hnX,Jnu hn,DiaM ia n la Washington* Ilyas and Mihai M. ATP Doina Zhang, produce Junhua Xu, and Chen energy photonic capture to mammalian mitochondria enable pigments chlorophyll Light-harvesting ARTICLE RESEARCH ß 388 2013 October 15 Accepted 2013; April 30 Received ATP ([email protected]) correspondence for USA. 10032, *Author increases NY York, New metabolite Ophthalmology, Center, Medical University same Columbia metabolite. light, the the to with that exposed mixed when not demonstrate tissues ATP We animal chlorophyll, of with tissues, of concentrations compared animal-derived higher metabolite and have mitochondria light-capturing mammalian a When mitochondria. isolated the are with in circulation, enriched be the incubated can enter and that tissues, can in show chlorophyll present We of into metabolites. light metabolites dietary of penetrate dietary potential consequences can the these that elucidate by to wavelengths absorption sought at We tissues. light spectrum absorb animal to visible ability Blakeslee, the the is retain and that in chlorophyll (Ferruzzi 1999) Dolphin, metabolites body, and of Ma the 2007; variety energy Inside a light-absorbing as into diet. this converted light-absorbing use their consume to through animals synthesize light Many chlorophyll synthesis. capture which ATP can for that endosymbiotically chloroplasts, molecules is co-evolved chlorophyll produce ATP have all catabolic of plants to a majority nearly respiration, However, adenosine-5 vast through In process. mitochondria as the the life. transported animals, in synthesized of and In (ATP). stored understanding triphosphate is our energy to organisms, environment fundamental the from energy is obtain organisms how Determining INTRODUCTION Mitochondria Light, ATP, sunlight. WORDS: from KEY pigments, directly energy chlorophyll We derive synthesis. to plant able ATP of are mitochondrial type too, consumption animals, in through chlorophyll step that slow suggest propose a as reduction Q, the Results coenzyme catalyzing mammals, of by diet. ATP in mitochondrial when modulate chlorophyll-rich swine exists molecules and energy a rats mice, into fed in accumulate light metabolites convert chlorophyll same the to demonstrate further We potential span. life in increase an with worm along the to fed elegans metabolite of same metabolite light-capturing The a light chlorophyll. with capture mixed also when can ATP synthesize mitochondria and organisms. mammalian that photosynthetic show of we Here form in the chloroplasts in energy chlorophyll-containing biological into sunlight adenosine-5 convert to However, ability planet. this the on source energy abundant most the is Sunlight ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,3839doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. ed oices nAPsnhssuo ih exposure, light upon synthesis ATP in increase to leads 9 tihsht AP stogtt elmtdto limited be to thought is (ATP) -triphosphate Caenorhabditis 9 - elfnto n ieo earth. These on Q. life normal and coenzyme aging, function of of understanding cell our reduction for the implications mitochondrial have catalyzing findings hypothesis modulate by the of chlorophyll stores with dietary ATP consistent in of span regulation are metabolites energy data that of presented life means The important animals. an dietary-derived median be through may capture metabolites energy the photonic that hypothesis extends elegans Caenorhabditis and concentrations, ossetwt eoodrutaestvt,o positively Koshland, or and metabolite and the presumably Goldbeter between ultrasensitivity, interaction coordinated by is a suggests described and is zero-order it as behavior binding, which threshold-sensitive cooperative with in This protein. one consistent a to with from environment change associated its a aqueous with reflects consistent fluorescence P-a, an in of environment change the abrupt in The change 2B). (Fig. reached plateau quickly a and fivefold, by abruptly, sheep P- increased mitochondria, fluorescence from of a concentration obtained the increasing fragments After 2A,B). mitochondrial (Fig. increasing of heart presence the of in amounts nm 675 at fluorescence P-a measured phosphorylation. oxidative by driven ATPproduction enhanced with consistent were Observations system. electrontransport the through occurring electron consumption oxygen with S1H–I), the consistent Fig. P-a material (supplementary of P-a-fueled with 57% and by consumption light- incubated Addition oxygen the not reduced S1G). azide, sodium mitochondria inhibitor, Fig. transport in levels material started consumption the (supplementary oxygen to of activity return rate mitochondrial P-a, to the added of and much measures concentrations too With ATP above S1E–G). no the Fig. had material of alone (supplementary light any P-a increased material or on Light upon effect mitochondria. (supplementary increased P-a-treated and in dark 1C) 1B) exposure (Fig. (Fig. 1A) the potential consumption (Fig. membrane oxygen P-a in Mitochondrial without of S1A–D). kept also Fig. mitochondria samples were mitochondria than dark light Control or red ATP to the that exposed more cells. mitochondria in P-a, reactions produce of kept animal presence by the and/or In in assayed. P-a, P-a without place from early mitochondria synthesized an take is of be normally metabolites it known can because most P-a chlorophyll however, chlorophyll, used tissues of We biological metabolite transparent. which to relatively and are 1950), (Aronoff, absorb strongly odmntaeta itr hoohl eaoie a modulate can metabolites chlorophyll dietary mitochondria that mammalian demonstrate isolated To in synthesis ATP Light-driven RESULTS sltdmuelvrmtcodi ntepeec frdlight chlorophyll red in the of synthesis presence of the ATP in effects mitochondria on ( liver the mouse (P-a) examined isolated pyropheophorbide-a we metabolite levels, ATP l max odtriewehrPaascae ihmtcodi,we mitochondria, with associates P-a whether determine To 5 7 m,wihclrntp oeue uha P-a as such molecules chlorin-type which nm), 670 pnlgtepsr;spotn the supporting exposure; light upon ,

Journal of Cell Science EERHARTICLE RESEARCH 5 ofdneitra rm7t 5 a bandb itn h aat the to data the fitting by obtained a was with [ 65, 36, equation to of Hill 7 coefficient from Hill interval A confidence fragments. 95% mitochondrial of changed. concentration 1 peak) a the main for to interaction. intensity P-a–mitochondrial fluorescence shoulder the measured the of We of response wavelength, state (ratio steady longer curve Ultrasensitive a the (B) to of shifted shape and the increased and heart the P-a sheep fragments, of of mitochondrial intensity addition of fluorescence after addition and Upon before fragments. fragments. P-a mitochondrial mitochondrial of to spectra P-a Fluorescence of (A) binding Cooperative 2. Fig. oxidized for and light reduced to of percentage mitochondria the the measured to exposed and response minutes We in 10 light. of fragments red mitochondrial photoreduction to heart exposure the sheep show above catalyze the To in could form. metabolite reduced CoQ the mitochondrial P-a ubiquinol, the ubiquinone, and oxidation: that of form, states oxidized alternate the two in exist molecules increased steadily fluorescence instead, when P-a; observed S1J). of Fig. added material not were solution (supplementary (BSA) a was albumin serum In to sensitivity bovine of 1981). amounts threshold Koshland, increasing this and contrast, (Goldbeter fragments mitochondrial tim each * Ali for mitochondria. seconds. shown of 30 are at deviations batch added standard same was and ADP the Means assay. and with luciferase zero triplicate firefly time the at in ( using started run exposure measured was Light levels ATP. ATP P-a). experiment make relative (no The and to controls shown light to times compared capture at light to obtained to mitochondria were exposed liver and mouse (treated) isolated P-a allows with P-a incubated metabolite Chlorophyll 1. Fig. tpi eprto Cae 01.Temjrt fCoQ of majority The 2001). (Crane, respiration in step nrae ytood hti,oye osmto nrae hntelgtwstre n hntelgtwstre f,oye osmto eundt returned lin consumption black oxygen the off, of turned slope was the light on the turned When was slope). on. light same turned the the was When have light off. while lines tri the was measured gray for light when continuously two traces the increased was the average when consumption fluorescence the (i.e. slopes oxygen are safranin levels show shown is, and baseline Curves lines That zero mitochondria. Dotted twofold. 4 time of consumption. with by at batch treated oxygen increased added same mitochondria faster the was for denotes with Safranin line) triplicate slope dark. (black in Steeper the trace run oxygraph in was Representative kept experiment (C) The or runs. light. minutes the 2 under for remained light samples to exposed were P-a, Dy aaoi euto fcezm Q coenzyme of reduction Catabolic )udrdfeettetet smaue ysfai loecne oe loecneeul ihrmmrn oeta.Mtcodi,wt rwi or with Mitochondria, potential. membrane higher equals fluorescence Lower fluorescence. safranin by measured as treatments different under m) y 5 ax b /( c 10 b + emaue h xdto tt fCoQ of state oxidation the measured we , x b +fst.Ft( Fit )+offset]. R 2 :0.96. ): m 10 - ouinwieincreasing while solution P-a M (CoQ 10 sart limiting rate a is ) 10 10 P , .5frtetdvru oto ape.