Organophosphorus and carbamate pesticides: Molecular toxicology and laboratory testing.

Population and food requirements are increasing daily throughout the world. To fulfil these requirements application of pesticides is also increasing. Organophosphorous (OP) and Organocarbamate (OC) compounds are widely used pesticides. These pesticides are used for suicidal purposes too. Both inhibit Acetylcholinesterase (AChE) and cholinergic symptoms are mainly used for the diagnosis of pesticide poisoning. Although the symptoms of the intoxication of OP and OC are similar, recent research has described different targets for OP and OC pesticides. Researchers believe the distinction of OP/OC poisoning will be beneficial for the management of pesticide exposure. OP compounds produce adducts with several proteins. There is a new generation of OP compounds like glyphosate that do not inhibit AChE. Therefore, it's high time to develop biomarkers that can distinguish OP poisoning from OC poisoning.

Electrophoresis of Amplicons is a Better Method to Understand the Performance of Loop-mediated Isothermal Amplification Assay for Screening the Presence of Escherichia coli in Water.

SUMMARY: LAMP assay is widely used for detecting pathogens. We observed that the conventional and gradient polymerase chain reaction (PCR) could not detect the extracted Escherichia coli DNA; real-time PCR was able to detect up to a certain limit (10-8 bacterial dilution). At the same time, the LAMP assay could detect the bacteria at a much lower concentration (10-14 dilution). The results of the LAMP assay were evaluated using agarose gel electrophoresis and DNA binding dye (PicoGreen), but only gel electrophoresis gave reliable results. Therefore, we propose using electrophoresis-based amplicon detection to overcome the limitations of dye-based detection. We believe that this amplicon detection will go a long way in the screening of potable drinking water.

Molecular docking analysis of melamine with nuclear factor erythroid 2-related factor 2 and succinate dehydrogenase.

Melamine consumption causes oxidative stress by an unknown mechanism. Therefore, it is of interest to analyze the interaction of melamine with two important proteins involved in oxidative stress biology namely, nuclear factor erythroid 2-related factor 2 and succinate dehydrogenase. The molecular docking data shows the melamine binding with these two proteins at critical residues. These interactions can be logically perceived for the causation of melamine induced oxidative stress.

Spectrofluorimetric-based approach to screen urine contamination in drinking water: A step toward the development of screening method for leptospirosis.

Hygiene hypothesis and sanitization are two important pivots of modern civilization. The drinking water should be free from urine and stool contamination. Coliform test is popular for understanding feces contamination. However, understanding urine contamination in drinking water is a difficult task. On the other hand, urine contamination can cause disease like leptospirosis. It occurs mainly in animals and infects humans through contaminated water, food, and soil and causes serious consequences. Rat urine is the most common source of such disease outbreaks. Further, sophisticated laboratories with high-end technologies may not be present at the site of disease outbreaks. In this context, we have proposed a spectrofluorimetric approach to screen urine contamination in water. The screening method can sense up to 156 nl/ml of rat urine.

The Effect of Recombinant Human Erythropoietin on Bacterial Growth: A Dual-Edged Sword.

BACKGROUND: Hypererythropoietinemia is associated with common diseases like non-uremic anaemia where infection burden is high. Erythropoietin (EPO) is also given as therapy for anaemia associated with chronic kidney disease and cancer and in those who are at a higher risk of infections. EPO is known to have an effect on macrophages by which it helps in the growth of some intracellular pathogens. However, its direct role on bacterial growth is currently unknown. SUMMARY: Here, we investigated the direct effect of recombinant human erythropoietin (rhuEPO) on the growth of pathogenic Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. In silico experiments were designed to gain insight into the mechanisms. We found that 30 IU/L rhuEPO promoted the growth of E. coli and S. aureus and inhibited the growth of P. aeruginosa. In silico observations suggest that bacterial cell surface proteins may interact with the EPO and may cause the observed effects. KEY MESSAGE: It appears that some pathogens can explore EPO to proliferate and growth of others are inhibited by the same. The consequence of such observation is a matter of widespread concern for future research.

A novel fluorescence based assay for the detection of organophosphorus pesticide exposed cholinesterase activity using 1-naphthyl acetate.

