Redox Biology 2016: Redox enzymes and Redox physiology

Redox Biology 2016: Redox enzymes and Redox physiology


TODAY WE HAVE LISA RIDNOUR SHE GOT HER Ph.D. UNIVERSITY OF IOWA.>>THAT’S RIGHT.>>THEN YOU WENT TO ARIZONA.>>THEN WASH U. THEN SHE CAME TO NCI, NOW IN THE CANCER INVESTIGATION PROGRAM SO SHE WORKS UP IN FREDERICK. SHE DARED 270 AND GOT HER EARLY. PLEASED TO HAVE LISA TO ARRIVE IN MECHANISM DISEASE PROGRESSION.>>ALL RIGHT, FOLKS. TODAY, I’M GOING TO TALK ABOUT TWO DIFFERENT DISEASES, ACTUALLY. I’LL TALK MOSTLY ABOUT CANCER BUT I’M GOING TO TALK ALSO A LITTLE BIT ABOUT ALZHEIMER’S DISEASE WHERE WE KNOW THAT IN DISEASE PROGRESSION. TO START OUT TALKING ABOUT BREAST CANCER, BREAST CANCER REMAINS SECOND-LEADING CAUSE OF DEATH AMONG WOMEN WITH MALIGNANCIES. DIAGNOSIS IS GENERALLY DETERMINED BY THESE CLASSICAL INDICATORS — LET’S SEE. — THESE ON THE TOP OF THE SLIDE, BUT WE THINK THAT WE CAN IMPROVE DISEASE MANAGEMENT AND SURVIVAL BY IDENTIFYING NEW BIOMARKERS THAT COMPETE IN MANAGING THE DISEASE AND IMPROVING PATIENT SURVIVALS. AND SO, SINCE WE’RE GOING TO TALK ABOUT NOs, THE PAST 10-15 YEARS WE SAY THE LAB HAS SPENT A LOT OF TIME EXAMINING SIMILAR EFFECTS OF NIGH TRUST OXIDE AND CHEMICALS KNOWN FOR NIGH NITROOXIDE. AS CONCENTRATIONS BEGIN TO RISE, WE SEE STABILIZATION AND PHOSPHORYLATION OF KINASE AS WELL AS AKT. IN HIGHER LEVELS, WE SEE PEOPLE SEE PHOSPHORYLATION AND EVEN HIGHER LEVELS AROUND ONE STUDY SAVING NOTE THAT IS CONSISTENT WITH NITRO-STATED LEVELS. SO THESE INTERMEDIATE AND HIGHER LEVELS CAN BE PRODUCED BY THE INDUCIBLE MOUTH SEW PLAN MOUTH TWO. WE KNOW THAT THE COMPONENTS OF THE TUMOR MICRO AND BIOME, WE KNOW THE TUMOR MICRO ENVIRONMENT IS COMPLEX AND CONSISTS OF CANCER CELLS, IMMUNE CELLS AND FIE SEW BLASTS AS WELL AS EXTRA CELLAR MATRIX. ALL F OF THESE WITHIN THE ENVIRONMENT COMMUNICATES WITH EACH OTHER TO PROMOTE TUMOR PROGRESSION. REDOX BIOLOGY DERIVED AND COMMUNELATING FACTOR IN THESE COMMUNICATION PROCESSES. WHILE OVER THE YEARS WE’VE DETERMINED QUITE A LOT INTERCIG WILLING AND CANCER CELLS, LESS IS KNOWN ABOUT INTERSIGNALING IN EXTRA CELLARER MATRIX. WHEN I FIRST JOINED THIS GROUP, THAT WAS ONE OF THE FIRST QUESTIONS WE WERE INTERESTED IN ADDRESSING. SO BECAME INVOLVED WITH DAVE ROBERTS HERE AT NCI AND WE BEGAN A COLLABORATION LOOKING AT CROSS TALK SIGNALING EFFECTS BETWEEN NITROOXIDE AND SOUND RESPONDING ONE WHICH WAS LARGE EXTRA CELLAR MATRIX PROTEIN AND IT’S GENERAL HLY THOUGHT TO BE ANTIANT KNOW GENIC WHERE IN CONTRACT WE KNOW NO IS COANGIOGENIC. WHAT THIS PROJECT DETERMINED — ALSO WE USED FUNCTIONAL ASSAYS INCLUDING UX PROLIFERATION AND VASCULAR GROWTH MUS KAL CUE LAR ASSAY TO LOOK AT NEGATIVE EFFECTS. WHAT