()Mtcodilmmrn potential membrane Mitochondrial (B) samples. control versus treated for 0.05 0mnts(nr ) nteasneo ih,n reduction a no was there light, Likewise, 4). CoQ proteins of (entry of mitochondrial occurred lack absence reduction the no denaturing the heat, Upon with In 3). (entry 2). occurred (entry minutes 10 CoQ ta. 03.I h rsl sltdmtcodi fragments, mitochondria isolated freshly CoQ the the In all nearly 2013). al., et xoe h upnint ih,4%o CoQ of and 46% P-a light, with to mitochondria suspension the the incubated exposed we when However, ih,Mt 2–14 2 None 46 Mito 5 Light, Q coenzyme light, the P-a, increase Mito not CoQ denatured 75 did reduced light, times employing of reaction P-a, atmosphere, Longer percentage anaerobic mitochondria. Mito of an dark, amount All under same P-a, conditions. minutes the listed 10 the for Mito under were Glutamate/malate, used reactions were Mito mitochondria light, heart P-a, Sheep mitochondria. Mito, 6 5 4 3 conditions 2 Reaction 1 number Condition CoQ of Photoreduction 1. Table rprto sd(nr ) eatiueti ‘background this attribute metabolites, chlorophyll We endogenous of 6). actions the mitochondrial (entry to the reduction’ on used P-a, depending added observed, of preparation was absence mitochondrial reduction the that In 2–14% showing P-a. a organize 2A,B, and Fig. sequester in proteins with consistent data are fluorescence observations the These 5). (entry mitochondria of tmltdAPsynthesis ATP stimulated Tbe1 nr ) ncmaio,a oiiecnrl we control, CoQ of positive reduction the 75% a a kept yielding and as dark, the glutamate/malate comparison, in with suspension In mitochondria the 1). energized entry 1, (Table nr sarneo ausosre o w ifrn mitochondrial different two for observed each. values experiments of two range preparations. for a results is are 6 1–5 Entry Entries HPLC. by quantified were m -.Telgtwstre no f ttetmsidctdb h arrows. the by indicated times the at off or on turned was light The P-a. M 10 yhg efrac iudcrmtgah HL)(Qu (HPLC) chromatography liquid performance high by ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal 10 euto ihCoQ with reduction 10 a xdzdi h omo ubiquinone. of form the in oxidized was 10 A T ytei nmuelvrmitochondria liver mouse in synthesis ATP (A) dt o hw) bqioeadubiquinol and Ubiquinone shown). not (data 10 10 sa al vn nlight- in event early an is - n ih nteabsence the in light and P-a , onyeQ coenzyme in reduction Percent 6 6 6 6 10 1 1 2 2 a reduced was 10 within point. e plicate quots thout 389 o e

Journal of Cell Science EERHARTICLE RESEARCH 390 171.7 and treated, error were: standard (CI) with P-a intervals with (means incubated confidence rates not 95% synthesis was that faster ATP 35% homogenate a [relative control light. at a 670-nm ATP than mouse to synthesized rate them treated homogenates brain exposed we treated and tissues, The P-a could with whole light homogenates in and brain production metabolites ATP chlorophyll influence whether homogenates determine tissue rodent To in synthesis ATP Light-driven (see spectroscopy fluorescence below). by chlorophyll, dietary detect light-absorbing to of Distribution able were we which the seen. than be smaller cannot are thus standard errors and and standard markings peak Means however, with triplicate. calculated, in correlated were done production errors ATP were and Peak Experiments P-a minutes. absorption. with 20 P-a the treated for and samples light line) in to (dotted production exposed P-a ATP of for spectrum tested the absorption wavelengths than the smaller of are Overlay * (B) errors seen. samples. standard be cannot the shown thus control, are and the markings errors line For standard point. and time Means the each with brains. for triplicate measured homogenized in was of run aliquots was batch experiment the same The in assay. ATP luciferase firefly Relative the shown. using times the at with withdrawn incubated were Homogenates exposure. ADP light ATP. with make tissue homogenate to brain brain light mouse capture allows to P-a homogenates metabolite Chlorophyll 3. Fig. 6 - n xoe olgtsatn ttm eo lqoswere Aliquots zero. time at starting light to exposed and P-a P , .5frtetdvru untreated versus treated for 0.05 A T ytei nmouse in synthesis ATP (A) 6 . (CI: 8.1 esrdb h raudrte65n ek hnaiaswere chlorophyll animals when 408, peak, as known nm diminished, at 675 fluorescence of the maxima red under This area with that the nm. by ring: 607 measured to and chlorin 562 intact 535, similar 504, an was with metabolites 4B) (Fig. peak means bopinadamsinseta(rnf,15) aeythey Namely signature 1950). have (Aronoff, spectra chlorins, admission both and metabolites, absorption its and chlorophyll Chlorophylls dietary light-absorbing of Distribution bobsrnl ( strongly absorb e loecnea 7 mwe xie iha410-nm a with excited oral Brain when exhibited 15.4 mice diet. nm after 675 these treated, from chlorophyll-rich [brain: tissue at extracts light a 4C) animal fluorescence (Fig. mice fat red in and fed 4A) present (Fig. we were and/or consumption, metabolites chlorophyll dietary mammals whether in its examine molecules To 1950). endogenous (Aronoff, from chlorins at differentiate emissions fluorescence intense demonstrate iia eut splmnaymtra i.S2E–F). Fig. material (supplementary gave results (HPLC) similar chromatography liquid luciferase high-performance the and both assay by ATP of Quantification S2C–E). (supplementary homogenates Fig. material heart and two-to- lens adipose, by in equal production increased of light it 690-nm dark, and 3B). the 630 (Fig. 500, in energy to kept exposure concentration during sample the fivefold ATP same produced the the P-a example, to and by of For 670 absorbed Wavelengths by ATP. increased energy. strongly in (630, total increase more red same largest were brain the and that with P-a-treated all nm) light the (500 light, ATP nm) exposed greenish 690 in we Increasing to production, increase homogenates observed. ATP same was enhance the P-a elicited S2A,B). added Fig. P-a material of (supplementary concentrations of ATP amount in concentrations the increase the and between correlation linear 111.3 control, 154.6–188.7); nadto obanhmgnts - loehne ATP enhanced also P-a homogenates, brain to addition In to necessary was P-a by absorption photon that demonstrate To 6 ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal s.d.); , 6fl uigepsr o60n ih;relative light; nm 670 to exposure during 16-fold P , e .1.Teectto pcrmo hs675-nm this of spectrum excitation The 0.01]. asdo hoohl-ihad-ordiets. -poor and chlorophyll-rich on raised A (D) diets. rich 675 and from chlorophyll-poor extracts fed fat mice abdominal Representative of (C) spectra diet. fluorescence from chlorophyll-rich extract a brain a fed of mice nm) 675 at (emission excitation spectrum a Representative fed (B) a animals diet. fed treated chlorophyll-rich animals six control and six diet of chlorophyll-poor total peak a Relative for nm. areas 410 at excitation brain following of extracts spectra fluorescence Representative (A) tissues. chlorophyll- in in fluorescence results metabolite-like chlorophyll Dietary 4. Fig. < 000M 50,000 6 0n loecneiaeo kne mice skinned of image fluorescence 10-nm 6 . ( 6.7 6 . C:9.–3.) i.3] No 3A]. Fig. 92.5–130.0); (CI: 9.1 2 1 n cm 5 ) oto:4.2 control: 6); 2 1 )at , 6–7 mand nm 665–670 , 7 m which nm, 675 6 . ( 2.6 n 5 6;

Journal of Cell Science niaieo oriaint ea o.Arepresentative A were ion. peaks metal of group a this to in of coordination spectra shown the of as addition, indicative 1944), In 5B. (Rabinowitch, Fig. chlorophylls demetalated 1 9–3)adcnrlbc a a,3 2–6] h red The (25–46)]. 35 pad, fat pad, fat back back control treated and (60–100); brain, (97–132) 82 treated brain, 116 minimum were: brackets control and gray in (97–138); deviation mean shown 118 standard value 4D; with gray [Fig. areas maximum diet was boxed and the chlorophyll-poor than fluorescence chlorophyll in a fed value imaging; animals given of animals brains fluorescence and in bodies using the in seen could stronger fluorescence Red be weeks. 2 also for diet chlorophyll-free a given ARTICLE RESEARCH pcr telctoso h bobnemxm n h Soret-to- absorption the and The maxima tail, P-a. absorbance as Q phytyl the 23– such of the locations group, (the without times carboxylate Compounds at spectra one metabolites retention least chlorophyll similar detected. at eluting had the with minutes of were 30 those compounds and to minutes 23 40–46 of between similar eluting and not contained groups minutes both chromatograms 30 Two extracts (similar that plasma illustrated). metabolites solvent representative and extracts eluting light. chlorophyll-derived a 410-nm fat the with shows excited Rat when in the fluorescence 5A of 675-nm compounds Fig. displayed source with the fluorescence. elucidate chromatogram to 675-nm HPLC data by red analyzed with further (MacDonald were reports 2006). consistent al., literature et Tang and mitochondria, 1999; 2A,B, al., Fig. et 675-nm the the in was did the P-a summarized in as that of suggests protein observation concentrated of This much homogenate. milligram liver as whole per isolated metabolite(s) 2.3-fold intensity, fluorescent fluorescence contained these by from mitochondria measured isolated mitochondria As in and homogenates. fluorescence homogenates 675-nm liver relative whole the measured we fluorescence. consistent the mitochondria, of intestines, source and the gut being chlorophyll the dietary in with enriched was fluorescence y a n lsaetat rmrt e hoohl-ihdiets chlorophyll-rich fed rats from extracts plasma and Fat to localized was fluorescence red the whether determine To bn ais fti ru fcmonswr ossetwith consistent were compounds of group this of ratios) -band i.5,soigardsitdSrtbn,abu hfe Q shifted blue a band, Soret in shifted shown red is a metabolite showing metalated 5C, presumably Fig. a such of spectrum n Soret-to-Q a and eaoie smaue yfursec spectroscopy fluorescence the there by whether incorporated determine To worms measured S3A). Fig. P-a, material as (supplementary with al., et their incubation Lagido metabolite, 2008; to al., Upon et proportional Lagido is 2009; 2001). al., that et luminescence (Lagido used pools a we ATP harvesting emit system, by luciferin model ATP our with in As decline luciferase-expressing synthesis. 