Factors affecting the growth and toxin content of key cyanobacteria species in a changing world.

Cyanobacteria are ubiquitous in marine, freshwater and terrestrial ecosystems. In freshwaters, under certain conditions cyanobacteria can form super abundant blooms. Many cyanobacteria taxa produce secondary metabolites, amongst these are many potent toxins (cyanotoxins), which can present substantial risks to human, animal and environmental health. Where risk mitigation strategies are implemented, they are usually based on cyanobacteria cell counts. However, not all cyanobacteria taxa produce toxins, and the drivers for cyanotoxin production and their eco- physiological role remain unknown. Thus, risk management strategies may over or under estimate risks. A predicted increase in the incidence of toxin producing cyanobacterial blooms has been proposed, linked to climate change driven water temperature increases and eutrophication. Consequently, better understanding of factors modulating cyanobacterial toxicity would assist in more proactive and effective water management. In the first part of this study the effects of biotic and abiotic factors on the growth and toxicity a recently isolated strain of Microcystis sp. (CCAP1450/17) and M. aeruginosa reference strain (PCC7806) were examined. Strains responded similarly under controlled laboratory batch culture systems. Highest cell densities and growth rates were observed at medium light intensity (36 μmol of photons m-2 s-1) compared to high (117 μmol of photons m-2 s-1) and low (15 μmol of photons m-2 s-1) light intensities. Nitrogen was an obligate requirement for microcystin production, but phosphorous was not, indicating that nitrogen eutrophication conditions (caused by agricultural run- off etc) would increase the risk of toxic blooms. Toxin (microcystin) concentrations were positively correlated with cell density, but microcystin synthesis was independent of growth rates under nitrogen replete conditions. Furthermore, smaller cells contained higher levels of toxins. Cellular microcystin content was significantly higher at 20°C compared to 25 & 30°C questioning the paradigm that increased water temperatures (caused by climate change) will favour toxic bloom formation. Although the role of microcystin is not clear, these data indicated that under nitrogen abundant conditions microcystins may perform an eco-physiological function, which is reduced under nitrogen deprivation and/or when cells are rapidly dividing. In the second part of the study time series monitoring of cyanobacteria taxa, toxin concentrations and a range of environmental parameters was undertaken in two connected freshwater reservoirs over a twelve-month period. Cyanobacterial cell counts exceeding UK threshold levels of >20,000 cells mL-1 were recorded on four occasions. Toxins were detected in both reservoirs, concentrations were significantly higher in lake 2 (not stocked with fish) and did not correspond with highest levels of Microcystis cells, indicating that cyanobacterial species other than Microcystis were producing microcystins. Microcystin levels did not exceed the WHO medium health threshold of 20 µg L-1 although low threshold values (1.0 µg L-1) were detected in 16% of samples. Monitoring data indicated complex bottom-up and top-down control mechanisms in the moderation of cyanobacterial taxa abundance and population structure, the latter potentially mediated by the presence of omnivorous fishes. Application of basic general linear modelling to the dataset indicated that approximately 60-65% of the variability could be explained by combined independent abiotic and biotic variables indicating the future applicability of this approach. These results confirmed complex interactions between biotic and abiotic factors in both laboratory and field conditions but were broadly suggestive of additive effects with respect to microcystin production. Although the functional role of microcystins remains unclear it was considered possible that microcystins perform an eco-physiological function, perhaps conferring an advantage over non-toxic strains under nitrogen rich conditions. In environmental monitoring multiple species of cyanobacterial were implicated in toxin production, but cell numbers alone were not directly proportional to toxin levels supporting the proposition that management strategies based on cell counting alone may not be indicative of risk. Trophic relationships that influence cyanobacterial population dynamics and toxin production require further study to generate data to inform predictive models, but this series of studies suggested several avenues for further study which will provide water quality managers with improved tools to enable efficient, active management of water bodies

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

Synchrotron Macro ATR-FTIR Microspectroscopy for High Resolution Chemical Mapping of Single Cells

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called “hybrid” macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 μm and 100 μm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science

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

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation

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

Listening to women: experiences of using closed-loop in type 1 diabetes pregnancy

Introduction: Recent high-profile calls have emphasized that women's experiences should be considered in maternity care provisioning. We explored women's experiences of using closed-loop during type 1 diabetes (T1D) pregnancy to inform decision-making about antenatal rollout and guidance and support given to future users. Methods: We interviewed 23 closed-loop participants in the Automated insulin Delivery Among Pregnant women with T1D (AiDAPT) trial after randomization to closed-loop and ∼20 weeks later. Data were analyzed thematically. Results: Women described how closed-loop lessened the physical and mental demands of diabetes management, enabling them to feel more normal and sleep better. By virtue of spending increased time-in-range, women also worried less about risks to their baby and being judged negatively by health care professionals. Most noted that intensive input and support during early pregnancy had been crucial to adjusting to, and developing confidence in, the technology. Women emphasized that attaining pregnancy glucose targets still required ongoing effort from themselves and the health care team. Women described needing education to help them determine when, and how, to intervene and when to allow the closed-loop to operate without interference. All women reported more enjoyable pregnancy experiences as a result of using closed-loop; some also noted being able to remain longer in paid employment. Conclusions: Study findings endorse closed-loop use in T1D pregnancy by highlighting how the technology can facilitate positive pregnancy experiences. To realize fully the benefits of closed-loop, pregnant women would benefit from initial intensive oversight and support together with closed-loop specific education and training. Clinical Trial Registration number: NCT04938557