WE FOUND WAS THAT AT LOW FLEX NO WE SEE INCREASED PROLIFERATION WHICH ABOUT ONE MICROMOLAR AND IN THE PROCESS OF INDUCING THAT THROUGH THAT PROLIFERATION, WE SEE NO-INCLUSED SUPPRESSION OF FONDING ONE AT VERY LOW LEVELS OF NITROOXIDE. SIMILARLY IN THE X PLANT ASSAY, WE SEE THAT IN THE CONTROL TISSUE, UM, YOU SEE A SMALL AMOUNT OF VASCULAR CELLS MIGRATING OUT AND AWAY FROM THE PERIMETER OF THE TISSUE, BUT IF YOU ADD INHIBITOR L-NAME YOU BLUNT THE OUTGROWTH OF THESE VASCULAR CELLS. IN CONTRAST, IF WE ADD A LITTLE BIT OF TRY TRO OXIDE TO — NITROOXIDE TO THESE TISSUES WE SEE ABUNDANT OUTGROWTH OF VASCULAR CELLS. THIS IS VERY CONSISTENT IN THIS INCLUSED RESPONSE ASSAY. — INDUCED RESPONSE ASSAY. THIS GOES ON TO SHOW THAT ALL THESE PROCESSES CAN BE INHIBITED BY THE ADDITION OF ENDOGENOUS RESPONDING ONE. SO IN SUMMARY, THIS PROJECT WENT ON, IT BECAME A FRUITFUL COLLABORATION AND WENT ON TO GENERATE A NUMBER OF VERY NICE PAPERS FOR DAVE ROBERTS’ GROUP. ANOTHER GROUP OF EXTRA CELLAR MATRIX PROTEINS THAT THEY WERE INTERESTED IN EXAMING FOR MATRIX AND POWER PROTEIN. THESE ARE SE KROOED CRETED PROTEASES, THEY CAN PROCESS COMPONENTS OF EXTRA CELLAR MATRIX AND THE REASON WE WERE INTERESTED IN THEM IS BECAUSE THE A ACID SITE CONTAINED VIOLATES WHICH TARGETED BY NITROGEN SPECIES. SO WE FIRST STARTED EXAMINING REGULATION AND ENDOGENOUS INHIBITORS [INDISCERNIBLE] IN MACROPHAGE CELLS THAT WE EXPOSED LOW DOSES OF NO STERNO. WE SEE INCREASE IN ACTIVITY AND WE ALSO SEE SUPPRESSION OF SECRETION AT THESE LOWER LEVELS. SO WE TOOK THE CONDITIONED MEDIA FROM THESE CELLS WHERE WE SAW PEAK ACTIVATION OF NPs AND WENT ON AND PUT THIS MEDIA ON TISSUE EXPLANT A ASSAY AND WHAT WE FOUND WAS THAT THE MEDIA FROM CELLS THAT WERE TREATED WITH MICROMOLARS SHOWED OUTGROWTH OF VASCULAR CELLS AWAY FROM THE PERIMETER OF THE THE TISSUE BED. THEN WHEN WE WENT ON TO KNOCK DOWN MNP 9 WE SAW THIS PROCESS WAS DEPENDENT UPON MNP 9 ACTIVITY. THIS WAS DEMONSTRATED THAT THE WAS IN PART BY DEPENDENT AND INVOLVED ONE SECRETION OF TIP ONE AND INCREAMNP 9 ACTIVITY. WE WANTED TO GO ON AND LOOK AT THIS REGULATION PROCESS AND FIGURE OUT RELEVANT MODELS. SO WE PROCEEDED TO EXAMINE THESE E EFFECTS IN CANCER AND ALL RECIPES. BEFORE WE TALKED ABOUT WHAT WE FOUND IN CANCER I’LL TELL YOU WHAT WE FOUND IN ALZHEIMER’S MODELS. THIS MODEL WAS DEVELOPED AT DUKE UNIVERSITY BY A COLLABORATOR OF OURS, AND SHE MADE THIS MOUTH THAT OVEREXPRESSES ATP PROTEIN WHICH IS THE TRANS MEMBRANE PROTEIN AND WHEN IT’S CLIPPED IT CAN MAKE A BETA. OKAY. SO THESE MICE, SHE THEN CROSSED THESE MICE WITH THREE KNOCKOUT ANIMALS. SHE HAD MICE THAT HAD FUNCTIONAL MOUSE TWO AS WELL AS MICE THAT LACKED MOUSE TWO IN ALL CELL TYPES