1999; this ATP firefly al., offset for would et could worm energy (Braeckman the it light that ATP if hypothesized cellular longer We live in 2002). al., drop et a Braeckman is As there organism. complex age, a in production ATP used stimulated we Next, in synthesis ATP Light-driven n eua 99,rsetvl,i h rat. (Halloran the D in and respectively, 2006) al., 1979), et DeLuca, (Tovar K reported and vitamins that soluble measured than fat higher of the two-times amount for and The five- was diet. absent metabolite was chlorophyll-poor total peak a 665-nm fed The animals diet. nm in chlorophyll-rich 0.05 665 a of fed at concentration rats chlorophyll-a-derived plasma two 52,000 of a estimated of typical we is coefficient pheophytins, which extinction 1987), an (Lichtenthaler, Using nm. recorded 665 mice were to unique 2013) not and were rats. uptake al., and metabolites that chlorophyll fed et suggesting of S2G), swine distribution (Mihai Fig. of material diets extracts (supplementary fat rich from pheophytin-a. as chlorophyll chromatograms such P-a, HPLC esterified Similar an or group, acid carboxylic (polarity In hexanes (pheophytin-a). index with partitioned standard index compounds of that chlorophyll-a these to times addition, retention demetalated similar had minutes the 46 and 40 between eqatfe oa lo imnsfo asta bobdat absorbed that rats from pigments blood total quantified We 5 5 .) hslte hrceitci ossetwt ako a of lack a (polarity with consistent acetonitrile is and characteristic latter hexanes This 5.8). with mixed when 0.1) ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal .elegans C. y a enasge oamtltdporphyrin. metalated a C to Spectrum assigned chlorophyll. been of has metabolites of those with r h aiso h oe ad rud40n,t the to nm, 400 around band, center Soret the Q the in of Numbers ratios nm. the in are maxima peak above are Numbers respectively). peaks (b–d, of A spectra in peaks Absorption labeled (B–D) below. shown absorption are corresponding spectra For the peaks. letters, minor with several peaks with peaks along major observed Five are shown. are ring, chlorin metabolites a its possessing and chlorophyll fluorescence, of 675-nm characteristic displayed that chromatogram, compounds the acetone only In the HPLC. and to acetone subjected with chlorophyll- concentrate extracted a fed was rat diet a rich of from grams tissue 2.5 adipose extract. abdominal adipose an of tissue. chromatogram adipose in chlorophyll present of are metabolites Light-absorbing 5. Fig. bn ai of ratio -band y ada rud65n.Alsetaaeconsistent are spectra All nm. 655 around at band .elegans C. .elegans C. oeaut h fet flight- of effects the evaluate to , .Tecmonseluting compounds The 1. hc pnincubation upon which , A HPLC (A) .elegans C. y m -band Min 391

Journal of Cell Science uiecnesga ftewrskp ntedr rmta of that the from dark subtracted the in we whether kept animals, worms determine the light-exposed To of signal groups 6A,B). in luminescence Fig. both increased in in ATP the (summarized luminescence luciferin in worms other measured the We containing of kept periodically zero. and light and plates time 660-nm dark at to group 96-well luminescence one two exposed worm plated into then we measured light, We worms to substrate. response of in stores groups ATP in changes were ARTICLE RESEARCH 392 (A) P-a. ATP. with generate incubated to not light worms from capture results to show worms lines gray enables concentrations; treatment P-a 6. Fig. methods. additional was by It quantified was off. ATP ATP was Thus the actual of pools. or light kinetics expression, the the luciferase reflected reaction, time signal luciferase–luciferin persistent the this did time whether during which constant intensity unclear at increase luminescence off, further turned remained the not was However, light signal the ceased. intensity after luminescence measurement hour elevated 1 luminescence for The in persisted kept experiment). (i.e. were the worms the throughout when dark levels ATP on to the metabolite effect exposed The no when increase. had no ATP alone showed in worms P-a control increase given whereas were significant light, that statistically Worms 6C). a (Fig. had light to exposed worms the otosa h aelgtepsr.()Tesm xeieta ecie nD u sn 0dyodworms. perfor 10-day-old was using experiment but Each D, shown. in times described the as for experiment light same to The exposed (E) worms exposure. of light group shown. same are different the deviations a at standard represents and controls point averages 12; time of Each sets luciferin. triplicate t (D) of of and B. Da each addition in zero for shown. the worms time shown times to are after at relative deviations the began standard A at exposure and in measured Means light worms pate. was for P-a; 96-well increase Luminescence a ATP of zero. Percentage wells (C) time 12 dark. at in plated light worms to (B) of runs. sets exposed separate separate and 12 luciferin of experiments with triplicate incubated represent were worms Luciferase-expressing light. nvivo In eltm T eesi om eti h ak h aeeprmn si ntesm 6wl lt,bttewrswr eti the in kept were worms the but plate, 96-well same the in A in as experiment same The dark. the in kept worms in levels ATP real-time , nvivo in T eeswr eemnda h ie hw nec ru fwrsb esrn omluminescence worm measuring by worms of group each in shown times the at determined were levels ATP P vle fStudent’s of -values lc ie hwrslsfo om nuae ihPaa h indicated the at P-a with incubated worms from results show lines Black nvivo In nvivo In nraei T hn5dyodad1-a-l dl worms light. additional adult to 10-day-old exposed and and metabolite 5-day-old the fed when group an were found a ATP We to 6D,E). in substrate (Fig. luciferin increase luminescence the measuring adding and by worms minutes zero, of for 45 time intervals of at 15-minute total and at determined a and substrate, was exposure, luciferase-expressing status luciferin light before ATP the immediately plated light. without to plate them we 96-well exposed a synthesis, into worms ATP stimulated i.SD.Ti bevto scnitn iha nraei ATP in increase an with material consistent (supplementary is worms observation This untreated S3D). as on in Fig. effect light, rates no to had respiration exposed light the However, when consumption. respiration oxygen in by were measured increase 13% methods a exhibited Both the HPLC in S3B,C). or ATP the assay Fig. with quantified luciferase consistent material firefly and the ATP (supplementary either their using extracted homogenate worms, the eltm T oioig ruso om eeicbtdwt rwithout or with incubated were worms of Groups monitoring. ATP real-time , eltm T eesi -a-l om eetakddrn xoueto exposure during tracked were worms 1-day-old in levels ATP real-time , efrhrcnimdthe confirmed further We light whether determining of means alternative an As nadto oa nraei T,wrstetdwt P-a with treated worms ATP, in increase an to addition In t tssaeas hw,rpeetn h infcnecmae ihthe with compared significance the representing shown, also are -tests ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal xvivo ex nvivo in ehd.Atrlgttetet elysed we treatment, light After methods. T measurements. ATP nvivo in nraei T sn two using ATP in increase ethree he e in med ta

Journal of Cell Science rae om xiie,o vrg,lwrlvl fROS. of levels w lower average, difference on exhibited, the worms and treated although treated using fact, measured in as In exposure, equivalent light the of were hous 2 with 5 (ROS) during worms species accordance untreated oxygen th in reactive Despite phosphorylation, data. mitochondrial oxidative through ARTICLE RESEARCH n one tec iepitfrec flask. each for of withdrawn point total were time A population, each microscope. total at light the counted a of and of 1–2% aid representing the worms, give with 60–100 to determined plate movement, 96-well their worms/ml. a of 500 in at placed culture and liquid withdrawn , in were grown aliquots were counting, Worms For analysis. span A. life for used for values the confidence of 95% plots f are span exposed parentheses Life in those (B) Numbers versus (CI). P-a. light intervals with to treated exposed not and but light P-a to with treated worms of increase spans light and P-a 7. Fig. 7A,B). (Fig. light to group the exposed the but than of P-a, span with life with 95% treated median treated logistic longer group not of 17% was two-parameter The a survival that had a 1998). light fit al., and to P-a et accurately data count (Vanfleteren each to the population at fitted known alive fraction and function, the time plotted To interval. we verses the of daily half-life, deaths midpoint the the and a at intervals identical obtain occurred 3-day under have in to to kept 2- assumed were at light were otherwise made but were of red given Counts light, not 5 conditions. to to were day exposed worms Control or worms at cycle. P-a light:dark Beginning the worms hours hours. Adult hours:19 exposed 24 1979). 5 al., for we liquid Mitchell et P-a and Mitchell adulthood, in with al. 1980; incubated taken et al., Gandhi et were were of (Gandhi method al. measurements the et span to could according Life P-a cultures by absorption life. energy photonic prolong whether tested span next life We extend to harvesting Light ts.Eprmnswr u ntilct.TeL otwsue stm zero time as used was molt L4 The triplicate. in run were Experiments -test. 9 0wrsprwl;tewrswr crdda raieo h basis the on alive or dead scored were worms the well; per worms 10 ,7 9 dclrfursi ictt splmnaymtra i.S3E). Fig. material (supplementary diacetate -dichlorofluorescin .elegans C. nraei T,telvl of levels the ATP, in increase e sntsaitclysignificant, statistically not as einlf span. life median P vlei rman from is -value A einlife Median (A) oa aeo lcrntransfer: electron of rate total cytochrome reduce to electrons ebaesae eeaigatasmmrn oeta sdto ATP-synthase. used enzyme potential inner the trans-membrane the drive flow, a into electron generating matrix this mitochondrial space, of the membrane result from pumped a are As protons oxygen. molecular to electrons cytochrome cytochrome to electrons etaserdb eaoieo hoohl oCoQ to chlorophyll of these metabolite with a would by mitochondria electrons transferred mechanism, in proposed be light the ATP In by then metabolites. driven dietary P-a, be as would such by synthesis metabolite, catalyzed chlorophyll be if the photoactivated could that ubiquinone hypothesized a is We mitochondrial photochemically 1963). of photosynthesis al., a reduction et the by during (Witt can plastoquinone, a chlorophyll step quinone, molecules excited the primary al., chlorophyll-type of et a reduction Okayama that 2002; Indeed, known al., 1967). et is (Chesnokov ATP quinones it photoreduce influence could as metabolites levels, chlorophyll which in pathway rae om eeaie(7 lv;20da)atr1 days, 15 above. consistent after experiments dead), 163 dead) cross-sectional alive; the 200 (111 worms with P-a- alive; control the the (175 of the of 41% 47% compared alive versus Result: and were days. day worms 15 per plate, after hours treated 96-well alive 5 a and for dead into light percentage red worms to non-P-a-treated them P-a and exposed with P-a- adult treated old not animals of S4B,C). decrease Fig. gradual worms that material a (supplementary to of approaching lead light lifespan however, batches of threshold, with in amount certain different the a span Increasing past with life P-a S4B–E). reproducible Fig. median material was increased (supplementary were in worms P-a The median the better when P-a. and in reversed was with survive decrease light by treated This on nematodes brought 1965). span that life (Thomas, darkness reports complete in S4B), with alone Fig. material treatment life (supplementary accordance on 10% Light by effect S4B). span no life had Fig. decreased light, material of (supplementary absence the span in alone, treatment P-a ri,hatadln ooeae;()hmgnzddc a;and fat; duck homogenized mouse (3) (2) homogenates; mitochondria; lens and mouse heart isolated brain, (1) of: incubation Upon Q coenzyme of Photoreduction DISCUSSION lcrn r sdt euemtcodilCoQ mitochondrial reduce to These respectively. used succinate, are and electrons NADH from II) electrons (complex reductase extract succinate and mitochondrial I) (complex ATP, reductase synthesize NADH To phenomenon. the demonstrate These this perhaps concentrations. of tissues generality animal ATP of increased variety a to in observations able was exposure light live (4) oetal c seeto oos hogotmammalian all Throughout could donors. acid, electron ferrous ascorbic as compounds, and act sulfhydryl Many NADPH milieu. potentially dienols, NADH, mitochondrial as the compounds, such in present molecules, oxidant chemical V h iohnra bqio ol n ordc ubiquinone, the reduce reasoned CoQ We to synthesis. of ATP maintain reduction and increased to in considered result pool, be should which ubiquinol can II mitochondrial and I the complexes of roles major (Kro bqio terdcdfr fCoQ of form reduced (the ubiquinol red h po qain fKro of equation’ ‘pool The eas xmndlf pnlniuial.W lcd6-day- placed We longitudinally. span life examined also We gradKigneg 93.Bsdo hseuto,the equation, this on Based 1973). Klingenberg, and ¨ger suiunn euto and reduction ubiquinone is .elegans C. ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal c xds cmlxI) hc liaeydntsthe donates ultimately which IV), (complex oxidase iharpeettv eaoieo chlorophyll, of metabolite representative a with , 10 ol eaptnilse nterespiratory the in step potential a be could c euts cmlxII,wihue the uses which III), (complex reductase gradKignegdsrbsthe describes Klingenberg and ¨ger c V hc htlsteeetosto electrons the shuttles which , obs V 5 10 ox V .Uiunlsute the shuttles Ubiquinol ). ox suiunloxidation ubiquinol is V red /( V ox 10 + ,resultingin V red 10 ,where ), rma from , 393

Journal of Cell Science hc ih aedvlpdt aeavnaeo hsphotonic this of implications. advantage far-reaching pathways, take have may of to energy, developed identification al., have Thus, 1988). et might 2000). al., Barun which al., 1931; et et Reed, Zourabian excite (Chance would and 2007; that calf cm (Bachem light 25 of metabolites the wavelengths chlorophyll penetrate in of bathed can is muscle tissue nm Adipose 800 and an and tissue penetrate al., 630 et through or Wan can between 2001; Photons Sun al., nm et 600–700 1981). (Bearden 1964). attenuation of magnitude three-to-five al., of only range orders with et wall the abdominal Vanbrunt 4-cm-thick over approximately 1970; Daigle, normal light al., and under room et Massopust brain 1956; Menaker of entire Harman, wavelength 1961; the and a reach (Berry at readily illumination light can book of light printed 50% animals, skull a only the of attenuate read bone , scalp temporal comfortably the your the humans, illuminating to In and light 1997). you of al., et amount allow (Benaron the day would clear brain a On day. clear CoQ rnpr usrts uha ltmt n aae(i.1A; hypothesis. (Fig. mechanistic this malate confirm evidence to and further needed However, glutamate is S1A–C). electron Fig. as added material of P-a such supplementary absence mitochondria the and/or substrates, isolated in ATP light in transport in II, with increase and an consistent observed I we Also complexes of 1). upstream (Table acting P-a and light hsi stmtn oseuaeta aml oss conserved energy. possess photonic mammals harness that to speculate light-to-energy to mechanisms of tempting 1976). is form (Krasnovsky, is it photosynthesis primitive Thus into Photosensitized molecules a evolved body. that chlorophyll-type be conversion the to excited hypothesized in from widely present tissue been transfer every have electron sunlight almost from inside light deep red of photons evolution, ARTICLE RESEARCH itr-eie ln imns itei nw bu the about known is 394 Little pigments. by absorption is plant capture energy dietary-derived photonic for pathway potential A animals in chlorophyll Dietary ( tissues wavelengths and animal-derived intensity in of < energetics light in with P-a, initiated changes of poorly presence the observed yet, In 2010). as we al., by, et cellular (Hashmi production stimulate mechanisms to energy defined thought and/or (Hashmi is light conditions metabolism red of energy to variety investigated Exposure a 2010). been treat al., has to et and intervention Passarella 2005), clinical to al., 2010; a reported et as al., Wong-Riley been et 1984; has al., nm (Hashmi et 700 processes and biological 600 modulate between light red Intense light of effect The the be with line In could the binding. chlorophyll CoQ protein quinone, the through from organizing ubiquinol oxidized and by derivative or transfer, the disproportionation by of Back-electron to inhibited metabolite content ubiquinone. one and photoreduced or ubiquinol of ubiquinone sequence of above molecule one the molecule total Ubisemiquinone give repeating to by 1994). disproportionation ubiquinol the by Albracht, to reduced and of be can Jong which 2–3% (De ubisemiquinone, mitochondria yield transfer for place charge would take the accounts protonation from could Escape and transfer solution. complex in Electron or possible. proteins through is state singlet ubiquinone this of quenching by Oxidative (*1). state singlet excited 7 m(ihe ta. 97 a ta. 91.I small In 1981). al., et Wan 1977; al., et (Eichler nm 670 0.8 htectto fclrpyladdrvtvspoue the produces derivatives and chlorophyll of Photoexcitation 10 10 6 . W/m 0.2 a eue hnioae iohnrawr xoe to exposed were mitochondria isolated when reduced was htrdcinhptei,w bevdmitochondrial observed we hypothesis, photoreduction 2 htcnb found be can that ) nvivo in nvivo in hnotor na on outdoors when < 7 mat nm 670 n htpyia rprisadtu a ar u similar out carry demonstrate data can Our 1970). structure thus al., a in et and (Gradyushko fed similar properties photochemistry are swine is photophysical molecules to and plants and remain Chlorin-type in metabolites rats found elucidated. the of of ones be structures to plasma the similar However, and are significant. that fat diet several the chlorophyll-rich of in to identification derivative in re-metalated The metal actively chemistry. metabolites a coordination was of in pigment presence part the the take that true, suggests If tissue 5). fat bonded (Fig. metabolites metal chlorophyll fat a of rat to presence from the pigments with Dolphin, extracted consistent from and is data from Ma absorbance magnesium 2007; of Our Blakeslee, loss 1999). and about (Ferruzzi environment bring chlorophyll acidic to Fernandes thought the The is 2003). 2000; stomach Flaoyen, al., the and of et Scheie 2007; (Egner al., demetalated et been have animals might energy, phenomenon. photonic general molecules, ambient a absorbing chlorophyll-derived chlorophylls be of of capable plant that are diets suggests in which Data the rich S2G). we from Fig. diet material Here, sequestration mice, supplementary a 5; in tissues. administered 4, molecules (Figs swine human chlorin-type and in of rats accumulation metabolites the chlorin-type observed chlorophyll its dietary or of pharmacodynamics and pharmacokinetics etrudrtnigo hi hraoyaisand pharmacodynamics their homeostasis, of chlorophyll energy If needed. in understanding be be involved will P-a. pharmacokinetics may better be of to response a binding found threshold are mitochondrial mitochondria metabolites the by with that the P-a regulated with of suggesting consistent mode also fragments, was binding levels response after ATP cooperative on/off P-a, ATP, observed This added in fall. much increase Increasing to too a began P-a. same with fitted However, to the exposure. better response elicited light P-a graded P-a of a of presence than concentrations the rather in on/off, light S2A,B). binary Fig. by material stimulation (supplementary linear ATP not was by P-a added P-a cells. of from animal most in formed as place be take metabolites, normally can other that chlorophyll reactions into of P-a distributed an Indeed, metabolites transformed to P-a. known be been chlorin lead the have may for can demonstrated absorption could as chlorophyll photon ATP in where increase of body the metabolites throughout dietary that ih f60n aeeghta eertstehmnbody, human the penetrates that wavelength nm yields 670 of Light span life and stores ATP vdnefrsc raiain vns,t prahtert of observed rate the our approach to so, Indeed, is Even photon fragments organization. catch. such mitochondrial of for with photon evidence area binding cooperative thus, cross-sectional organisms, positively effective and, photosynthetic of anticipate the in absorption might Organization systems increase similar one antenna would energy. structures, chlorophyll such, supramolecular additional to As into of metabolites second. chlorophyll only per amounts absorb to photons 2000), negligible expected al., 1970; few et be 1931; al., Zourabian would a et 1981; 1997; Reed, metabolite al., Menaker al., et chlorophyll 1977; and et Wan al., each 1964; Benaron et (Bachem al., 2001; Eichler et al., nm Vanbrunt 1988; 670 et al., Bearden 2003) et at 2007; Flaoyen, Chance al., and flux (Egner Scheie et photon 2007; Barun body al., the the et Fernandes and in 2000; derivatives al., et chlorophyll of concentrations odt,terpre hoohl eaoie sltdfrom isolated metabolites chlorophyll reported the date, To hr eainhpbtenteices nAPadteamount the and ATP in increase the between relationship There , 3ka/o (1.18 kcal/mol 43 ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal 6 10 2 22 clpoo) ie estimated Given kcal/photon).

