Performance evaluation of a dual-flow recharge filter for improving groundwater quality.

A dual-flow multimedia stormwater filter integrated with a groundwater recharge system was developed and tested for hydraulic efficiency and pollutant removal efficiency. The influent stormwater first flows horizontally through the circular layers of planted grass and biofibers. Subsequently, the flow direction changes to a vertical direction so that water moves through layers of pebbles and sand and finally gets recharged to the deep aquifers. The media in the sequence of vegetative medium:biofiber to pebble:sand were filled in nine proportions and tested for the best performing combination. Three grass species, viz., Typha (Typha angustifolia), Vetiver (Chrysopogon zizanioides), and St. Augustine grass (Stenotaphrum secundatum), were tested as the best performing vegetative medium. The adsorption behavior of Coconut (Cocos nucifera) fiber, which was filled in the middle layer, was determined by a series of column and batch studies.The dual-flow filter showed an increasing trend in hydraulic efficiency with an increase in flowrate. The chemical removal efficiency of the recharge dual-flow filter was found to be very high in case of K+ (81.6%) and Na+ (77.55%). The pH normalizing efficiency and electrical conductivity reduction efficiency were also recorded as high. The average removal percentage of Ca2+ was moderate, while that of Mg2+ was very low. The filter proportions of 1:1 to 1:2 (plant:fiber to pebble:sand) showed a superior performance compared to all other proportions. Based on the estimated annual costs and returns, all the financial viability criteria (internal rate of return, net present value, and benefit-cost ratio) were found to be favorable and affordable to farmers in terms of investing in the developed filtration system.

Performance evaluation and modelling studies of gravel--coir fibre--sand multimedia stormwater filter.

A horizontal flow multimedia stormwater filter was developed and tested for hydraulic efficiency and pollutant removal efficiency. Gravel, coconut (Cocos nucifera) fibre and sand were selected as the media and filled in 1:1:1 proportion. A fabric screen made up of woven sisal hemp was used to separate the media. The adsorption behaviour of coir fibre was determined in a series of column and batch studies and the corresponding isotherms were developed. The hydraulic efficiency of the filter showed a diminishing trend as the sediment level in inflow increased. The filter exhibited 100% sediment removal at lower sediment concentrations in inflow water (>6 g L(-1)). The filter could remove NO3(-), SO4(2-) and total solids (TS) effectively. Removal percentages of Mg(2+) and Na(+) were also found to be good. Similar results were obtained from a field evaluation study. Studies were also conducted to determine the pattern of silt and sediment deposition inside the filter body. The effects of residence time and rate of flow on removal percentages of NO3(-) and TS were also investigated out. In addition, a multiple regression equation that mathematically represents the filtration process was developed. Based on estimated annual costs and returns, all financial viability criteria (internal rate of return, net present value and benefit-cost ratio) were found favourable and affordable to farmers for investment in the developed filtration system. The model MUSIC was calibrated and validated for field conditions with respect to the developed stormwater filter.

NPPB structure-specifically activates TRPA1 channels.

TRPA1 channels have been found to play an important role in mammalian pain sensation, especially when the pain is caused by chemicals on site of inflammation. A large number of structurally diverse chemicals are found to activate TRPA1 channels, implicating a potential chemosensor in neuronal nociception. Identification of the channel activation by cysteine modification through covalent chemical reaction provides arguments for the diversity of the agonist structures. However, it is largely unknown how nonreactive compounds activate TRPA1 channels. Here, we report that NPPB, a classic Cl(-) channel blocker, potently activated human TRPA1 channels overexpressed in mammalian HEK-293 cells. This effect was confirmed in Ca(2+) imaging assay, patch clamp whole cell and single channel recordings. The NPPB response was quick, fully reversible and replicable, contrary to the effect of covalent modification by AITC. The mutagenesis studies revealed a refreshed look at several mutations known to be critical for the actions of AITC and menthol. The blocking profile of NPPB on these mutants showed that the NPPB activation was similar to that of FTS and different from AITC and menthol. The results indicated a possible close interaction between S5 and N-terminal domains of the channel. We also tested a group of NPPB analogs on TRPA1 channel activities. The results demonstrated that NPPB activation was tightly associated with chemical structure. None of the single chemical group was sufficient to activate the channel, indicating that NPPB activated TRPA1 through a structure-specific mechanism.