THAT MAKE IT, OKAY. SO WHAT SHE FOUND WAS THAT THE MICE THAT LACKED FUNCTIONAL MOUSE TWO HAD A LOT OF INSOLUBLE PLAQUE. WHERE AS, MICE THAT CONTAINED FUNCTIONAL MOUSE TWO DID NOT, AND CERTAINLY WHEN SHE LOOKED AT THE STATE OF THE NEURONS IN THESE ANIMALS, MICE THAT HAD MOUSE TWO DID NOT EXHIBIT NEURONS BUT THE MICE THAT LACKED MOUSE TWO DID HAVE QUITE A LOT OF DEAD NEURONS WHEN THEY COMPARED TO A CONTROL. SO ONE THING THAT WE KNOW FROM THE LITERATURE IS THAT A BETA IS A SUB STRAIGHT FOR MNP 9 AND THIS WAS DEMONSTRATED BY A GROUP AT WASHINGTON UNIVERSITY AND THEY SHOWED THAT ACTIVE MNP 9 COULD CLIP A BETA AT THESE SPECIFIC SITES IN THIS SEQUENCE, SO WE BEGAN TO EXAMINE WHETHER OR NOT NO REGULATION OF MNP 9 AND ONE MAY BE ATTRIBUTED TO THIS DIFFERENCE IN GENOTYPES OF MOUSE. SO THE FIRST THING WE DID WAS WE LOOKED AT MNP 9 AND TENT ONE EXPRESSION IN THESE MICE AND WE COULD SEE THAT IN THE ANIMALS THAT LACKED FUNCTIONAL MOUSE TWO, THEY HAD LOWER LEVELS OF MNP 9 EXTREGS, AND THEY ALSO HAVE HIGHER LEVELS OF TENT ONE EXPRESSION. WE EXAMINED MNP 9 AND TENT TWO LEVELS IN THESE MICE AND THEY DID NOT APPEAR TO HAVE ANY DIFFERENCE IN THEIR EXPRESSION LEVELS. SO THEN WE WENT ON AND LOOKED AT SIGNOGRAPHY OF THESE MICE, WE USED GELATIN SIGNOGRAPHY ASSAY. THIS WOULD MEASURE MNB 9 AND MNP 2. THE ANIMALS THAT HAVE FUNCTIONAL HAVE HIGHER MNP ACTIVITY IN THESE THE TISSUES. THIS ACTIVITY SEEMS TO BE RESTRICTED TO MNP 9 BECAUSE WE DON’T SEE — REALLY, WE SEE MINIMAL LEVELS OF MNP 2 IN THESE ANIMALS. SO UNFORTUNATELY, THIS PART OF THE ASSAYS WASN’T ABLE TO GIVE US A DIFFERENCE IN ACTIVITY LEVELS, SO WE WENT ON AND DEVELOPED AN ANTIBODY-BASED MRN A ASSAY TO EXAM THE ACTIVITY IN THESE TISSUES. AND SO WE MADE AN ANTIBODY THAT RECOGNIZED A FRAGMENT OF MNP 9 CLIPS INSOLUBLE A BETA. MANY ENZYMES CAN BE GREAT SOLUBLE BETA, BUT MNP 9 IS THE ONLY ENZYME THAT CAN LOOK THE ENSOLUBLE FORM. IT CAN CLIP IT AT THESE POINTS IN THE SEQUENCE AND WE MADE AN ANTEAGAIN FOR THE ONES 16 FRAGMENT. THIS ANTIBODY WAS EXTENSIVELY CHARACTERIZED USING MASS SPECK. WE IMMUNO PRECIPITATED MATERIAL FROM MNP 9 DESTRAITED INSOL BETA AS WELL AS THE WHOLE GRAIN HA KNOWLEDGE NATE OF THE MICE THAT CONTAINED NOS TWO AND WE ALSO USED A BETA ONE TO 16 THAT WE PURCHASED. WHAT YOU CAN SEE IS THAT THESE MRNs ARE ALL THE SAME AND THAT TELLS US THAT THIS PROGRESSMENT IS ABUNDANT IN THESE ANIMALS AND WE CAN MEASURE IT. AND SO WE DID THAT IN THE BRAINS OF THE MICE WHICH IS SHOWN ON THIS TOP PANEL HERE, PANEL A, AND WHAT THIS SHOWS US IS THAT MICE THAT CONTAIN FUNCTIONAL NOS TWO HAVE A LITTLE BIT MORE THAN TWO-FOLD INCREASE IN THE AWAY TA 16 FRAGMENT AND WE USE THAT AS AN INDEX OF MNP 9 ACTIVITY AND THESE