Journal of Cell Science ute eerhwl enee odsigihbtenteabove the between distinguish mechanisms. to needed possible be will research Further T ytei oee yND rFADH or NADH by powered synthesis ATP ARTICLE RESEARCH ta. 03 n h osmto fgenvegetables green Veer in of outcomes van’t health 2007; overall consumption better Blakeslee, with and correlated the are Ferruzzi 2000) and al., 1992; Levandovski et 2013b; al., al., John 2013) et et 2013; Kent (Block al., 2013a; Lambert, al., and et et Kent (Dhar 2004; exposure al., et sun increased Both Conclusion h agrqeto s o uho nices nAPi enough is ATP difference? in biological increase an a of make much perhaps to how increase, which green. the is: by of question amount mechanism larger in animals measured the the the or increase of biology turn increased Regardless fundamental is an ATP span). in to life changes measure in and added we (extention absorption light systems, be upon model ATP to in have Nevertheless, would pigment ogtr xdtv aae hc sas ehns hthas that increase mechanism to a also shown have is against been protecting which might by damage, P-a method oxidative alternative long-term ubiquinol, an lipid by producing span potent life by of increased offset a Indeed, photoreduction be the is Q. might from generated increase coenzyme ubiquinol ubiquinol, ROS in As any increase an 1990) ROS. by the al., in et to from (Frei increase due here antioxidant data an ROS and with show in 2013) data al., increase not published et an our (Qu the of plasma However, extension blood oxygen. result in life-span singlet a resulting of Thus be generation ROS. oxygen, might of a here to metabolites oxygen, seen singlet absorption, energy of photon transfer generation Upon can 2007). chlorophyll al., in et is span Schulz (ROS) life species increase oxygen reactive to in thought increase an example, For when out. span life increase animal. to adult 2007) an that al., to an interventions given et few observed (Petrascheck a known only we This that are are note why there We 2002). ATP. restriction, in for caloric increase al., an besides to account response et in span might life Braeckman in increase timing 1999; in al., difference et day to compared and (Braeckman 50% ATP genetic as of much zero to level as by the begin ATP drop adulthood, through reportedly can of increase consumption stores 4 oxygen day to ATP by hatching when example, able For time decline. were a from we at adulthood here, during studies contrast, synthesis most By However, engineering. 2010). ATP al., entire et worm’s decrease Raamsdonk the (Van across span expenditure early achievedlife energy be slow may total a observations span decreasing life of Other increased by that coupling 2003). hypothesis the al., a to et be led have (Lee might longevity there and that metabolism and (Dillin 2002) development during al., status et metabolic the by determined be that suggesting of span studies life elegans the are increases synthesis ATP there and metabolism may that limiting span life given in contradictory, increase The seem enhancement. vision in al., role also modulation a et ATP played that broad Washington suspect a we regulate processes, 2006; biological can of ATP al., range Because 2007). et vision al., (Isayama et enhance Washington model 2004; to mouse chlorophyll capture a that energy using photonic observed enabled previously metabolites We processes. fundamental biological affect light significantly these can of metabolites presence dietary the in absorbing light that suggesting span, life median naias ramn ihPaadlgtbt nrae T and ATP increased both light and P-a with treatment animals, In lentv ehnsso iesa xeso antb ruled be cannot extension life-span of mechanisms Alternative thsbe rpsdta h iesa fti ommight worm this of span life the that proposed been has It . .elegans C. .elegans C. iesa Ihie l,2004). al., et (Ishii span life Hilre l,2010; al., et (Heidler 2 ufcetP-a sufficient , .elegans C. do C. oss eaoi ahast eieeeg ietyfrom directly energy conclusions. these derive confirm should also to studies animals Additional pathways in sunlight. organisms, span photosynthetic metabolic life and possess plants of up to in extension resulting the similarly 17% biology in fundamental (3) a and alters to P-a; light by P-a, absorbed of wavelengths presence of light to exposure cetfc okod L nbfe 020Mmnio,00 M 0.02 mannitol, KH Fisher M M (0.250 (Thermo 0.003 A assay KCl, buffer protein M in 0.01 (Bradford) IL) HEPES, Plus Rockford, Coomassie Scientific, the by r eusee noaia ise;()i h rsneo the of isolated presence in in and the ATP homogenates in tissue in increase mitochondria, an animal (2) The is there tissues; source? P-a, metabolite animal chlorophyll energy into abundant sequestered to molecules are them this chlorophyll-type light-sensitive enable of (1) that that: demonstration advantage animals, pathways greater metabolic most the have from take of Throughout energy animals photonic Do planet. in organs bathed sun. this been internal have on is humans, the including source Sunlight evolution, energy incomplete. mammalian abundant be work most might Our commonly vegetables. the explanations green are these and from benefits exposure suggests sunlight antioxidants These from D of aging. vitamin consumption in of increase an diseases to attributed of variety a muto nietsnih htsoetruhlbrtr windows laboratory through shone that sunlight minimum ( indirect a preparing using of by off, exposure turned amount light lights minimized laboratory prior we with experiments, light, samples/experiments all to For samples UT. exposing organs Logan, to several Scientific, March that Frontier clear level from a the to obtained on than measured 30–60 less exposed is we was and that are City used light York red light New of in red level afternoon of the than intensity less The times NE). Lincoln, Biosciences, itr 50 3,60 9 mwt ulwdhhl aiu FH)of interference (FWHM) maximum pass half full-width band with and nm halogen 690 transformer W 670, variable 632, 300 [500, a a filters either with experiments, all equipped for lamp used were sources light Two procedures General METHODS AND MATERIALS 5m ia)admlt 25m ia)a esrdwt an with measured Canada]. as ON, final) Kingston, Inc., mM of (2.5 concentration Systems a final a Qubit malate with at Mitochondria from preparations and electrode only glutamate oxygen mouse final) used using III/II, isolated and [state mM 4.0 (5 We above 2007) existing ratio weeks. control al., to respiratory according minimum 2 et centrifugation of (Frezza differential procedures minimum by a mitochondria Harlan for liver by supplied IN) diet rodent purified (Indianapolis, chlorophyll-poor, mitochondria a liver fed mouse were Mice isolated in monitoring ATP Continuous density power Luminous bulb. light LED nm, 660 0.8 W, to set 1.70 was a or nm] 10 .0 GA gm at cdpo S,p .)were 7.4) pH BSA, 0 acid–poor at fatty minutes 30 for mg/ml 1 P-a with EGTA, incubated M 0.001 xoe olgtad()wr nuae ihu - n o exposed not and P-a without incubated were (3) light. and to light to exposed ihde aebe ecie rvosy(ia ta. 2013). al., et chlorophyll- (Mihai a previously fed described Swine, been used. River, have were diet Charles g Teklad, rich Harlan 300 (ICR, 344, weighing (Fisher Mice rats IN), and Indianapolis, g University. 22–28 weighing Columbia MA) Wilmington, of Committee Use ia ocnrto)adte 250 then and concentration) final lcdi iewlso 6wl lt o xouet ih troom at light to exposure for 20 plate 96-well times, a various of At wells temperature. nine in placed auatrrsisrcin.Cnrl eetetdi h aeway, 0 the determined same at to the were incubated according in (1) levels CA) treated were: ATP Carlsbad, were they except (Invitrogen, and Controls kit instructions. X-100), manufacturer’s commercial Triton a 1% with and EDTA 150 to . nmlpoooswr prvdb h ntttoa nmlCr and Care Animal Institutional the by approved were protocols Animal .0 W/m 0.001 m yi ufr(0m rs H75 0 MNC;1mM 1 NaCl; mM 100 7.5; pH Tris, mM (10 buffer lysis l ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal 6 2 ). . W/m 0.2 nvivo in 2 yohohriea(-,9%prt)was purity) 95% (P-a, Pyropheophorbide-a . smaue yaL-5Algtmtr(LI-COR meter light LI-250A a by measured as m m lqoso hssseso were suspension this of aliquots l lqoswr ihrw,added withdrawn, were aliquots l ˚ ihu - son,()not (2) (shown), P-a without C < 2 PO ˚ gpoenm sdetermined as protein/ml mg 1 .APwsadd(. mM (0.5 added was ADP C. .elegans C. 4 .05MMgAc M 0.0015 , .elegans C. ugssthat, suggests upon , 2 ? H 395 2 O,