High-throughput screening for Kv1.3 channel blockers using an improved FLIPR-based membrane-potential assay.

Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates.

Rb+ efflux assay for assessment of non-selective cation channel activities.

ABSTRACT Transient receptor potential (TRP) channels have been found to play important roles in cellular physiology and hold promise as therapeutic targets. These channels activate in response to a variety of chemical or physical stimuli and conduct non-selective cation currents (NSCC). Due to their unique activation properties, application of automated electrophysiology to measure the channel activity has been difficult. Using HEK293 cells stably expressing human TRP channels, hTRPC6 and hTRPA1, we developed and validated a high-throughput Rb(+) efflux assay for NSCC channels. The assay was performed in cell-based 96-well format. A significant increase in Rb(+) efflux can be detected upon channel activation by specific agonists, confirming that both TRPC6 and TRPA1 channels are permeable to Rb(+) ions. The agonists induced Rb(+) efflux can be blocked by known channel blockers and selected compounds from our high-throughput screening (HTS) hits. The assay is suitable for HTS with Z' factors of 0.53 and above. We also tested the Ca(2+) effect on channel activities in this assay. Both TRPC6 and TRPA1 channels were found to be inhibited by increasing the concentration of Ca(2+) in the assay buffer. However, Ca(2+) significantly reduced the potency of allyl isothiocyanate (AITC) on TRPA1 but did not affect the potency of carbochol on TRPC6. Using this assay for secondary confirmation screen, we successfully identified and confirmed the positive hits as TRPC6 inhibitors.

Selective Kv1.5 blockers: development of (R)-1-(methylsulfonylamino)-3-[2-(4-methoxyphenyl)ethyl]-4-(4-methoxyphenyl)-2-imidazolidinone (KVI-020/WYE-160020) as a potential treatment for atrial arrhythmia.

Atrial fibrillation is the most prevalent form of cardiac arrhythmia. Current treatments extend the atrial effective refractory period by nonselective blockade of cardiac ion channels. An alternative approach selectively targeting the Kv1.5 ion channel offers the opportunity for therapeutic benefit with decreased risk of adverse cardiovascular events. KVI-020 (4g) successfully demonstrated antiarrhythmic efficacy in a canine arrhythmia model, and these findings support its utility as an antiarrhythmic agent.

Evaluation of emergency medicine trainees' ability to use transport equipment.

OBJECTIVE: To assess the skills of advanced emergency medicine trainees in the use of commonly used transport equipment and to determine what teaching has been undertaken and what level of confidence trainees have in their ability to use the equipment tested. METHODS: This is a prospective multicentre observational study examining equipment use. Three pieces of equipment were examined: the infusion pump, defibrillator-monitor and transport ventilator. A questionnaire and a series of practical tasks were used. Advanced trainees from Victorian ED were enrolled as participants. Outcomes included proportion of participants able to perform all tasks successfully, subjective registrar confidence with use of equipment and departmental training practices. Eleven ED were represented, covering metropolitan tertiary, metropolitan peripheral and regional hospitals. RESULTS: There were 47 participants enrolled in the study. The range of advanced training years of participants was 1-5+. Of the 47 participants, 39 (83%; 95% CI 72.2-93.7%) had been involved as medical escorts. Eighteen participants (38.3%; 95% CI 24.4-52.2%) described some form of teaching for equipment use. The median level of confidence of participants (on a 5-point scale) was 4 (25-75% interquartile range 3-4), which correlates with a moderate degree of confidence. Of 47 participants, 7 (14.9%; 95% CI 4.7-25.1%) rated themselves as not confident or somewhat under-confident (i.e. 1 or 2 on the scale). Of 47, 12 passed overall (25.5%; 95% CI 13.1-38%) and 8 completely failed (17.0%; 95% CI 6.3-27.8%) the skill testing. CONCLUSION: When tested in isolation, there appears to be deficits in the equipment skills of advanced ED trainees who are expected to be proficient in transport medicine.

A spectrophotometric method to quantify linear DNA.

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.