DATA SUGGEST THAT THESE ANIMALSES DO HAVE HIGHER MNP 9 ACTIVITY, WHEREAS IN THE ABSENCE OF NOS TWO, IT’S REDUCED. SO WE WENT AHEAD AND LOOKED AT HUMAN ALZHEIMER’S BRAIN AND HUMAN CONTROL BRAIN AND WE SEE THE SAME TWIN IN THESE TISSUES AS WELL. SO TOGETHER THESE DATA TELL US THAT PERHAPS NITRIC OXIDE IS PROTECTING AGAINST DISEASE PROGRESSION OF A ALZHEIMER’S, IN PART, BY INCREASED MNP 9 ACTIVITY AND LOWERING TENT ONE WHICH FACILITATED THE DEGRADATION AND CLEARANCE OF ANDROID FAT TO NON-TOXIC PEPTIDE FRAGMENTS. NOW WE’LL TALK ABOUT CANCER, AND SO ON THIS SLIDE ANYTHING THAT IS HIGH LIGHTED IN GREEN ARE CANCER TYPES THAT HAVE PUBLISHED THE ABILITY OF NOS TWO TO PREDICT PROGNOSIS. AMONG THEM ARE BREAST CANCER, WHICH YOU CAN SEE ON THE RIGHT SIDE OF THE SLIDE, AND SO WE WENT ON TO EXAMINE NOS TWO AS A PREDICTOR OF BREAST CANCER SURVIVAL. WE DID A COHORT STUDY WITH NCI FREDERICK AND WHAT THEY SHOWEDED IN THIS STUDY WAS THAT NOS TWO DID, IN FACT, PREDICT SURVIVAL IN PATIENTS THAT HAD BR 2 BREAST CANCER WHICH WAS A AGGRESSIVE FORM OF BREAST CANCER. IT DID NOT PREDICT SURVIVAL IN THE LESS. INTERESTINGLY, AMONG THE PATIENTS THAT BECOMES TO DISEASE, HIGH NOS TWO EXPRESSING GROUP, 92% OF THOSE PATIENTS EXPRESSED ELEVATED TUMOR NOS TWO. SO ANOTHER IMPORTANT THING THAT I WANTED TO SHOW FROM THIS WORK IS THAT NOS TWO EXPRESSION DID CORRELATE POSITIVELY WITH BRAVE THREE. IT ALSO CORRELATED POSITIVELY WITH 253 MUTATION AS WELL AS CD 31 WHICH IS AN INDEX OF VASCULATURE. SO IN THIS STUDY, THEY USED LASER CAPTURE OF HIGH NOS TWO AND LOW NOS TWO TUMOR TISSUES AND DID A GINA RAY ANALYSIS AND FOUND SIGNATURE OF ABOUT 44 GENES THAT WERE UP REGULATED IN TUMORS THAT HAD HIGH NOS TWO. WE WERE INTERESTED IN A SET OF THESE PROTEINS INCLUDING IL 8 WHICH IS BASIL-LIKE MARKER AND INVOLVED IN PROLIFERATION, ANGIOGENESIS AND CELL MIGRATION. IN ADDITION, THE PEAK HEARING IS ALSO LIKE MARKER 108 A IS COAL RECEPTOR AGONIST INVOLVED IN INFLAMMATORY PROCESSES AND CD 44 IS A STEM CELL MARKER IN THESE WERE ALL ELEVATED. INTERESTINGLY, I THINK PROTEIN [INDISCERNIBLE], THIS H IS VERY INTERESTING TO ME BECAUSE THIS TELLS ME BASICALLY CATALYZED RECYCLING OF SIT LIEN WHICH IS ALSO A BY-PRODUCT OF THE NOS-MEDIATED OXIZATION OF L LARGENEN. — — KEEPING LEVELS HIGH ENOUGH IN THE TUMORER TO GENERATE NO. THAT WAS VERY INTERESTING. ONE THING YOU DON’T SEE ARE MNP 9 AND TEMPS ON THIS TABLE, BUT THERE ARE SOME OTHER CONNECTIONS HERE BECAUSE MNP 9 HAS BEEN SHOWN TO PROCESS IL 8 TO INCREASE ITS BIOACTIVITY AND ALSO CD 44 CAN SERVE AS A DOCKING PORT FOR MNP 9 SO IT ARE RESIGNED TO IT AND IT’S BEEN IDENTIFIED AS LEADING EDGE OF MIGRATING CELLS AND IT’S BEEN SHOWN IN SOME STUDIES TO BE