Degradation and adsorption of synthetic DNA water tracers in environmental matrices.

Synthetic DNA tracers are gaining interest as tools for tracking contamination pathways and hydraulic connections in surface water and groundwater systems. However, few quantitative data exist that describe DNA tracer degradation and adsorption in environmental matrices. We undertook laboratory experiments to quantify the degradation of multiple double-stranded DNA tracers in stream water, groundwater, and domestic and dairy-shed effluent, and adsorption to stream sediments, soils, coastal sand aquifer media and alluvial sandy gravel aquifer media. Faster DNA tracer degradation seemed to be associated with high bacterial concentrations in the liquid phase. Overall, the degradation of the 352 base pair (bp) DNA tracers in the aqueous phase was significantly (P = 0.018) slower than that of the 302 bp DNA tracers. Although the tracers' internal amplicon lengths were similar, the longer non-amplified flanking regions of the 352 bp tracers may better protect them from environmental degradation. Thermodynamic analysis suggests that longer flanking regions contribute to greater tracer stability. This finding may explain our previous field observations that 352 bp tracer mass reductions were often lower than 302 bp tracer mass reductions. The 2 sets of DNA tracers did not differ significantly regarding their adsorption to stream sediment-stream water or aquifer media-groundwater mixtures (P > 0.067), but the 352 bp tracers showed significantly less adsorption to soil-effluent mixtures than the 302 bp tracers (P = 0.005). The DNA tracers' adsorption to soil-effluent mixtures was comparatively less than their adsorption to the aquifer media-groundwater and stream sediment-stream water mixtures, suggesting that DNA tracers may compete with like-charged organic matter for adsorption sites. These findings provide insights into the fate of DNA tracers in the environment. The DNA tracers' degradation rate constants determined in this study for a range of environmental conditions could assist the design of future field investigations.

Impact of a computerized decision support tool deployed in two intensive care units on acute kidney injury progression and guideline compliance: a prospective observational study.

BACKGROUND: Acute kidney injury (AKI) affects a large proportion of the critically ill and is associated with worse patient outcomes. Early identification of AKI can lead to earlier initiation of supportive therapy and better management. In this study, we evaluate the impact of computerized AKI decision support tool integrated with the critical care clinical information system (CCIS) on patient outcomes. Specifically, we hypothesize that integration of AKI guidelines into CCIS will decrease the proportion of patients with Stage 1 AKI deteriorating into higher stages of AKI. METHODS: The study was conducted in two intensive care units (ICUs) at University Hospitals Bristol, UK, in a before (control) and after (intervention) format. The intervention consisted of the AKIN guidelines and AKI care bundle which included guidance for medication usage, AKI advisory and dashboard with AKI score. Clinical data and patient outcomes were collected from all patients admitted to the units. AKI stage was calculated using the Acute Kidney Injury Network (AKIN) guidelines. Maximum AKI stage per admission, change in AKI stage and other metrics were calculated for the cohort. Adherence to eGFR-based enoxaparin dosing guidelines was evaluated as a proxy for clinician awareness of AKI. RESULTS: Each phase of the study lasted a year, and a total of 5044 admissions were included for analysis with equal numbers of patients for the control and intervention stages. The proportion of patients worsening from Stage 1 AKI decreased from 42% (control) to 33.5% (intervention), p = 0.002. The proportion of incorrect enoxaparin doses decreased from 1.72% (control) to 0.6% (intervention), p < 0.001. The prevalence of any AKI decreased from 43.1% (control) to 37.5% (intervention), p < 0.05. CONCLUSIONS: This observational study demonstrated a significant reduction in AKI progression from Stage 1 and a reduction in overall development of AKI. In addition, a reduction in incorrect enoxaparin dosing was also observed, indicating increased clinical awareness. This study demonstrates that AKI guidelines coupled with a newly designed AKI care bundle integrated into CCIS can impact patient outcomes positively.

A 2-step synthesis of Combretastatin A-4 and derivatives as potent tubulin assembly inhibitors.

A series of combretastatin derivatives were designed and synthesised by a two-step stereoselective synthesis by use of Wittig olefination followed by Suzuki cross-coupling. Interestingly, all new compounds (2a-2i) showed potent cell-based antiproliferative activities in nanomolar concentrations. Among the compounds, 2a, 2b and 2e were the most active across three cancer cell lines. In addition, these compounds inhibited the polymerisation of tubulin in vitro more efficiently than CA-4. They caused cell cycle arrest in G2/M phase further confirming their ability to inhibit tubulin polymerisation.