Journal of Cell Science notesetoee.Teedo h ie pi al a positioned power light was experiments, cable these optic to Prior fiber mixture. the reaction bulb the of light above fiber LED end nm cm a The used 660 1 a we spectrometer. from exposure, light the light direct For into and capture ml. to 1 guide spectrometer Japan) of light fluorescence optic volume Kyoto, a as reaction Scientific, Measurements final inside Horiba a 2005). A placed with Yvon, cuvette al., Jobin buffer ml et HORIBA 3 (Feldkamp (Fluorormax-4, in a in al. monitored made et were Feldkamp was by potential described membrane Mitochondrial measurement potential Membrane ARTICLE RESEARCH 396 relatively contain they because CoQ mitochondria of heart amounts sheep large used status redox We Q coenzyme of Analysis tris(hydroxymethyl)aminomethane isolated M were Mitochondria 0.1 ice. 250 sucrose, by in surrounded stored was and M which 0.25 7.5, laboratory pH the of at to to (Tris) transported bath were due hearts a the complications hour and animal in 1 the potential within of started death minimize isolation the Mitochondrial of import. to P-a of chain, rates variable respiratory access direct the P-a using allow heart(s) to to sheep fragments mitochondrial 1 described used and previously We 2 1’. from as ‘Procedure occasions separate prepared two on were 1967) hearts (Smith, sheep from Mitochondria ultrasensitivity zero-order of Analysis of concentration a were ON, at Reactions Kingston, mitochondria instructions. Inc., manufacturer’s oxygen with Systems the an run to Qubit using according ml; measured Canada) (OX1LP-1 was cuvette consumption electrode oxygen cable. Mitochondrial optic fiber measurement the consumption Oxygen of end the from cm 1 measured was CoQ ni s.Tetae iohnraehbtdarsiaoycnrlrtoof ratio control respiratory a , exhibited mitochondria thawed The use. until ih500 with xdto faypoue bqio.P a de (25 added was Pa ubiquinol. produced any of oxidation cytochrome tc rtedntrdsseso a sda bv npaeof place give in to above adjusted as were used reactions All to was solution. triglycerides, suspension stock denatured chain mitochondrial the the medium 50 Q, or reactions coenzyme and these stock 10% sucrose For was containing A. A starch, buffer solution buffer food Q in modified Switzerland), coenzyme solution products, water-soluble Nutritinal stock (DSM a Q ALL-QTM adding coenzyme by prepared a mitochondria, added faie(ouk n otzk 1966). Wojtczak, concentrations and increasing (Bogucka by azide azide inhibited during of Sodium progressively consumption is water. oxygen respiration in 3 IV): solution state (complex stock oxidase a cytochrome from inhibits M 0.005 plastic of the concentration at directed was LED 660-nm chamber. a methacrylate)] exposure, [poly-(methyl light For A. buffer 0 Mteaoe 0m EE–O H77 0m C,1mM 1 KCl, mM at 10 BSA 0.1% 7.7, and pH EDTA HEPES–KOH mM mM 1 EGTA, 10 trehalose, mM 300 eto c ni s.Frratos 50 reactions, 500 For to use. added until ice on kept .2 aaei rsbfe tp .Frmtcodildenaturing, mitochondrial For 7. pH at buffer a Tris For in 200 Q. malate coenzyme in added M (4) 0.125 with 10 and added the but we mitochondria; above in mitochondria control denatured (2) positive added the heat light; of with of repeated absence (3) absence the light we P-a; the added in LED controls, of (1) absence two negative following: minutes The the 10 between For changing for and sequence DMSO. tube temperature. samples argon the room the with in irradiated at placing purged We solution described). mixtures by (previously tube, bulbs initiated stock test reactions a mg/ml to 1.3 the added was a suspension from concentration) 25 lcdi aha 70 at bath a in placed ,idctn iohnra fragmentation. mitochondrial indicating 1, o niiino eprto,sdu zd a de tafinal a at added was azide sodium respiration, of inhibition For m m 10 /lsokslto nehnl niyi id oteQ the to binds A Antimycin ethanol. in solution stock g/ml fsokmtcodilsseso a ugdwt ro and argon with purged was suspension mitochondrial stock of l eo ais rznmtcodi eetae t37 at thawed were mitochondria frozen ratios, redox m ufrAt raeamtcodilsokslto,wihwas which solution, stock mitochondrial a create to A buffer l c m euts cmlxII,teeyihbtn h upstream the inhibiting thereby III), (complex reductase fbfe ,cnann 0.5 containing A, buffer of l m lqosa ocnrto of concentration a at aliquots l 10 ˚ o iue.Frcnrlratoswithout reactions control For minutes. 5 for C hc xeie nlss o vlainof evaluation For analysis. expedited which , m fasokslto f02 glutamate/ M 0.25 of solution stock a of l m ftewtrslbeCezm Q Coenzyme water-soluble the of l 2 m fti tc upninwas suspension stock this of l 80 ˚ Ymgcie l,2007) al., et (Yamaguchi C m /latmcnAfo a from A antimycin g/ml , < 0m fpoenm in protein/ml of mg 60 gpoenm in protein/m’ mg 1 ˚ n diluted and C m final M i ieof site este 0mntsatrdahb eaiainadhmgnzdtefat the homogenized and decapitation by 4 death at after minutes 30 then less lp fwihi eotda h eaieAPsnhssrate. synthesis the ATP line, production relative a the ATP to as fitted lysis. reported was is which after time, which minutes this of and 50 during slope triplicate increase and in linear a 5 run showed were between Reactions obtained samples. data liver for above described (80 homogenate opouehmgntso os ri,tefotllb was lobe frontal (Sartorius homogenizer the S Potter brain, a 4 at mouse of Germany) Goettingen, strokes AG, of two using homogenates homogenized homogenates produce brain mouse in To synthesis ATP of Analysis ooeie 2 go ri o1m ufrC .5m sucrose, Dounce mM a 0.15 C: with buffer homogenized ml 1 was to MgCl mouse mM brain 5 a EDTA, of mM 0.5 of mg (20 brain homogenizer wavelength entire light of The effect the of Analysis ( duck a from fat visceral adipose removed duck in We concentrations ATP of Analysis (KONTES homogenized were homogenates mice heart and from lens mouse Lenses in synthesis ATP of Analysis lqo a ae rmterato itr n a de o200 to added was and mixture reaction the measured from taken as was mixture aliquot reaction the in Q coenzyme HPLC. of by amount same the w rus n ru a eti h ak hl h te a exposed was W/m other 0.8 the at while nm dark, (671 the light in red kept to was group one groups: two (70 P-a added then We 60M 4640 ufr 10 buffer. NY). instructions Island, manufacture’s Grand the luciferase- Technologies, to a according (Life using kit determined quantification were ATP concentrations based ATP minutes. 20 for eto 40- a took We lqoswr ae rmec ihadAPwsmaue using measured and was (Ally literature ATP the in and described as dish HPLC, 1992). by Park, each or assay from luciferase taken the were aliquots itr a xoe ordlgt(7 ma . W/m 0.8 at nm (671 light red sucrose, to exposed mM was (0.15 mixture 1 phosphate, buffer and sodium 50 mM) mM assay at (1 7.5 HEPES) chloride, ATP magnesium mM in mM 2 5 pestle) EDTA, mM glass 0.5 with grinder %btnli ehnla . lmnt.TeHL ounwas column HPLC The ml/minute. 0.6 at acid, methanol acetic been glacial in 50 have an 3% used acetate butanol we conditions sodium Briefly, 1% 5% 2011). and HPLC of al., r.p.m. et consisting was HPLC. Qu elutent 15,000 2009; isocratic by al., at solution et minutes analyzed (Qu previously 2 The reported phase for antioxidant. organic centrifuged minute, the an 1 for as vortexed 100 hydroxytoluene/ml and acid butylated perchloric M 0.4 (800 T sa ufrwr de no40 into added were buffer assay ATP .W de 10 added We C. yteroln bopinsetauigetnto ofiinsof coefficients extinction using was spectra ubiquinone absorption for concentrations M nm online ubiquinone 14,200 275 and and their ubiquinol ubiquinol relative by for determined nm We 290 used. at set detector a xoe ordlgtadAPwsdtrie sdsrbdabove described as determined kit. was ATP ATP based and luciferase light a red using to exposed was apeo c o or ete de 10 added then We hour. 100- 1 Five for stock). ice on sample 6wl lt n xoe olgtfr4 iue.Te,20- Then, minutes. 