To measure the mitochondrial ROS production, freshly isolated mitochondria (25-?g protein) were suspended in 50 ?l of HMS medium (10 mM HEPES pH 7.4, 225 mM mannitol, 75 mM sucrose), and added in triplicate to 96-well, black flat-bottomed plates. The reaction was started with addition of 50 ?l of 2X reaction buffer (20 mM Tris-HCl at pH 7.4, 500 mM sucrose, 2 mM EDTA) containing 66-?M amplex red and 0.2U/ml horseradish peroxidase. Mitochondria were energized with complex I or complex II substrates, and amplex red oxidation by ROS to fluorescent resorufin was measured for three minutes at Ex563nm/Em587nm on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). Standard curve was prepared with H2O2 (50 nM–5 ?M) .
To measure the H2O2 levels, 50 ?l of tissue lysates (100 ?g) or cell lysates (1 x 104 cells) were added in triplicate to flat-bottom (dark-walled) 96-well plates. Then 100 ?l of reaction mixture containing 0.05 M sodium phosphate, pH 7.4, 33 ?M amplex Red, and 0.1 U/ml HRP was added. The plates were incubated for 30 min in dark, and ROS levels were recorded as above.
The level of 3-nitrotyrosine (3-NT) in heart tissue lysates was measured by using an ELISA kit (ab116691, Abcam, Cambridge MA). Protein carbonyls in tissue homogenates and plasma were measured by a colorimetric protein carbonyl assay (cat#10005020, Cayman Chemical, Ann Arbor MI). Malonyldialdehydes (MDA) provide a measure of lipid peroxidation products, and were measured in tissue lysates and plasma samples by a TBARS assay (10009055, Cayman Chemical). Concentration of lipid peroxides was calculated as an MDA equivalent using the extinction coefficient for the MDA–TBA complex of 1.56? 105 M?1 cm?1 at 532 nm.
Total antioxidant capacity was assessed by using lag time by antioxidants against the myoglobin-induced oxidation of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) with H2O2 (709001, Cayman Chemical). Briefly, 20 ?l of plasma samples (diluted 1:20, v/v) or heart homogenates (15 ?g) were added in triplicate to 96-well plates, and mixed with 90 ?l of 10 mM PBS (pH 7.2), 50 ?l of myoglobin solution, and 20 ?l of 3 mM ABTS. Reaction was initiated with H2O2 (20 ?l) and change in color monitored at 600 nm (standard curve: 2–25 ?M trolox).
To measure mitochondrial stress, cardiac myocytes (104/well) were incubated with Tc and/or PJ34, and then loaded for 30 min with 5 ?M MitoSOX red (detects mitochondrial ROS, Ex498nm/Em598nm) or 10 ?M JC-1 (5,5?,6,6?-tetrachloro-1,1?,3,3?-tetraethylbenzimidazolylcarbocyanine iodide). Cells were washed, and JC-1 red aggregates (Ex560nm/Em595nm) vs. green monomers (Ex485nm/Em/535nm) ratio recorded by using a Spectra MaxR M2 microplate reader. The fluorescent probes were purchased from Invitrogen/Molecular Probes.
After all, they pulled this trick off in 2015 with the reveal of Fall...
Apart from this, another person has been tested positive for the infection in Kozhikode, PTI said.
To check further spread of the virus, directions have been given to officials to observe and monitor all those persons who had come in touch with the Nipah patients and are likely to be affected.
Reopening of schools in the two districts had earlier been postponed to June 5.
"Till such time the report from the NIV in Pune is received it can not be confirmed whether it was a case of Nipah virus or not", the spokesman said. The objective was to avoid crowding of people, the official said.
Two new cases of the deadly brain-damaging Nipah virus have been confirmed in the southern Indian state of Kerala, where another two people were killed by the virus, a health official said on Thursday. Even the health department in Kerala has issued advisory for people travelling to the state and urged travellers to be extra cautious while visiting Kozhikode, Malappuram, Wayanad, and Kannur districts, according to Business Standard.
The central experts from the National Centre for Disease Control are continuing to evaluate the situation and taking necessary measures, said a press release quoting the minister. Her body fluids were sent to Manipal medical college for test on Friday.
In Kolkata, a 28-year-old soldier from Kerala, who was posted at Fort William, Kolkata, died of suspected Nipah virus last week.
Director of Health Services Dr R.L. Saritha said the condition of the afflicted persons remained unchanged.
Necessary protective gears, including masks and gloves, have been provided to the hospital staff.
Earlier, cases of Nipah virus were reported from Siliguri in 2001 and Nadia in 2007 in the eastern state of West Bengal and around 47 deaths were reported. Two people, who died of Nipah infection, had undergone treatment at this hospital.
Large-scale awareness campaigns are also being held in the two districts.
The two new Nipah cases in Kerala take the total number of confirmed cases to 17, Kozhikode district medical officer V. Jayashree told Reuters.
Following the death of a superintendent of Kozhikode district court complex due to Nipah, the district bar association asked the collector to stop the functioning of the district court for the time-being..