Assessing changes in nitrogen contamination in groundwater using water aging: Waikato River, New Zealand.

Groundwater and surface water contamination by nitrogen can produce serious health issues particularly regarding the contraction of methemoglobinemia in infants; prevention of this disorder is a main reason for regulating drinking water quality in New Zealand. To assist the management of water quality in the Upper Waikato Catchment in the light of growing intensification of nitrogen producing land uses, a regional groundwater flow and contamination transport model was developed to assess probable future nitrogen concentrations. Calibration of the steady state model was achieved with a root mean square error of 4.7% for groundwater levels and 7.9% for gains and losses in river flow within the catchment. Model performance was assisted by relationships determined between SiO2 concentrations in groundwater and water age. Modelling coupled with water aging indicates that the mean residence time of contributing water in the Upper Waikato Catchment ranges from 5 to 101 years with an average of 50 years. Some 77% of surface water is sourced from groundwater which is likely to convey the predominant contribution to the total nitrogen load. Nitrogen concentrations are increasing but the long-term total load is difficult to estimate owing largely to lack of information about historical land use and the degree of attenuation of nitrogen. The model and results obtained may be employed to provide information on water travel time distributions, appropriate source location and surface water receiving reaches in rivers and streams all of which is important and necessary for effective water quality management in the Catchment.

Water tracking in surface water, groundwater and soils using free and alginate-chitosan encapsulated synthetic DNA tracers.

Investigating contamination pathways and hydraulic connections in complex hydrological systems will benefit greatly from multi-tracer approaches. The use of non-toxic synthetic DNA tracers is promising, because unlimited numbers of tracers, each with a unique DNA identifier, could be used concurrently and detected at extremely low concentrations. This study aimed to develop multiple synthetic DNA tracers as free molecules and encapsulated within microparticles of biocompatible and biodegradable alginate and chitosan, and to validate their field utility in different systems. Experiments encompassing a wide range of conditions and flow rates (19 cm/day-39 km/day) were conducted in a stream, an alluvial gravel aquifer, a fine coastal sand aquifer, and in lysimeters containing undisturbed silt loam over gravels. The DNA tracers were identifiable in all field conditions investigated, and they were directly detectable in the stream at a distance of at least 1 km. The DNA tracers showed promise at tracking fast-flowing water in the stream, gravel aquifer and permeable soils, but were unsatisfactory at tracking slow-moving groundwater in the fine sand aquifer. In the surface water experiments, the microencapsulated DNA tracers' concentrations and mass recoveries were 1-3 orders of magnitude greater than those of the free DNA tracers, because encapsulation protected them from environmental stressors and they were more negatively charged. The opposite was observed in the gravel aquifer, probably due to microparticle filtration by the aquifer media. Although these new DNA tracers showed promise in proof-of-concept field validations, further work is needed before they can be used for large-scale investigations.

Achieving unbiased predictions of national-scale groundwater redox conditions via data oversampling and statistical learning.

An important policy consideration for integrated land and water management is to understand the spatial distribution of nitrate attenuation in the groundwater system, for which redox condition is the key indicator. This paper proposes a methodology to accommodate the computational demands of large datasets, and presents national-scale predictions of groundwater redox class for New Zealand. Our approach applies statistical learning methods to relate the redox class determined on groundwater samples to spatially varying attributes. The trained model uses these spatial variables to predict redox status in areas without sample data. We assembled the groundwater sample data from regional authority databases, and assigned each sample a redox class. A key achievement was to overcome the influence of sample selection bias on model training via oversampling. We removed additional bias imposed by imbalances in the predictor variables by applying a conditional inference random forest classifier. The unbiased trained model uses eight predictors, and achieves a high validation performance (accuracy 0.81, kappa 0.71), providing good confidence in model predictions. National maps are provided for redox class and probability at specified depths. Feature importance rankings indicate that reducing conditions are associated with poorly-drained soils, and to a lesser extent, high hydrological variability, low elevation, and low-permeability lithology. These conditions are common in New Zealand's coastal and lowland plains, where artificial drainage is required to make land suitable for production. The spatial extent of reduced groundwater increases with depth, suggesting a shallow influence of soil infiltration or mobile organic carbon, and a deeper influence of lithological electron donors. Our model provides unbiased predictions at a scale relevant for environmental policy development and legislation. Identifying where the ecosystem service provided by denitrification can be utilised will enable spatially targeted interventions that can achieve the desired environmental outcome in a more cost-effective manner than non-targeted interventions.