Acetylcholinesterase (AChE) is the primary target of organophosphorus pesticides (OPs). Ellman's method using Acetylthiocholine (ATCh) is the standard approach for the detection of AChE activity. Though ATCh is a popular substrate, it has certain drawbacks as well. Because of these limitations, there is a need for the development of reliable and rapid assays for determination of AChE activity in cases of OP poisoning. In the present work, we have used 1-Naphthyl acetate (1-NA) as a fluorogenic substrate for the estimation of AChE activity of human erythrocytes. It is well known that due to inter-individual variation in AChE activity, the baseline value cannot be correctly predicted. Therefore, using 1-NA, we have developed a rapid, sensitive and baseline free assay for the estimation of AChE activity. The assay is based on reactivation and fluorescence quenching using a cocktail of oximes for the determination of cholinesterase activity in a post-exposure sample. Moreover, it is free from interference due to oximolysis which is an established limitation of ATCh. We feel that such an assay using 1-NA has the potential to be explored at the point of care for rapid detection of OP poisoning.

1-Naphthyl acetate: A chromogenic substrate for the detection of erythrocyte acetylcholinesterase activity.

Erythrocyte acetylcholinesterase (AChE) is a preferred biomarker for the detection of organophosphorus poisoning. Acetylthiocholine (ATCh) is the most popular substrate for the detection of AChE activity. However, oximolysis is a prominent feature with ATCh. In this context, we have searched alternative substrates for AChE using in silico tools for screening of a better substrate. The in silico approach was performed to understand the fitness and the Total Interaction Energy (TIE) of substrates for AChE. The alternative substrates for AChE were screened in terms of high Goldscore and favorable TIE in comparison to acetylcholine (ACh)-AChE complex and other relevant esterases. Among the screened substrates, 1-Naphthyl acetate (1-NA) exhibited the most favorable interaction with AChE in terms of highest TIE and corresponding high Goldscore. The Molecular Dynamic (MD) simulation of the 1-NA-AChE complex showed a stable complex formation over a period of 5 ns. The results obtained in the in silico studies were validated in vitro using pure erythrocyte AChE and hemolysate. We observed 1-NA to be a better alternative substrate for AChE than ATCh in terms of lower Km value. Its specificity appeared at least similar to ATCh. Therefore, we propose that 1-NA can be an attractive chromogenic substrate for the measurement of AChE activity, and it possess the potential to detect organophosphorus pesticide (OP) poisoning.

A color-reaction-based rapid screening for null activity of butyrylcholinesterase: a step toward point-of-care screening for succinylcholine apnea.

Succinylcholine apnea happens in cases of null butyrylcholinesterase activity after administration of preintubation succinylcholine. So far, there is no such popular test that can rapidly screen null butyrylcholinesterase activity from plasma. Development of a novel method for rapid screening of null butyrylcholinesterase activity of plasma samples was the objective of the current work. Dichromate reagent was added to 1-naphthol, 2-naphthol, phenol, and para-nitrophenol in separate aliquots and watched for the color formation. Plasma samples preincubated with and without selective butyrylcholinesterase inhibitor were mixed with 1-naphthylacetate and watched for color development after addition of dichromate reagent. Fitting of 1-naphthylacetate at the active site of butyrylcholinesterase was analyzed by using tools of computational biology. It was seen that 1-naphthol formed color with dichromate reagent in a concentration-dependent manner. Other phenols did not form color with dichromate reagent even at 500-µm concentrations. Plasma sample with and without selective butyrylcholinesterase inhibitor (tetra isopropyl pyrophosphoramide) was distinguishable by color formation when incubated with 1-naphthylacetate, followed by the addition of dichromate reagent. In silico analysis also showed that 1-naphthylacetate fitted well at the active site of butyrylcholinesterase. The developed method may be used for rapid screening for null butyrylcholinesterase activity at point of care.

Acute organophosphorus poisoning.

Acute organophosphorus poisoning continues to be a detrimental problem and a potential cause of mortality especially in developing countries. Inhibition of acetylcholinesterase enzyme is the main mechanism of toxicity of such pesticides and measurement of acetylcholinesterase activity is the commonly used laboratory diagnosis approved for the purpose. It is now proved beyond any doubt that early intervention is beneficial for cases of acute organophosphorus poisoning and, therefore, considerable current interest has been generated for development of point of care testing tool for screening of the same. However, to the best of our knowledge so far the matter is not reviewed from the view of point of care testing tool development. In this paper, this subject is reviewed highlighting the methodological aspects and point of care testing tool development in the context of organophosphorus poisoning.