ASSOCIATED WITH INCREASED METASTASES. OKAY. UM, SO, OTHER GROUPS HAVE ALSO DEMONSTRATED THAT MNP 9 PREDICTS OVERALL SURVIVAL AND RELAPSE-FREE SURVIVAL. TEMP TENT ONE DOES AS WELL. TENT ONE IS GENERALLY THOUGHT OF AS AN INHIBITOR FOR MNP 9 BUT IT DOES HAVE OTHER MNP-INDEPENDENT FUNCTIONS. WE ASKED THE QUESTION OF WHETHER OR NOT NO MIGHT ELUCIDATE PROTUMOR EFFECTS BETWEEN MNP 9 AND TENT ONE. TO DO THIS, WE WENT BACK TO STE FAN’S COHORT AND WE STAINED FOR TEMP ONE EXPRESSION IN THESE TUMOR TISSUES AS WELL AS PD 63 WHICH IS A CELL SURFACE PROTEIN WHICH IT BINDS TO. WHAT YOU SEE IS THAT THESE TUMORS — THESE ARE JUST TWO EXAMPLES — WE SEE DIFFERENTIAL EXPRESSION IN THESE TUMORS. SOME HAD HIGH, SOME INTERMEDIATE, AND SOME HAD LOW OR NO LEVELS OF TENT ONE AND CD 63. WE EXAMINED THE SURVIVAL EFFECTS OF TENT ONE IN OUR MODEL AND SHOW THAT TENT ONE DOES PREDICT SURVIVAL IN OUR COHORTS, AND WHEN WE STRATIFIED FOR TIEING OFF TWO, THIS EFFECTS SEEMED TO BE RESTRICTED TO TUMORS THAT ALSO EXPRESSED KINASE TWO BECAUSE TEMP ONE DID NOT PREDICT SURVIVAL IN TUMORS THAT EXPRESS LOW NOS TWO. WE THOUGHT THAT THIS MIGHT BE TELLING US SURVIVAL EFFECTS OF HIGH NOS TWO AND HIGH TENT ONE MAY BE MECHANISTICALLY INTERRELATED. WHAT MIGHT A COMMON SIGNALING PATHWAY BE? AKT SIGNALING MAY BE COMMONLY SHARED AS A MECHANISM. SO AGAIN, WE KNOW FROM THE LITERATURE THAT TENT ONE COMBINES TO CD 63 TO INITIATE PROSURVIVAL SIGNALING THAT GOES TO PAP KINASE, AKT BAD PHOSPHORYLATION. WE ALSO KNOW FROM PREVIOUS STUDIES THAT FROM STEFAN’S LAB THAT NOS TWO CORRELATES WITH PHOSPHOR AKT AND THAT IS SHOWN BY THIS ODD RATIO OF 4.49. RATIO DESCRIBES STRENGTH AND SOESHUATION BETWEEN TWO MOLECULES AND THE ODDS OF THAT EVENT OCCURS IN ONE GROUP COMPARED TO ANOTHER. HUMANS THAT HAVE HIGH NOS TWO AND HIGH TAKT EXHIBIT AN ODDS RATIO OF THAT 4.5. WE THEN STRATIFIED FOR HIGH AND LOW TENT ONE AND ALSO EXPRESSED HIGH TENT ONE RATIO GOES FROM 4.5 TO NEARLY 13. IN CONTRAST, LOW TENT ONE EXPRESSED, ODDS RATIO GOES DOWN BY NEARLY HALF. SO WE THEN LOOKED AT TENT SNF TEMP] CORRELATION WITH NOS TWO AND FOUND THAT TEMP ONE ONLY CORRELATED WITH NOS TWO AND CR NEGATIVE PATIENTS. SO WE WENT FORWARD WITH OUR CULTURE EXPRERMENTS USING ER NEGATIVE BREAST CANCER CELLS AND WE TREATED THESE CELLS WITH AN INNER DONOR DETAINER AND WE FOUND THAT AT HIGH CONCENTRATIONS OF NO, WE SEE INCREASED AKT PHOSPHORYLATION AND BAD PHOSPHORYLATION, BUT WHEN WE KNOCKED OUT TEMP ONE, THESE PHOSPHORYLATION PATTERNS WERE DIMINISHED AND WE ALSO KNOW THAT ON THE BOTTOM OF THE FIGURE WHERE WE PRODDED NORMALIZED PHOSPHORYLATION OF AKT TO TOTAL AKT AND PHOSPHOR