The effects of denitrification parameterization and potential benefits of spatially targeted regulation for the reduction of N-discharges from agriculture.

Diffuse nitrate leaching from agricultural areas is a major environmental problem in many parts of the world. Understanding where in a catchment nitrate is removed is key for designing effective land use management strategies that protect water quality, while minimizing the impact on economic development. In this study we assess the effects of spatially targeted nitrate leaching regulation in a basin with limited knowledge of the complexity of chemical heterogeneity. Three alternative nitrate reactivity spatial parameterizations were incorporated in a catchment-scale flow and transport model and used to evaluate the effectiveness of four possible spatially targeted regulation options. Our findings confirm that denitrification parameterization cannot be numerically determined based on model inversion alone. Detailed field based characterization using physical and geochemical methods should be considered and incorporated in the numerical inversion scheme. We also demonstrate that there are potential benefits of implementing spatially targeted regulation compared to spatially uniform regulation. Focusing regulation in areas where nitrate residence time is short, such as riparian zones or areas with low natural N-reduction, results in greater reduction of N-discharges through groundwater. Significantly improved efficiencies can be expected when delineation of management zones considers the chemical heterogeneity and groundwater flow paths. These improved efficiencies are achieved by adopting management rules that regulate land use in discharge sensitive areas, where leaching changes contribute the most to the catchment nitrate discharges. In our case study, regulation in discharge sensitive zones was twice as efficient compared to other management options.

A baseline assessment of emerging organic contaminants in New Zealand groundwater.

Emerging organic contaminants (EOCs) are manufactured compounds, used for a range of purposes, that are a rising concern for freshwater quality, human and aquatic health. Their occurrence in groundwater has been demonstrated in several international surveys. We conducted the first baseline survey on EOC occurrence in New Zealand groundwater, using a wide-screening approach (723 compounds) and a novel stratified to mean residence time (MRT) randomised design to inform future monitoring. A total of 61 sites were sampled: 51 baseline sites from the State of the Environment (SOE) network in the Waikato region and 10 targeted sites located in the vicinity of known EOC sources for comparison. EOCs were detected at 91% of the baseline sites at concentrations ranging from 0.1 to 11,000 ng·L-1. Multiple groups of EOCs were encountered: pesticides (48 compounds), pharmaceuticals (11), industrial (10), preservatives/food additives (3) and personal care products (1). Similar diversity and concentration range of EOCs were observed at the targeted sites, with the addition of drugs of abuse and life-style compounds. EOC detections occurred across young (1-11 yrs. MRT), intermediate (11-50 yrs. MRT) and old (50-250 yrs. MRT) groundwaters with higher concentrations and more types of EOCs detected at sites in the youngest age category. Concentrations of the 73 compounds detected at baseline sites were comparable to those found in overseas groundwaters with 28 compounds measured at concentrations greater than the EU maximum admissible concentration for pesticides. We used the survey results to: review current pesticide monitoring; propose complementary monitoring; identify potential EOC groundwater tracers and identify compounds for which cost-effective national laboratory capability is needed. The Waikato survey results demonstrated ubiquitous occurrence of unmonitored, unregulated EOCs in groundwater and limitations in using targeted approaches to establish monitoring.

Tubulin-binding dibenz[c,e]oxepines: Part 2. Structural variation and biological evaluation as tumour vasculature disrupting agents.

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αß-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Rates of organ donation in a UK tertiary cardiac arrest centre following out-of-hospital cardiac arrest.

AIM: To ascertain the rate of successful organ donation (OD) within patients who sustained an out of hospital cardiac arrest (OHCA) with initial return of spontaneous circulation (ROSC) and survival to hospital admission, but whom subsequently do not survive to hospital discharge. METHODS: A retrospective audit of ambulance service and hospital databases from January 2010 to January 2015 was undertaken in a United Kingdom tertiary-referral regional cardiac arrest centre. Crude denominator data for cardiac arrests was obtained from the regional ambulance service; the ICU database was interrogated for OHCA patient admissions and outcomes. Patients who died were cross-referenced against the local Organ Donation service database. RESULTS: Five hundred and fourteen {514} patients were admitted to ICU following OHCA over this five year period. Two hundred and forty-one {241} patients (47%) survived to hospital discharge and 273 (53%) died of whom 106 (39%) were referred to a Specialist Nurse for Organ Donation (SNOD). The conversion rate after the family was approached was 64%. Twenty-eight {28} patients proceeded to donation and 25 patients (24%) successfully donated at least one organ. On average, a patient proceeding to donation provided 1.9 organs. CONCLUSIONS: A proactive, systematic approach to OD in OHCA patients can provide a good conversion rate and substantial number of donors. Most donations occur after death from circulatory criteria. There is a positive socio-economic benefit with nearly £4m in savings to the health service within the next 5 years potentially being realised during this period by liberating patients from dialysis.