Fully automated closed-loop glucose control compared with standard insulin therapy in adults with type 2 diabetes requiring dialysis: an open-label, randomized crossover trial

Funder: CB was supported by a grant from The Novo Nordisk UK Research FoundationFunder: LB was supported by a grant of the Swiss Society for Endocrinology and a grant of the Diabetes and Swiss Kidney Foundation.Funder: Supported by National Institute for Health Research Cambridge Biomedical Research Centre.Abstract: We evaluated the safety and efficacy of fully closed-loop insulin therapy compared with standard insulin therapy in adults with type 2 diabetes requiring dialysis. In an open-label, multinational, two-center, randomized crossover trial, 26 adults with type 2 diabetes requiring dialysis (17 men, 9 women, average age 68 ± 11 years (mean ± s.d.), diabetes duration of 20 ± 10 years) underwent two 20-day periods of unrestricted living, comparing the Cambridge fully closed-loop system using faster insulin aspart (‘closed-loop’) with standard insulin therapy and a masked continuous glucose monitor (‘control’) in random order. The primary endpoint was time in target glucose range (5.6–10.0 mmol l−1). Thirteen participants received closed-loop first and thirteen received control therapy first. The proportion of time in target glucose range (5.6–10.0 mmol l−1; primary endpoint) was 52.8 ± 12.5% with closed-loop versus 37.7 ± 20.5% with control; mean difference, 15.1 percentage points (95% CI 8.0–22.2; P < 0.001). Mean glucose was lower with closed-loop than control (10.1 ± 1.3 versus 11.6 ± 2.8 mmol l−1; P = 0.003). Time in hypoglycemia (<3.9 mmol l−1) was reduced with closed-loop versus control (median (IQR) 0.1 (0.0–0.4%) versus 0.2 (0.0–0.9%); P = 0.040). No severe hypoglycemia events occurred during the control period, whereas one severe hypoglycemic event occurred during the closed-loop period, but not during closed-loop operation. Fully closed-loop improved glucose control and reduced hypoglycemia compared with standard insulin therapy in adult outpatients with type 2 diabetes requiring dialysis. The trial registration number is NCT04025775

Paediatric patient safety and the need for aviation black box thinking to learn from and prevent medication errors

Since the publication of To Err Is Human: Building a Safer Health System in 1999, there has been much research conducted into the epidemiology, nature and causes of medication errors in children, from prescribing and supply to administration. It is reassuring to see growing evidence of improving medication safety in children; however, based on media reports, it can be seen that serious and fatal medication errors still occur. This critical opinion article examines the problem of medication errors in children and provides recommendations for research, training of healthcare professionals and a culture shift towards dealing with medication errors. There are three factors that we need to consider to unravel what is missing and why fatal medication errors still occur. (1) Who is involved and affected by the medication error? (2) What factors hinder staff and organisations from learning from mistakes? Does the fear of litigation and criminal charges deter healthcare professionals from voluntarily reporting medication errors? (3) What are the educational needs required to prevent medication errors? It is important to educate future healthcare professionals about medication errors and human factors to prevent these from happening. Further research is required to apply aviation’s ‘black box’ principles in healthcare to record and learn from near misses and errors to prevent future events. There is an urgent need for the black box investigations to be published and made public for the benefit of other organisations that may have similar potential risks for adverse events. International sharing of investigations and learning is also needed

Assessing the effect of closed-loop insulin delivery from onset of type 1 diabetes in youth on residual beta-cell function compared to standard insulin therapy (CLOuD study): a randomised parallel study protocol.

INTRODUCTION:Management of newly diagnosed type 1 diabetes (T1D) in children and adolescents is challenging for patients, families and healthcare professionals. The objective of this study is to determine whether continued intensive metabolic control using hybrid closed-loop (CL) insulin delivery following diagnosis of T1D can preserve C-peptide secretion, a marker of residual beta-cell function, compared with standard multiple daily injections (MDI) therapy. METHODS AND ANALYSIS:The study adopts an open-label, multicentre, randomised, parallel design, and aims to randomise 96 participants aged 10-16.9 years, recruited within 21 days of diagnosis with T1D. Following a baseline mixed meal tolerance test (MMTT), participants will be randomised to receive 24 months treatment with conventional MDI therapy or with CL insulin delivery. A further 24-month optional extension phase will be offered to all participants to continue with the allocated treatment. The primary outcome is the between group difference in area under the stimulated C-peptide curve (AUC) of the MMTT at 12 months post diagnosis. Analyses will be conducted on an intention-to-treat basis. Key secondary outcomes are between group differences in time spent in target glucose range (3.9-10 mmol/L), glycated haemoglobin (HbA1c) and time spent in hypoglycaemia (<3.9 mmol/L) at 12 months. Secondary efficacy outcomes include between group differences in stimulated C-peptide AUC at 24 months, time spent in target glucose range, glucose variability, hypoglycaemia and hyperglycaemia as recorded by periodically applied masked continuous glucose monitoring devices, total, basal and bolus insulin dose, and change in body weight. Cognitive, emotional and behavioural characteristics of participants and parents will be evaluated, and a cost-utility analysis performed to support adoption of CL as a standard treatment modality following diagnosis of T1D. ETHICS AND DISSEMINATION:Ethics approval has been obtained from Cambridge East Research Ethics Committee. The results will be disseminated by peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER:NCT02871089; Pre-results