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    ROS production, oxidative stress markers, and antioxidant capacity

    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) [8].

    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.

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    Suspect in Jeremy Thorpe scandal 'may be alive'

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    Cyclist threatens driver with knife after accidental collision
    The attacker managed to breakthrough one of the vehicle's windows, and the driver subsequently fled the auto in fear of his life. The Volkswagen nearly hits the cyclist while trying to pull out into traffic which appears to start the confrontation.
    6/02/2018

    Al Qaeda Calls Out Saudi Arabia For Involving Themselves With WWE
    Almost a dozen prominent activists were arrested last month, just weeks before the ban on women driving is due to be lifted. This issue was Vogue's first-ever edition exclusively dedicated to Saudi Arabia and Her Royal Highness' first cover.
    6/02/2018

    Google reportedly not renewing contract for Project Maven drone program
    The email also said that the budget for the program was expected to eventually expand to 0 million. Although tech companies often chase contracts in the U.S. defense sector, Google's involvement in.
    6/02/2018

    BBC launches Ultra HD and VR trials ahead of World Cup
    These trials add to the comprehensive coverage the BBC has lined up for the World Cup across TV, Radio and Digital. The showpiece soccer/football (delete as appropriate) matches now confirmed for the service include Portugal vs.
    6/02/2018

    Hotel valet parks a guest’s Porsche … under another vehicle
    The SUV had to be propped up with poles in order to free both the valet and the crushed Porsche. The hotel insurance is expected to cover the cost of the damage.
    6/02/2018

    Mattis slams China on South China Sea island weaponization
    Last month, China landed bomber aircraft on islands and reefs in the sea as part of a military training exercise. Trump said that the June 12 talks in Singapore with the North's leader, Kim Jong-un, were back on.
    6/02/2018

    Arm brings laptop-esque power to your smartphone
    TechDay 2017 brought the Arm Cortex-A75 and the Mali-G72, while 2016's TechDay brought the Cortex-A73 and the Mali-G71. The 7nm, 3GHz new Cortex A76 has 40 percent better power efficiency compared to the Cortex A75 CPU at 2.8GHz in 10nm.
    6/02/2018

    United Nations postpones vote on protecting Palestinians: diplomats
    Israel said some 70 rockets and mortar bombs were fired from Gaza and three of its soldiers were wounded by shrapnel. Meanwhile, a fragile calm returned to Gaza Strip on Wednesday.
    6/02/2018

    Sean Hannity: Joy Reid Should Keep Her Job
    Reid and MSNBC have yet to respond amid growing pressure from the public for an explanation. She made no mention of the hacker defense when she issued her statement on Friday.
    6/02/2018

    Radio 1 breakfast show host Nick Grimshaw quits as ratings fall
    He announced the news to the country on his show this morning, as the BBC revealed Greg James will take over. This week he became the second-longest serving breakfast show presenter in the station's history.
    6/02/2018

    MI basketball coach John Beilein interviewed for Detroit Pistons job, per report
    MI made it to the 2013 final as well under Beilein, this time losing to the Louisville Cardinals. The Pistons are searching for a new head coach after firing Stan Van Gundy this offseason.
    6/02/2018

    Jeep To Offer Baby Model Below The Renegade
    One slide of the company's presentation (below) hinted that more off-road-oriented Cherokee Deserthawk was also on the way. Deserthawk may be a bid to grow the Jeep brand in the Middle East, where dune-bashing is practically a national sport.
    6/02/2018

    Samsung adds 18 more AR Emoji expressions
    As a result Samsung's execs decided to make a small design change that will delay the launch day by approximately two weeks. When Samsung revealed that the Samsung Galaxy S9 would feature animated Emoji characters, the world groaned.
    6/02/2018

    Harvey Weinstein faces new rape allegation, suit
    He has pleaded not guilty to charges of rape and criminal sexual acts in NY , and faces up to 25 years in prison on those charges. Almost 100 women have now accused Weinstein of crimes ranging from sexual harassment to assault and rape going back decades.
    6/02/2018

    Facebook is getting rid of its Trending section next week
    In a surprising Friday news dump, Facebook announced it will remove the Trending news section from its website and app. It's unusual for a site that's used for search, like Facebook, to not manage a trending section.
    6/02/2018

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    Manchester Airport'Everyone knows who done it': Holidaymaker claims whole neighbourhood knows who vandalised car with 'No Parking' paint.
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