40 for light to exposed and plate 96-well a n T eeswr eemnda bv sn luciferase-based a using above as determined were kit. 200- ATP levels with lysed ATP were and mixture the of 6 oqatf eaieuiunn n bqio ocnrtos 50 a concentrations, ubiquinol and ubiquinone relative quantify To er ise(0m)wshmgnzda bv n1m T assay ATP ml 1 in above as homogenized was mg) (20 tissue Heart ˚ . m -8 . ,10A 100 u, 2.6 C-18, mm, 2.1 wtotbfe)i os-itn otrEvhe homogenizer. Potter-Elvehjem loose-fitting a in buffer) (without C m fa1 gm tc ouin.Tehmgnt a iie into divided was homogenate The solution). stock mg/ml 10 a of l 2 1 2 ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal m cm 1 fPa( M n 10 and mM) (1 P-a of l cm –1 m m lqo ftehmgnt n de tt 940 to it added and homogenate the of aliquot l 2 fAPsok(0m)t 100 to mM) (10 stock ADP of l t20n nehnlfruiunl(etre l,1959). al., et (Lester ubiquinol for ethanol in nm 290 at m m m 1 otoso h upninwr de oec elof well each to added were suspension the of portions l - fo Msoki MO n lcdthe placed and DMSO) in stock mM 1 a (from P-a l )wsaddt ufrA(920 A buffer to added was l) t25n nehnlfruiunn and ubiquinone for ethanol in nm 275 at m ufrprln.W de 1 added We lens. per buffer l m fa33m/lsokslto)adADP and solution) stock mg/ml 3.3 a of l 2 . 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Journal of Cell Science ramn ih1 aC/. aHslto Emn ta. 1979) with al., culture et liquid (50 (Emmons a by solution to population NaOH transferred the M and from NaOCl/0.5 isolated yolk 1% were egg with eggs on treatment Worm population 1995). mixed (Krause, a plates expanded we populations, synchronous (Omni homogenizer hand-held a ( with Homogenizer homogenized Micro was fat abdominal diets of respective devoid 2013). al., diet et purified (Mihai has previously a diet chlorophyll-rich described was swine been The diet Teklad). (Harlan control chlorophylls dietary The chlorophyll-a. weight oayeaoao n esseddtesml n500 in sample a the with re-suspended ethanol the and evaporated evaporator and rotary separated centrifugation, by material 5 ywih prln afo upeetpoue from produced supplement to food equivalent is [a which spirulina 1983)], contained weight (Ciferri, IN) Indianapolis, cyanobacteria by Teklad, (Harlan 15% diet extracts chlorophyll-rich tissue in The fluorescence red of Analysis ARTICLE RESEARCH wnyfu or eoeteeprmn,tecluewssltinto split was culture worms/ml. the 10,000 of experiment, density the a at before culture hours liquid Twenty-four in grown were Worms A fluorescence, Method such no exhibited P-a given uptake. worms. not metabolite the confirming were in whereas that fluorescence, spectra worms chlorophyll-derived control fluorescence signature had the the we worms determined uptake, in Treated to worms and P-a the it confirm suspended medium To P-a, adding containing fresh hours. medium by 24 culture the of metabolite away minimum washed a chlorophyll for P-a medium culture the administered We in monitoring ATP Real-time 1988). (NGM) (Wood, medium using growth previously nematode (Nunc) 2001). on described al., agar maintained et were been Lagido animals has 2009; Briefly, al., husbandry Sciences, et Molecular Medical (Lagido Nematode of of UK) Institute (Department Aberdeen, Aberdeen Lagido of Foresterhill, Cristina University Dr Biology, from Cell and gift a were Worms General Maestro a with imaged were Animals vivo In ethanol, absolute of ml 10 added to then chlorophyll- We a sample oil. given clear the been were a had cooled give that to samples swine diet, or rich The rats from above, evaporator. directly. described for measured rotary vortexed and chloroform was a the ml 3 sample and with in centrifugation the resuspended by evaporated and precipitated was added acetone material was Insoluble minute. ml) 1 (40 acetone grade kne ordc nefrnefo knatfursne(Bouchard were autofluoresence animals skin the al.; from 2007). et interference al., Bouchard et reduce by to described skinned as MA), Hopkinton, h aesb etiuain ermvdteehlaeaelayer, acetate ethyl the 300 in removed film resulting the We separated re-suspended and and centrifugation. acetate minute NaCl ethyl 1 saturated the by for of evaporated sample ml the layers 1 vortexed to acetate, plasma the ethyl of ml ml 10 4 and added we plasma, For ethanol. faslt tao.Tesmlswr hnue o PCadUV a A and and 100 detector HPLC (PDA) u, a array 2.6 for photodiode pump, C18, 600 2998 used a a with detector, then system fluorescent were HPLC 2475 MA) samples (Milford, Waters The A spectroscopy. ethanol. absolute of vr1 iue.A 5mnts h lwwsicesdt . ml/minute. 0.5 50:50 to increased a A:B was 100:0, flow with to the ml/minute linearly minutes, changed 0.3 35 was At of which minutes. minutes, 15 rate 5 over flow for A:B a of a at mixture used B). eluted (solvent acid We 0.1% were formic to 0.1% and recorded. containing Compounds set water alcohol and was isopropyl emission A) (solvent 10% and 700 acid containing formic nm acetonitrile and 410 of 275 to phase between mobile set Absorbance was Excitation nm. HPLC. 675 for used was o loecnesetocp,fv isec eegvnthese given were each pigs five spectroscopy, fluorescence For o PCadU pcrsoy eetatd25gaso a,as fat, of grams 2.5 extracted we spectroscopy, UV and HPLC For m /l n mhtrcnB(0.1 B amphotericin and g/ml) imaging .elegans C. dlibitum ad ˚ ,150 .coli E. maintenance m 6 ) miItrainl ensw A,HPLC GA), Kennesaw, International, Omni H), 2 .0m oun(hnmnx orne CA) Torrance, (Phenomenex, column mm 2.10 20 o ek.Woebano – rm of grams 2–7 or brain Whole weeks. 2 for ˚ tanO5 safo ore oobtain To source. food a as OP50 strain o 0mnts eltdteinsoluble the pelleted minutes, 30 for C .elegans C. m TM /l opeemedium). complete g/ml; .coli E. nVv mgn ytm(CRi, System Imaging In-Vivo tanO5,carbenicillin OP50, strain m fabsolute of l , .5 by 0.15% m l xoe oLDlgtcnee t60n t1 at were nm group 660 experimental at centered each light from LED worms to the exposed of half measurements, .5 rtnX10adDlcfrn(100 D-luciferin and 2009; DMSO, 1% al., 6.5, X-100 pH by et at Triton The buffer (Lagido stores reader. phosphate plate 0.05% literature citric ATP a a in the was recorded assay was buffer to luminescence luminescence To and according 2001) group wells. al., well, et experimental Lagido 12 each Each of to plate. added minimum 96-well a a 100 of luminescence, into well plated a was into plated were n - eermvdb he eetdwse ihM n h worms 50 the buffer. and M9 M9 with worms/ml washes 500 repeated to three resuspended by removed were P-a and lesug D ormv odaduasre - n eupne at Scientific, resuspended and (IPM 50 P-a buffer unabsorbed worms/ml. given and M9 3000 were food with remove worms to washed Control MD) groups. were Eldersburg, treated Worms the solution vehicle. stock to DMSO P-a added a were of amounts DMSO varying in and groups treatment and control otiigwrstetdwt ayn ocnrtoso - n control and 100 P-a zero, of each time concentrations made, At were varying worms. plates with 96-well treated into identical worms divided Four plate. group containing 96-well experimental a each of with wells above, 12 as plated were Worms B Method n ramnsgop.Tetetetgopwsicbtdfr2 hours 24 for incubated was control a group into 12 treatment at divided with The then culture groups. medium, liquid complete treatments in and in worms/ml synchronized 500 al., of et were (Schulz density al. worms et Schulz Three-day-old by described 2007). as quantified was formation in ROS formation ROS of Analysis were 1 at at suspension nm measured M9 660 this was in respiration of and resuspended aliquots chamber 25 and respiration One-ml P-a the with into worms/ml. excess times a transferred and 10,000 three at bacteria at washed remove culture were buffer to Animals liquid buffer medium. in M9 complete worms 1977; in hours adult Dusenbery, 24 One-day-old and of 2007). (Anderson density al., described et as Zarse electrode Inc.), oxygen Systems Clark-type a (Qubit using measured was consumption Oxygen of Analysis P-a. without vehicle 50,000 for DMSO at 100 in buffer P-a incubated M9 was in with group re-suspended control incubated and The worms/ml. buffer were M9 with culture washed hours, liquid 24 in worms adult One-day-old vitro every 100 In performed of were addition the assays by ATP minutes and 45 light for to minutes 15 exposed were plates and o nuae ihP-a. with incubated not oln ae o 5mntst yetewrs(ra-azand 1992). Park, (Artal-Sanz and HPCL (Ally by the protocols worms or using established instructions measured to was manufacturer’s according the lysate the the to in according ATP lyse and assay rpm luciferase to centrifugation 15,000 at by minutes cleared minutes 5 was for solution 15 resulting in The them 2009). for we placed Tavernarakis, and ATP, nitrogen water measure liquid To the boiling nitrogen. from liquid tubes centrifuge into tubes the six placed minutes, removed were 30 group and 15 each the at and Then, from nitrogen light. liquid red in to placed exposed were tubes group remaining each from tubes