape

Landscape features of anthropogenic or natural origin can influence organisms\u27 dispersal patterns and the connectivity of populations. Understanding these relationships is of broad interest in ecology and evolutionary biology and provides key insights for habitat conservation planning at the landscape scale. This knowledge is germane to restoration efforts for the New England cottontail (Sylvilagus transitionalis), an early successional habitat specialist of conservation concern. We evaluated local population structure and measures of genetic diversity of a geographically isolated population of cottontails in the northeastern United States. We also conducted a multiscale landscape genetic analysis, in which we assessed genetic discontinuities relative to the landscape and developed several resistance models to test hypotheses about landscape features that promote or inhibit cottontail dispersal within and across the local populations. Bayesian clustering identified four genetically distinct populations, with very little migration among them, and additional substructure within one of those populations. These populations had private alleles, low genetic diversity, critically low effective population sizes (3.2-36.7), and evidence of recent genetic bottlenecks. Major highways and a river were found to limit cottontail dispersal and to separate populations. The habitat along roadsides, railroad beds, and utility corridors, on the other hand, was found to facilitate cottontail movement among patches. The relative importance of dispersal barriers and facilitators on gene flow varied among populations in relation to landscape composition, demonstrating the complexity and context dependency of factors influencing gene flow and highlighting the importance of replication and scale in landscape genetic studies. Our findings provide information for the design of restoration landscapes for the New England cottontail and also highlight the dual influence of roads, as both barriers and facilitators of dispersal for an early successional habitat specialist in a fragmented landscape

Spatiotemporal characteristics of atrial fibrillation electrograms: a novel marker for arrhythmia stability and termination

Background: Sequentially mapped complex fractionated atrial electrograms (CFAE) and dominant frequency (DF) sites have been targeted during catheter ablation for atrial fibrillation (AF). However, these strategies have yielded variable success and have not been shown to correlate consistently with AF dynamics. Here, we evaluated whether the spatiotemporal stability of CFAE and DF may be a better marker of AF sustenance and termination. Methods: Eighteen sheep with 12 weeks of "one-kidney, one-clip" hypertension underwent open-chest studies. A total of 42 self-terminating (28–100 s) and 6 sustained (>15 min) AF episodes were mapped using a custom epicardial plaque and analyzed in 4-s epochs for CFAE, using the NavX CFE-m algorithm, and DF, using a Fast Fourier Transform. The spatiotemporal stability index (STSI) was calculated using the intraclass correlation coefficient of consecutive AF epochs. Results: A total of 67,733 AF epochs were analyzed. During AF initiation, mean CFE-m and the STSI of CFE-m/DF were similar between sustained and self-terminating episodes, although median DF was higher in sustained AF (p=0.001). During sustained AF, the STSI of CFE-m increased significantly (p=0.02), whereas mean CFE-m (p=0.5), median DF (p=0.07), and the STSI of DF remained unchanged (p=0.5). Prior to AF termination, the STSI of CFE-m was significantly lower (p<0.001), with a physiologically non-significant decrease in median DF (−0.3 Hz, p=0.006) and no significant changes in mean CFE-m (p=0.14) or the STSI of DF (p=0.06). Conclusions: Spatiotemporal stabilization of CFAE favors AF sustenance and its destabilization heralds AF termination. The STSI of CFE-m is more representative of AF dynamics than are the STSI of DF, sequential mean CFE-m, or median DF

Group-based trajectory models: assessing adherence to antihypertensive medication in older adults in a community pharmacy setting.

Antihypertensive medication nonadherence is highly prevalent, leading to uncontrolled blood pressure. Methods that facilitate the targeting and tailoring of adherence interventions in clinical settings are required. Group-Based Trajectory Modeling (GBTM) is a newer method to evaluate adherence using pharmacy dispensing (refill) data that has advantages over traditional refill adherence metrics (e.g. Proportion of Days Covered) by identifying groups of patients who may benefit from adherence interventions, and identifying patterns of adherence behavior over time that may facilitate tailoring of an adherence intervention. We evaluated adherence to antihypertensive medication in 905 patients over a 12-month period in a community pharmacy setting using GBTM, identifying three subgroups of adherence patterns: 52.8%, 40.7%, and 6.5% had very high, high, and low adherence, respectively. However, GBTM failed to demonstrate predictive validity with blood pressure at 12 months. Further research on the validity of adherence measures that facilitate interventions in clinical settings is required

Proverb interpretation in fluent aphasia and Alzheimer's disease: Implications beyond abstract thinking

This study compared proverb processing across three groups, i.e. patients with fluent aphasia (APH), patients with Alzheimer's Disease (AD), and normal control subjects (NC). Proverb stimuli were used to examine the effects of group membership and proverb familiarity in two presentation formats (i.e. spontaneous versus multiple-choice) on performance. The sensitivity of cognitive measures as predictors of ability to interpret proverbs was also investigated. In relation to NC subjects, patients with fluent APH exhibited significant difficulty formulating responses for familiar and unfamiliar spontaneous proverbs, whereas patients with AD demonstrated lower performance only on the unfamiliar proverbs. On the multiple-choice paradigm, however, patients with APH exhibited minimal difficulty. Conversely, the patients with AD manifested significant problems selecting the correct abstract response for familiar proverbs. With regard to predictors, language was relevant to familiar proverb interpretations and to proverbs presented in the spontaneous format. Cognition was a sensitive predictor for unfamiliar proverb interpretations and to the multiple-choice format. Deficits on the proverb tasks are discussed with reference to the potential breakdown of underlying linguistic and cognitive processes. The present data support the diagnostic value of proverbs in elucidating brain- behaviour relationships