TU[INDISCERNIBLE] AND THE CELLS THAT HAD HIGH TEMP ONE AND CELLS THAT DID NOT. WE WENT ON TO LOOK AT THE EXPRESSION, WHETHER OR NOT NO IS INDUCING EXPRESSION IN TEMP ONE OF THESE CELLS AND WE COULD NOT IDENTIFY SIGNIFICANT INCREASE AT THESE DONOR CONCENTRATIONS IN TEMP ONE EXPRESSION. P WE DO SEE SIGNIFICANT INCREASE IN PROTEIN SECRETION AT 100 MIE I KRO MOLAR, BUT THIS ISN’T — — THAT INDUCED PHOSPHORYLATION OF THE PROTEIN. WE BEGAN TO THINK THAT WE WERE LOOKING AT THIS WRONG BECAUSE THIS IS ACTUALLY MEASURING THE LEVELS OF TEMP ONE SECRETE INTO THE MEDIA BUT IF IT HAS TO FIND BIND TO THE CELLS AT CELL SURFACE PRO TAN, YOU KNOW, MAYBE WE’RE, YOU KNOW, NOT LOOKING AT THE RIGHT PLACE HERE. SO WE THEN LOOKED IN THE LITERATURE AND FOUND A PAPER — INTERESTING PAPER — WHERE THEY MADE A MU TRANT FORM OF TEMP ONE THAT WOULD NOT INHIBIT ACTIVE NNC 9 AND FOUND THIS MUTANT PROTEIN ACTUALLY BOUND TO A HIGHER DEGREE TO CD 63 THAN WILD-TYPE PROTEIN. IN THEIR SYSTEM, IT DID AFFECT THE PHENOTYPE OF THEIR SYSTEM. SO WE ASKED THE QUESTION OF WHETHER OR NOT NO CAN MODULATE TEMP ONE FUNCTION AND TUFRP ONE CD 63 COLOCAL LIE IIZATION. TO DO THIS WE DID A STUDY OF CONTROL AND NO-TREATED NDP 31 CELLS AND TEMP ONE IS IN GREEN AND CD 63 IS SHOWN IN RED. YOU CAN SEE THAT THE FLOAT THAT GOT NO, THERE’S A LOT MORE GREEN INDICATING THAT TEMP ONE IS STUCK TO THE SURFACE OF THESE CELLS AND THEN THERE DOES APPEAR TO BE SOME COLOCALIZATION WITH CD 63 AND TEMP ONE AS SHOWN BY THESE OWE LEISH AND ORANGISH REEDS ON THE BOTTOM PANELS HERE. WE KNOW THAT TEMP ONE CAN — WE KNOW THAT NO CAN CAUSE NIGH GRAGS EVENTS IN PROTEIN SO WE TREATED REKOFSH NANT TEMP ONE PROTEIN WITH THESE SAME CONCENTRATION OF NO DONOR AND THEN WE DRIED TO PROTEIN DOWN AND PUT ON MASS SPECK AND SEE THAT THERE IS, INDEED, A 45 MATCH SHIFT IN THE PROTEIN THAT WE EXPOSED TO NO. THIS IS CONSISTENT WITH NIGH TRAGS EVENTSES AND WE IDENTIFIED TWO NIGH TRAGS EVENTS ON TWO RESIDUES IN THIS PROTEIN. WE WENT BACK AND IMMUNOPRECIPITATED NC TWO 31 WITH TEMP ONE AND THEN BLOTTED WITH 3 MP AND WE SEE THERE’S AN INCREASE IN NITROTYROSINE IN THE CELLS TREATED WITH NO, AND THERE SEEMS TO ALSO BE AN INCREASE IN PULL-DOWN OF CD 63 IN THESE AS WELL. SIMILARLY, WE WENT BACK AND WE LOOKED AT DI 3 CHAIN SIGNALING IN THESE CELLS AND WE SEE THAT NO INDUCES INCREASE IN TO 3 K PHOSPHORYLATION AT 500 MICROMOLAR WHICH IS REDUCED WHEN WE KNOCKED OUT TEMP ONE. SO TOGETHER, THESE DATA TELL US THAT, UM, TEMP ONE PREDICTS POOR SURVIVAL IN PATIENTS WITH HIGH NOS TWO TUMOR EXPRESSION, THAT ELEVATED TEMP ONE AUGMENTS NOS TWO TAKP SOESHUATION IN THE BREAST TUMORS AND THAT IF WE SILENCED TEMP ONE WE CAN SUPPRESS NO-INDUCED ASIMILAR PA TICK SIGNALLING IN BREAST CANCER CELLS. THEN AN INTERESTING PAPER CAME OUT ABOUT SIX