Microwave-mediated enzyme-linked immunosorbent assay procedure

Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), human interleuken 1β (IL-1β), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diagnosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters.     

Use of Infrared Camera to Understand Bats’ Access to Date Palm Sap: Implications for Preventing Nipah Virus Transmission

Pteropus bats are commonly infected with Nipah virus, but show no signs of illness. Human Nipah outbreaks in Bangladesh coincide with the date palm sap harvesting season. In epidemiologic studies, drinking raw date palm sap is a risk factor for human Nipah infection. We conducted a study to evaluate bats’ access to date palm sap. We mounted infrared cameras that silently captured images upon detection of motion on date palm trees from 5:00 pm to 6:00 am. Additionally, we placed two locally used preventative techniques, bamboo skirts and lime (CaCO₃) smeared on date palm trees to assess their effectiveness in preventing bats access to sap. Out of 20 camera-nights of observations, 14 identified 132 visits of bats around the tree, 91 to the shaved surface of the tree where the sap flow originates, 4 at the stream of sap moving toward the collection pot, and no bats at the tap or on the collection pots; the remaining 6 camera-nights recorded no visits. Of the preventative techniques, the bamboo skirt placed for four camera-nights prevented bats access to sap. This study confirmed that bats commonly visited date palm trees and physically contacted the sap collected for human consumption. This is further evidence that date palm sap is an important link between Nipah virus in bats and Nipah virus in humans. Efforts that prevent bat access to the shaved surface and the sap stream of the tree could reduce Nipah spillovers to the human population.     

Fatal Outbreak from Consuming Xanthium strumarium Seedlings during Time of Food Scarcity in Northeastern Bangladesh

Background

An outbreak characterized by vomiting and rapid progression to unconsciousness and death was reported in Sylhet Distrct in northeastern Bangladesh following destructive monsoon floods in November 2007.

Methods and Findings

We identified cases presenting to local hospitals and described their clinical signs and symptoms. We interviewed patients and their families to collect illness histories and generate hypotheses about exposures associated with disease. An epidemiological study was conducted in two outbreak villages to investigate risk factors for developing illness. 76 patients were identified from 9 villages; 25% (19/76) died. Common presenting symptoms included vomiting, elevated liver enzymes, and altered mental status. In-depth interviews with 33 cases revealed that 31 (94%) had consumed ghagra shak, an uncultivated plant, in the hours before illness onset. Ghagra shak was consumed as a main meal by villagers due to inaccessibility of other foods following destructive monsoon flooding and rises in global food prices. Persons who ate this plant were 34.2 times more likely (95% CI 10.2 to 115.8, p-value<0.000) than others to develop vomiting and unconsciousness during the outbreak in our multivariate model. Ghagra shak is the local name for Xanthium strumarium, or common cocklebur.

Conclusions

The consumption of Xanthium strumarium seedlings in large quantities, due to inaccessibility of other foods, caused this outbreak. The toxic chemical in the plant, carboxyatratyloside, has been previously described and eating X. strumarium seeds and seedlings has been associated with fatalities in humans and livestock. Unless people are able to meet their nutritional requirements with safe foods, they will continue to be at risk for poor health outcomes beyond undernutrition.     

The protein fluorescence and structural toolkit: Database and programs for the analysis of protein fluorescence and structural data

Protein fluorescence is a powerful tool for studying protein structure and dynamics if we have a means to interpret the spectral data in terms of protein structural properties. Our previous research successfully provided this support through the development of individual software modules implementing the algorithms for fluorescence and structural analyses. Now we have integrated the developed software modules, introduced a new program for the assignment of tryptophan residues to spectral-structural classes, and created a web-based toolkit PFAST: protein fluorescence and structural toolkit: http://pfast.phys.uri.edu/. PFAST contains three modules: (1) FCAT is a fluorescence-correlation analysis tool, which decomposes protein fluorescence spectra to reveal the spectral components of individual tryptophan residues or groups of tryptophan residues located close to each other, and assigns spectral components to one of five previously established spectral-structural classes. (2) SCAT is a structural-correlation analysis tool for the calculation of the structural parameters of the environment of tryptophan residues from the atomic structures of the proteins from the Protein Data Bank (PDB), and for the assignment of tryptophan residues to one of five spectral-structural classes. (3) The last module is a PFAST database that contains protein fluorescence and structural data obtained from results of the FCAT and SCAT analyses.     