Three-dimensional imaging and uptake of the anticancer drug combretastatin in cell spheroids and photoisomerization in gels with multiphoton excitation.

The uptake of E -combretastatins, potential prodrugs of the anticancer Z -isomers, into multicellular spheroids has been imaged by intrinsic fluorescence in three dimensions using two-photon excited fluorescence lifetime imaging with 625-nm ultrafast femtosecond laser pulses. Uptake is initially observed at the spheroid periphery but extends to the spheroid core within 30 min. Using agarose gels as a three-dimensional model, the conversion of Z(trans)→E(cis) via two-photon photoisomerization is demonstrated and the location of this photochemical process may be precisely selected within the micron scale in all three dimensions at depths up to almost 2 mm. We discuss these results for enhanced tissue penetration at longer near-infrared wavelengths for cancer therapy and up to three-photon excitation and imaging using 930-nm laser pulses with suitable combretastatin analogs.

Anticancer phototherapy using activation of E-combretastatins by two-photon-induced isomerization.

The photoisomerization of relatively nontoxic E-combretastatins to clinically active Z-isomers is shown to occur in solution through both one- and two-photon excitations at 340 and 625 nm, respectively. The photoisomerization is also demonstrated to induce mammalian cell death by a two-photon absorption process at 625 nm. Unlike conventional photodynamic therapy (PDT), the mechanism of photoisomerization is oxygen-independent and active in hypoxic environments such as in tumors. The use of red or near-infrared (NIR) light for two-photon excitation allows greater tissue penetration than conventional UV one-photon excitation. The results provide a baseline for the development of a novel phototherapy that overcomes nondiscriminative systemic toxicity of Z-combretastatins and the limitations of PDT drugs that require the presence of oxygen to promote their activity, with the added benefits of two-photon red or NIR excitation for deeper tissue penetration.

Novel cyanocombretastatins as potent tubulin polymerisation inhibitors.

A series of novel cyanocombretastatins bearing a 3,4,5-trimethoxyphenyl moiety combined with a variety of substituted phenyl rings, were synthesised and their antitumour activity was evaluated. The Z-cyanocombretastatins were synthesised in a one-step protocol in high purity and yield. Fluoro, bromo, iodo, and derivatives with boronic acid and an ethyne function at meta position of the B ring were synthesised. In vitro MTT bioassays against human chronic myelogenous leukaemia (K562) and transfected breast adenocarcinoma (MDA NQO1) cell lines, revealed promising IC(50) inhibitory values in nanomolar range (<50 nM). Introduction of a nitrile function on the olefinic bond not only increased the cytotoxicity of the less active Z-isomers but rendered the analogues as moderate to potent inhibitors of tubulin polymerisation comparable to that of CA-4 (IC(50)=2.2 µM).

Fluorescence lifetime imaging of E-combretastatin uptake and distribution in live mammalian cells.

To investigate within live mammalian cells the uptake and disposition of combretastatins, fluorescence lifetime imaging was used with two-photon excitation (2PE). Combretastatin A4 (CA4) and analogues are potential anticancer drugs due to their ability to inhibit angiogenesis. E(trans)-combretastatins are considerably less active than the Z(cis)-combretastatins proposed for clinical use. However the E-combretastatins exhibit stronger intrinsic fluorescence with quantum yields and lifetimes that depend markedly on solvent polarity and viscosity. It is proposed that 2PE in the red and near-infrared tissue window may allow in situ isomerization of E-combretastatins to the more active Z-isomer, offering spatial and temporal control of drug activation and constitute a novel form of photodynamic therapy. In the present work we have characterised 2PE of E-CA4 and have used fluorescence lifetime imaging with 2PE to study uptake and intracellular disposition of E-CA4 and an analogue. The results show that these molecules accumulate rapidly in cells and are located mainly in lipidic environments such as lipid droplets. Within the droplets the local concentrations may be up to two orders of magnitude higher than that of the drug in the surrounding medium.

Tubulin-binding dibenz[c,e]oxepines as colchinol analogues for targeting tumour vasculature.