six zero, time luminescence. of recording the and buffer he ie ihdfeetppltoso worms. the of repeated and populations was experiment different dark The with light light. the times red dim three of in under outside performed kept procedures were experimental worms exposure All light. of to ATP group exposed signal experimental ATP worms foil. luminescence an of aluminium within the amount difference with the between The plate as periodically. the reported was covering recorded synthesized by was dark signal) the (luminescence in kept was ˚ o 0mntswiebigepsdt nLDlgtcnee at centered light LED an to exposed being while minutes 10 for C m nvivo in fec omsseso a lcdit 8cnrfg ue.At tubes. centrifuge 18 into placed was suspension worm each of l m T oioigin monitoring ATP - n h oto ru nDS eil.Bceilfood Bacterial vehicle. DMSO in group control the and P-a M ora fCl cec 21)17 8–9 doi:10.1242/jcs.134262 388–399 127, (2014) Science Cell of Journal , 6 .elegans C. T a sae slmnsec.Termiigthree remaining The luminescence. as assayed was ATP 000wrsm eeicbtdwt - (25 P-a with incubated were worms/ml 10,000 W/m 2 m flmnsec ufrcnann -uiei was D-luciferin containing buffer luminescence of l m 2 h oto ru a rae ntesm a but way same the in treated was group control The . fwr upninfo aho hs groups these of each from suspension worm of l xgnconsumption oxygen m flmnsec ufrwsaddt plate a to added was buffer luminescence of l .elegans C. .elegans C. m m 6 ftesseso from suspension the of l ) fe nta ATP initial After M). W/m 2 m fluminescence of l 2 h te half other the ; m )for M) 397

Journal of Cell Science E ih n loecnewsr-esrda . n or under hours red 5 to and previously. exposed 2.5 used then at those were re-measured to was plates equivalent emission fluorescence The conditions and nm. and 523 light excitation reader and LED at microplate 502 CA) M5 of Sunnyvale, SpectraMax wavelengths LLC, a Devices, in measured (Molecular was fluorescence the h olwn a dy5o dlho) om eeepsdt LED to exposed 1 were at worms nm worms. 500 adulthood), 660 5000 was of at of flasks 5 centered total reaction light (day a the all day therefore in given ml, following worms 10 was The of contained group flask density each control final into split worms/ml; The The then were three. DMSO. cultures or control in and two treated solution The alone. stock vehicle DMSO a from a 2 of in addition control prepared and after experimental Immediately were each for group. these measured were replicates animals; Five parallel. without diacetate dichlorofluorescin 50 ot tdy4o dlho,tecluewssltit oto and control into split was culture 12 the with treated larval adulthood, was fourth group of the experimental during The 4 groups. isolation, experimental egg day after At hours 35 molt. at added was final) 50 of concentration final from 2 prepared was mM buffer 100 M9 in a solution MO) Louis, St. (Sigma-Aldrich, ihu 2 without walls opaque with 100 plate 96-well A a bottom. of transparent wells the a to and added was group each ARTICLE RESEARCH 398 and Naval Science of Nanoscale Office the Navy, I.W.]; the to of N00014-08-1-0150 Department number the [grant by Research supported was work This Funding manuscript. the wrote and study the I.W. supervised studies. and distribution and designed metabolite-binding mitochondria conducted in D.M. measurements homogenates. and ATP tissue mitochondria conducted in J.Z. measurements ATP homogenates. and tissue worms, with studies conducted C.X. contributions Author interests. competing no declare authors The interests 15. Competing day on once done was at alive) commenced treatment (fraction light Scoring and 5. 4 day day at added was P-a Petrascheck, that and except (Solis literature 2011), the plates in described as microtiter measured was 96-well span Life in measurements span Life in grown al., progeny and et any prevent Mitchell harvested To temperature. 1980; room were 5-fluoro-2 al., at developing, culture Eggs et of liquid (Gandhi a modifications. method in al. darkness some the et to with Mitchell and according 1979) al. performed et were Gandhi measurements span Life studies Population analysis span Life eewtdanadpae na9-elpaet give aliquots to counting, plate 96-well For a experiment. in the placed of and withdrawn end were the until day every repeated oto esrmnswr odce ttesm ieudridentical accurately under ( time dropped fairly as same P-a of population the such concentration at The environment, the conditions. conducted span, life in were influence of can measurements changes food, control of 95% amount Because the and 1998). density of is worm temperature, model al., survival Prism two-parameter et The fit GraphPad (Vanfleteren CA). software to Jolla, a La the known to Inc., data using the Software, fitted we (GraphPad function and half-life, time the verses obtain logistic count To analysis. each two-parameter added span at used life alive We was fraction for counted. the zero not FUDR plotted time were as the and molt FUDR before L4 of the produced presence the eggs in Any small interval. from remained the hatched of midpoint that and the intervals at 2–3-day larvae occurred at have made to were assumed movement, Counts worms were flask. deaths 60–100 their each of for at of counted point total and basis time withdrawn A each were microscope. the population) total light on the a of alive 1–2% of (representing aid or the dead with scored determined were worms the pnsuisadi a o dutd(upeetr aeilFg S4F). Fig. material (supplementary adjusted not was it and studies span m fti ouinwr ietdit h upnin,rsligi a in resulting suspensions, the into pipetted were solution this of l 9 ,7 9 9 dclrfursi ictt n el otiig2 containing wells and diacetate -dichlorofluorescin ,7 9 dclrfursi ictt tc ouini DMSO. in solution stock diacetate -dichlorofluorescin 9 doyrdn FD)(im-lrc,120 (Sigma-Aldrich, (FUDR) -deoxyuridine m 6 .Adtoa otosicue worms included controls Additional M. 2W/m m M2 2 9 o or.Lgtepsr was exposure Light hours. 5 for 9 ,7 ,7 9 9 dclrfursi diacetate, -dichlorofluorescin dclrfursi diacetate -dichlorofluorescin , 5)truhu h life the throughout 75%) , 0wrsprwell; per worms 10 m MP- 9 m ,7 M 9 - mos .W,Kas .R n is,D. Hirsh, and R. M. Klass, W., S. H. Emmons, Lenz, and J. Knof, J., and A. Eichler, P. Hintz, E., M. Rogers, P., E. Snyder, H., K. Stansbury, A., P. Egner, lc,G,Ptesn .adSbr A. Subar, and B. Patterson, G., Block, J. P. Harman, and M. C. Berry, ier,O. Ciferri, P. M. Shurygina, and K. V. Cherkasov, A., G. Abukumov, and A., R. S. Greenfeld, Chesnokov, M., Fountain, K., McCully, J., Kent, S., Nioka, B., Chance, L. Wojtczak, and K. Bogucka, edap . rbe,A n eneg .M. J. Weinberg, and A. Kribben, T., Feldkamp, Fraser, H., Hsin, J., Lehrer-Graiwer, N., Arantes-Oliveira, L., A. Hsu, A., Dillin, W. G. Lambert, and K. P. A. Dhar, S. Albracht, and M. A. Jong, De L. F. Crane, eao,D . hog .F n tvno,D K. D. Stevenson, L. and C. W.-F. Lowery, Cheong, and A., P. V. D. Zharov, Benaron, D., J. Wilson, S. D., V. E. Ulashchik, Bearden, and V. A. Volotovskaya, P., A. Ivanov, V., V. Barun, G. Park, and A. Ally, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.134262/-/DC1 online available material Supplementary material Supplementary reka,B . otof,K,D ree .adVnltrn .R. J. Vanfleteren, and A. Vreese, De K., Houthoofd, P., B. Braeckman, R. J. Vanfleteren, and A. Vreese, De K., Houthoofd, R. P., Levenson, B. Braeckman, J., Mansfield, J., P. Dwyer, A., S. 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Journal of Cell Science eae,M,Rbrs . lit,J n newo,H. Underwood, and J. Elliott, R., Roberts, J. M., Menaker, H. Daigle, and Jr C., L. Massopust, ia,D . in,H,Bae,W . oao,A n ahntn I. Washington, and A. Romanov, S., W. Blaner, H., Jiang, M., D. Mihai, Whitaker, M., G. Paszkiewicz, A., D. Bellnier, J., Morgan, J., I. MacDonald, a .F n opi,D. Dolphin, and F. L. Ma, ihetae,H K. Hidalgo, H. and S. Lichtenthaler, C. Gama, A., Carissimi, B., Pfaffenseller, L. R., F. Levandovski, Crane, and C. Widmer, Y., Hatefi, L., G. R. Ruvkun, and Lester, J. Ahringer, S., R. Kamath, G., A. Fraser, Y., R. Lee, S., S. Lee, aio . cagn . lt,A,Ptit .adGoe,L A. L. Glover, and J. Pettitt, A., Flett, D., McLaggan, C., Lagido, A. L. Glover, and A. Flett, J., Pettitt, C., Lagido, odee,A n ohad .E,Jr E., D. Koshland, and A. Goldbeter, R. D. Sanadi, and W. J. Stiles, H., D. Mitchell, J., Santelli, S., Gandhi, L. Scorrano, and S. Cipolat, C., Frezza, N. B. Ames, and C. M. 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