Photochemical immobilization of proteins on microwave-synthesized photoreactive polymers

We report a rapid and versatile procedure for the preparation of photoreactive polymers and light-induced immobilization of proteins onto such polymers. Photoreactive controlled-pore glass, silica gel, glass slide, and polystyrene microtiter plate are prepared in 40-60s by microwave irradiation of the respective amino polymers and 1-fluoro-2-nitro-4-azidobenzene. Azido group, now part of the polymer, yields highly reactive nitrene under ultraviolet (UV) light at 365 nm. Thus, when photoreactive polymer and horseradish peroxidase or glucose oxidase are exposed to UV light, the reactive nitrene immobilizes the protein molecules in 10 to 20 min through covalent bonding. As nitrene has a property of inserting into C-H bond, the method may find potential applications for immobilization of biomolecules irrespective of their functional groups.     

Synthesis of ester-linked lithocholic acid dimers

Four lithocholic acid dimers were synthesised via esterification. The ester-linked dimer, 3-oxo-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), was obtained by condensation of methyl lithocholate with 3-oxo-5beta-cholan-24-oic acid. Borohydride reduction of this ester-linked dimer gave 3alpha-hydroxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta), which was acetylated to 3alpha-acetoxy-5beta-cholan-24-oic acid (cholan-24-oic acid methyl ester)-3-yl ester, (3alpha,5beta). Reaction of methyl lithocholate with oxalyl chloride yielded the oxalate dimer, bis(5beta-cholan-24-oic acid methyl ester)-3alpha-yl oxalate.     

Protective effect of alpha-tocotrienol against free radical-induced impairment of erythrocyte deformability

Alpha-tocotrienol (alpha-T3) has been suggested to protect cellular membranes against free radical damage. This study was done to estimate the effect of alpha-T3 on free radical-induced impairment of erythrocyte deformability by comparing it to alpha-tocopherol (alpha-T). An erythrocyte suspension containing 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) was forced to flow through microchannels with an equivalent diameter of 7 microm for measuring erythrocyte deformability. A higher concentration of AAPH caused a marked decrease in erythrocyte deformability with concomitant increase of membranous lipid peroxidation. Treatment of erythrocytes with alpha-T or alpha-T3 suppressed the impairment of erythrocyte deformability as well as membranous lipid peroxidation and they also increased erythrocyte deformability even in the absence of AAPH. In these cases, the protecting effect of alpha-T3 was significantly higher than that of alpha-T. We emphasize that higher incorporating activity of alpha-T3 into erythrocyte membranes seems to be the most important reason for higher protection against erythrocyte oxidation and impairment its deformability.     

Protective effect of quercetin against cigarette tar extract-induced impairment of erythrocyte deformability

Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the most abundant flavonol-type flavonoids rich in diet and suggested to possess a beneficial role in blood circulation. This study was conducted to know the effect of quercetin aglycone and one of its possible metabolite, quercetin-3-O-beta-D-glucuronide on cigarette tar extract-induced impairment of erythrocyte deformability. Erythrocyte suspension containing quercetin aglycone, quercetin-3-O-beta-D-glucuronide or quercetin-3-O-beta-D-glucoside was forced to flow through microchannels with equivalent diameter of 7 &mgr;m and its transit time was measured as an index of erythrocyte deformability using microchannel array method. Both quercetin aglycone and quercetin-3-O-beta-D-glucuronide, but not quercetin-3-O-beta-D-glucoside, substantially increased erythrocyte deformability indicating that the former two compounds affect the physicochemical state of erythrocyte by interacting with its membranes. Aqueous cigarette tar extract caused marked decrease in erythrocyte deformability with concomitant increase of membranous lipid peroxidation. In that case, quercetin aglycone suppressed the impairment of erythrocyte deformability as well as membranous lipid peroxidation. The same effect was found in quercetin-3-O-beta-D-glucuronide, eventhough its effect was lower than that of quercetin aglycone. Thus, not only quercetin aglycone but also its conjugate metabolite protects erythrocyte membrane from the damage of smoking by scavenging reactive oxygen species generated from cigarette tar. Intake of quercetin-rich food may be helpful to protect membranous damage in erythrocytes from smoking.