Various methoxy- and hydroxy-substituted dibenz[c,e]oxepines were prepared via the copper(I)-induced coupling of ether-tethered arylstannanes or the dehydrative cyclisation of 1,1'-biphenyl-2,2'-dimethanols, assembled using the Ullmann cross-coupling of ortho-bromoaryl carbonyl compounds. The dibenzoxepines were screened for their ability to inhibit tubulin polymerisation and the in vitro growth of K562 human chronic myelogenous leukemia cells. The most active was 5,7-dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol, whose tubulin inhibitory and cytotoxicity (IC(50)) values were 1 µM and 40 nM, respectively.

Synthesis and biological evaluation of 2- and 3-aminobenzo[b]thiophene derivatives as antimitotic agents and inhibitors of tubulin polymerization.

Two new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene molecular skeleton and its 3-amino positional isomer were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. Although many more 3-amino derivatives have been synthesized so far, the most promising compound in this series was 2-amino-6-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene, which inhibits cancer cell growth at subnanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.

Synthesis and evaluation of double bond substituted combretastatins.

A series of combretastatins substituted with epoxides, amides and small alkyl groups has been synthesised and evaluated for cytotoxicity and their ability to inhibit the assembly of tubulin. The methyl and ethyl substituted phenols 36, 44 have shown potent antimitotic effects whilst exhibiting reduced cytotoxicity.

Functional comparison of annexin V analogues labeled indirectly and directly with iodine-124.

We are interested in imaging cell death in vivo using annexin V radiolabeled with (124)I. In this study, [(124)I]4IB-annexin V and [(124)I]4IB-ovalbumin were made using [(124)I]N-hydroxysuccinimidyl-4-iodobenzoate prepared by iododestannylation of N-hydroxysuccinimidyl-4-(tributylstannyl)benzoate. [(124)I]4IB-annexin V binds to phosphatidylserine-coated microtiter plates and apoptotic Jurkat cells and accumulates in hepatic apoptotic lesions in mice treated with anti-Fas antibody, while [(124)I]4IB-ovalbumin does not. In comparison with (124)I-annexin V, [(124)I]4IB-annexin V has a higher rate of binding to phosphatidylserine in vitro, a higher kidney and urine uptake, a lower thyroid and stomach content uptake, greater plasma stability and a lower rate of plasma clearance. Binding of radioactivity to apoptotic cells relative to normal cells in vitro and in vivo appears to be lower for [(124)I]4IB-annexin V than for (124)I-annexin V.

Hydraulic constraints on the performance of a groundwater denitrification wall for nitrate removal from shallow groundwater.

Denitrification walls are a practical approach for decreasing non-point source pollution of surface waters. They are constructed by digging a trench perpendicular to groundwater flow and mixing the aquifer material with organic matter, such as sawdust, which acts as a carbon source to stimulate denitrification. For efficient functioning, walls need to be permeable to groundwater flow. We examined the functioning of a denitrification wall constructed in an aquifer consisting of coarse sands. Wells were monitored for changes in nitrate concentration as groundwater passed through the wall and soil samples were taken to measure microbial parameters inside the wall. Nitrate concentrations upstream of the wall ranged from 21 to 39 g N m(-3), in the wall from 0 to 2 g N m(-3) and downstream from 19 to 44 g N m(-3). An initial groundwater flow investigation using a salt tracer dilution technique showed that the flow through the wall was less than 4% of the flow occurring in the aquifer. Natural gradient tracer tests using bromide and Rhodamine-WT confirmed groundwater bypass under the wall. Hydraulic conductivity of 0.48 m day(-1) was measured inside the wall, whereas the surrounding aquifer had a hydraulic conductivity of 65.4 m day(-1). This indicated that during construction of the wall, hydraulic conductivity of the aquifer had been greatly reduced, so that most of the groundwater flowed under rather than through the wall. Denitrification rates measured in the center of the wall ranged from 0.020 to 0.13 g N m(-3) day(-1), which did not account for the rates of nitrate removal (0.16-0.29 g N m(-3) day(-1)) calculated from monitoring of groundwater nitrate concentrations. This suggested that the rate of denitrification was greater at the upstream face of the wall than in its center where it was limited by low nitrate concentrations. While denitrification walls can be an inexpensive tool for removing nitrate from groundwater, they may not be suitable in aquifers with coarse textured subsoils where simple inexpensive construction techniques result in major decreases in hydraulic conductivity.