MONTHS BEFORE WE PUBLISHED OUR PAPER AND BASICALLY THIS WORK THEY ALSO SHOWED IDENTIFIED HIGH LEVELS OF TEMP ONE NITRATINGS. THEY EXPOSED TO LPS SO YOU SEE BIG HIGH LEVELS OF TEMP ONE NITRATION. THEY WENT ON AND SHOWED THIS NITRATED TEMP WAS NOT ABLE TO BIND TO MMP 9 AND YOU SEE HIGH LEVELS OF MNP 9 ACTIVITY IN THESE DIAGRAM. INTERESTINGLY, THEY USED THE MOUSE MODEL OR A MATH MODEL TO PREDICT WHAT TYROSINE LEVELS RESIDENCE DUZ WOULD BE AND THEY PREDICTED THE SAME CAIRO SCENE RESIDUE WITH THEIR MATH MODEL THAT WE SHOWED BY MASS SPECK TROM PI. GOING BACK TO OUR COHORT, WE ALSO LOOKED AT MNP 9 SURVIVAL. IT DOES PREDICT POOR SURVIVAL. IT PREDICTS THE HAZARD RATIO GOES UP A LITTLE BIT IN TUMORS THAT HAVE LOW TEMP ONE EXPRESSION. TEMP ONE — MNP 9 DOES NOT PREDICT SURVIVAL IN TUMORS THAT HAVE HIGH TEMP ONE EXPRESSION. SO WE KNOW THAT MNP 9 IN ADDITION TO PROCESSING IL 8 AND INCREASING BIOACTIVITY, IT CAN ALSO INCREASE AVAILABILITY AND BEEN ASSOCIATED WITH INCREASING GENESIS AND THEN AGAIN DOWN HERE WHERE I TOLD YOU THAT IT’S BEEN SHOWN THAT IT CAN FWIEN CD 44 AND PROMOTE METASTASES. PATIENTS IN HIGH MP 9 AND CD 44 EXHIBIT INCREASE HAZARD RATIO OF 2.49. THAT MAYBE, YOU KNOW, THERE MAY BE A SIMILAR EFFECT IN OUR BREAST CANCER COHORT AS WELL AND INVOLVEMENT OF NOS TWO. SO, CAN NO ELUCIDATE PROTUMOR EFFECTS IN MNP 9 AND TEMP ONE. WE THINK WHAT THIS DATA MAY BE TELLING US IS NITRATION MAY SHIFT TEMP ONE SIGNALING AWAY FROM MNP 9 INHIBITOR FUNCTIONS TO FAVOR CD 63 PROSURVIVAL CIG ISSALING WHILE MAINTAINING MNP 9 ACTIVITY TO PROMOTE OVER PROHUMOR EFFECTS LIKE CELL MIGRATION AND METASTASES. SO WE WENT ON AND LOOKED AT WHETHER OR NOT WE COULD TARGET NOS TWO IN A MOUSE MODEL. SO WE INJECTED MICE WITH ND 231 SPIRALS THAT HAVE A TAG AND THAT WAY WE CAN LOOK AT FLUORESCENCE OF TUMORS IN THE MOUSE. THEN WE TREATED MICE WITH WATER THAT HAD AMY KNOW I NOS INHIBITORS. GROWTH RATE OF THESE TUMORS IS SIGNIFICANTLY LESS THAN THE CONTROL MICE THAT DID NOT GET THE INHIBITORS. YOU CAN SEE FLUORESCENCE OF THOSE TUMORS AS WELL OVER HERE. BUT THE MOST STRIKING OBSERVATION FROM THIS STUDY WAS THAT THE CONTROL MICE HAD MORE FLUORESCENCE THAN THE MICE — THEIR BRAINS HAD MORE AND THEIR LUNGS — WE DON’T SHOW THAT ON THIS SLIDE HERE BUT WE DID LOOK AT LUNGS — MICE THAT GOT TREATED THE IMMY KNOW GAUN KEEN HAD FAR LESS FLUORESCENCE IN THEIR BRAIN TISSUE WHICH INDICATED THAT THERE WAS LESS METASTATIC EVENT IN THE BRAINS OF THESE MICE. JULIE WENT ON AND LOOKED AT SOME OF OUR BIOMARKER EXPRESSION LEVELS IN THE MICE AND LOOKED AT CD 44, S 100 A 8 WHICH WE SHOWED IN OUR TABLES OF OUR TUMOR TISSUES, AND SHE ALSO LOOKED AT COX TWO, PLS 4, AGONIST, AND IL 6. IN ALL CASES, THE MICE