Seizure amelioration effect of omega-3 EPA and DHA fatty acid supplementation on patients with drug resistant epilepsy: a scoping review using the GOED clinical study database

Objectives: 70% of patients with epilepsy are successfully treated with one or a combination of anti-seizure medications (ASMs). The latter 30%, do not respond to ASMs and continue to have uncontrollable seizures. They are known to have drug-resistant epilepsy (DRE). There is an unmet need for non-pharmacological treatment options to be considered. The objective of this scoping review is to assess the seizure amelioration effect of omega-3 (n-3) polyunsaturated fatty acids (PUFAs): eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on patients with DRE from randomized human clinical trials (RCTs). Methods: The Global Organization for EPA & DHA Omega-3s (GOED) Clinical Study Database was used as the primary search tool to generate a list of RCTs using the following terms: seizures, seizure severity, drug resistant epilepsy, refractory epilepsy, and intractable epilepsy. Subsequently, PubMed and Google Scholar English databases were searched for publications using the additional keywords: eicosapentaenoic acid and docosahexaenoic acid. Results: Eleven RCTs published before October 2023 were included in this review. EPA and DHA formulations administered to patients ranged between 0.3-2.9g/day, with 90.9% of RCTs investigating the fatty acids at a comparable ratio. A supplementation period of 3 months or less was employed by 72.7% of RCTs. 54.5% of RCTs reported a positive outcome of at least 50% or more significant reduction in seizure frequency compared to placebo or baseline. 27.2% of RCTs reported significant seizure freedom in patients during or at the end of the supplementation period, compared to placebo. Positive outcomes regarding seizure severity remain inconsistent with only 18.1% of RCTs reporting a significant reduction compared to placebo or baseline. Conclusions: N-3 EPA and DHA have the potential to be a simple add-on treatment intervention for the management of uncontrollable seizures in DRE. Additional RCTs are required to determine the optimal dosage for patients, the effect of long-term treatment duration and separately enriched preparations of EPA/DHA on seizure frequency and severity

Specific protease activity indicates the degree of Pseudomonas aeruginosa infection in chronic infected wounds

Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics—a process associated with promoting antibiotic resistance. We analysed chronic wound fluid samples, which had been assessed for microbial content, using 20 different fluorescent labelled peptide substrates to determine whether protease activity correlated with the bacterial load. Eight of the peptide substrates showed significant release of fluorescence after reaction with some of the wound samples. Comparison of wound fluid protease activities with the microbiological data indicated that there was no correlation between bacterial counts and enzyme activity for most of the substrates tested. However, two of the peptide substrates produced a signal corresponding with the microbial data revealing a strong positive correlation with Pseudomonas aeruginosa numbers. This demonstrated that short fluorescent labelled peptides can be used to detect protease activity in chronic wound fluid samples. The finding that two peptides were specific indicators for the presence of P. aeruginosa may be the basis for a diagnostic test to determine wound colonisation by this organism

Escherichia coli contamination of the river Thames in different seasons and weather conditions

Contamination of public water ways with sewage represents a serious environmental and health risk. We monitored pollution of the river Thames by enumerating the indicator organism Escherichia coli. Samples were taken from a site in central London near Waterloo Bridge in different seasons. E. coli were quantified using a membrane filtration method, and correlated with the tidal variations of the river and meteorological data on rainfall and temperature. More frequent and severe incidents of pollution occurred in the autumn. Heavy rainfall resulted in sharp peaks of E. coli contamination that implies a potential increase of numbers of pathogenic micro-organisms. Sixty percent of all samples were found to be in excess of the accepted upper limit of pollution set by European Union (EU) legislation for bathing water. This study demonstrated that frequent sewage pollution of the Thames results in high numbers of E. coli and incidents of detectable levels of pathogenic bacteria demonstrating the need for regular monitoring of bacterial pollution

Optimisation of the detection of bacterial proteases using adsorbed immunoglobulins as universal substrates

Bacterial proteases, Type XXIV from Bacillus licheniformens and Type XIV from Streptomyces griseus, were used to investigate the utility and optimisation of a solid phase assay for proteases, using immunoglobulin proteins as substrates. Immunoglobulins IgA and IgG were adsorbed on to surfaces of ELISA plates and exposed to various levels of the bacterial proteases which led to digestion and desorption of proportional amounts of the immunoglobulins. The assay signal was developed by measuring the remaining proteins on the polystyrene surface with appropriate enzyme-labelled anti-immunoglobulin reagents. The assay was fully optimised in terms of substrate levels employing ELISA techniques to titrate levels of adsorbed substrates and protease analytes. The critical factor which influences assay sensitivity was found to be the substrate concentration, the levels of adsorbed immunoglobulins. The estimated detection limits for protease XXIV and XIV were 10 μ units/test and 9 μ units/test using IgA as a substrate. EC50 values were calculated as 213 and 48 μ units/test for each protease respectively. Using IgG as a substrate, the estimated detection limits were 104 μ units/test for protease XXIV and 9 μ units/test for protease XIV. EC50 values were calculated at 529 and 28 μ units/test for protease XXIV and XIV respectively. The solid phase protease assay required no modification of the substrates and the adsorption step is merely simple addition of immunoglobulins to ELISA plates. Adsorption of the immunoglobulins to polystyrene enabled straightforward separation of reaction mixtures prior to development of assay signal. The assay exploits the advantages of the technical facilities of ELISA technology and commercially available reagents enabling the detection and measurement of a wide range of proteases. However, the key issue was found to be that in order to achieve the potential performance of the simple assay, optimisation of the method was essential

Detection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate

A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatin–hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein–hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin–cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody–HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose–response curves for enzyme activities were measured within ranges of 0–550 µunits mL−1 for chymotrypsin, 0–12 µunits mL−1 for type IX, 0–35 µunits mL−1 for type XIV and 0–100 µunits mL−1 for type XXIV. The detection limits of the proteases studied were 89 µunits mL−1 for chymotrypsin, 0.26 µunits mL−1 for type IX, 5.8 µunits mL−1 for type XIV and 6.5 µunits mL−1 for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format

Characterization of bacterial proteases with a panel of fluorescent peptide substrates

Bacteria produce a range of proteolytic enzymes. In an attempt to detect and identify bacteria on the basis of their protease activity, a panel of protease substrates was investigated. Peptides conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC) are well-established substrates for measuring protease activity. Although peptide–AMC substrates are generally not specific for a single protease, a unique pattern can be achieved for both highly specific enzymes and those with a broader substrate range by comparing different peptide substrates. The panel of 7 peptide–AMC substrates chosen exhibited a unique pattern for nine microbial proteases. The selected peptides were used to determine protease activity in cultured strains of Pseudomonas aeruginosa and Staphylococcus aureus. A signal pattern obtained with peptides with arginine, lysine, and tyrosine in the P1 position characterized the bacterial protease activities in these samples. The kinetic parameters for the three best substrates for the P. aeruginosa sample were calculated. Further information about substrate specificity was gained by the selective use of protease inhibitors. The results presented show that peptide–AMC substrates provide a simple and sensitive tool to characterize protease activity in microbiological samples and that they have the potential to identify and distinguish different bacterial species