THAT GOT AMY KNOW GAUN DEAN HAD REDUCED EXPRESSION OF THESE BIOMARKERS. SO AN IMPORTANT QUESTION OF THOSE AND WITH YOU OF THE JCI PAPER OF THE COHORT, AND THEY WANTED TO KNOW IF NOS TWO WAS ACTUALLY MAKING NO, AND SO JULIE WENT ON TO LOOK AT HOW NOS TWO MIGHT BE REGULATED IN TUMORS AND SO SHE USED COMPONENTS OF THE TUMOR MICROENVIRONMENT INCLUDING STARVATION, WITHDRAWAL, SHE HIGH POX YEAH AND CYTOKINE WHICH CAN MIMIC CHRONIC INFLAMMATION IN TUMOR MICROENVIRONMENT, AND SHE FOUND THAT WHEN SHE JUST SIMPLY E TREATED THE CELLS WITH SERUM WITHDRAWAL WHEN COMPARED TO CONTROL SHE SAW HIGHER LEVELS OF NOS TWO PROTEIN EXPRESSION AND SHE ALSO QUANTIFIED NIGH TRITE LEVELS IN THESE SAMPLES AS WELL AND SHE SHOWED, I THINK ABOUT A TWO-FOLD INCREASE IN NITRATE LEVELS WHEN THE CELLS WERE EXPOSED TO SERL WITHDRAWAL. SERUM WITHDRAWAL. INTERFERON WAS VERY, VERY HIGH. WE TOOK THESE CELLS EXPOSED TO INTERFERON AND WITHDRAWAL AND SHE LOOKED AT TEMP EXPRESSION AND TEMP IS INDUCED DRAMATICALLY BY INTERFERON GAMMA AND WHEN SHE IPED TEMP OUT AND THEN PMP SHE SAW A LITTLE BMP STAINING WHICH WAS MAYBE REDUCED A LITTLE BIT IN THE PRESENCE OF AMY KNOW GAUN DEAN. AMY KNOW MAY BE INDICATION OF GAUN DEAN WITHIN THE KNEW TUMOR. INDUCE POST SURVIVAL SIGNALING. IT CAN INDUCE TUMOR GROWTH AND ME IT’S A — IT CAN REDUCE CHEMO RESISTANCE AS WELL. FROM MD ANDERSON. THEY LOOKED AT NOS TWO EX EXPRESSION IN PATIENTS NEGATIVE DISEASE AND SHE FOUND NOS TWO A ALSO PREDICTED BOTH SURVIVAL IN THESE PATIENTS AND THIS WAS VERIFIED IN ANOTHER IN TWO OTHER COHORTS THAT SHE EXAMINED. SO SHE WANTED TO TARGET NOS TWO IN MICE AND SHE GAVE THEM ANOTHER A DIFFERENT INHIBITOR, L MAIN WHICH IS A PAN INHIBITOR OF NOS. THESE ARE MP31 SEE KNOW GRAPHS. WE CAN SEE THE MICE THAT GOT THE NOS INHIBITOR HAS REDUCED STROKE AND THEY ALSO HAVE REDUCED LUMINESCENCE. THESE ARE TAGGEDED WITH ARE RESIF RACE. REDUCED LUMINESCENCE IN THE LUNGS OF THESE ANIMALS AS WELL. THIS STUDY SUPPORTS OURS THAT TARGETING NOS CAN REDUCE TUMOR BUT CAN ALSO REDUCE ME IT’S A SEES AS WELAS WELL. SHOWED IT’S SUPPRESSED IN THE ANIMALS THAT GOT L MAIN AND SHE PROPOSED THAT SIGNALING PATHWAY INVOLVING I KNOX GOING TO PGS BETA INCREASE STICK ONE WOULD PROMOTE TUMOR METASTISES. THIS IS JUST A SLIDE THAT, THIS IS NEW DATA THAT WE’VE HAD WHERE WE TREATED MP 231 CELLS WITH ARGENEN, AND ALSO TITRATED BACK IN DETAIN NO AND THE CELLS THAT GOT IMMUNOGAUN DEAN. WE JUST LOOKED AT TRAJECTORY OF DIE-LABELED CELLS. COMPAREDED TO CONTROLLED THERE’S A LOT MORE EVENT AND YOU CAN SEE LONGER TRAJECTORIES IN THE CELLS THAT GOT NO. THAT’S P PRELIMINARY DATA FOR A NEW PROJECT WE’RE TRYING TO START, BUT TOGETHER I THINK BOTH OF OUR STUDY AND THEIRS SUGGEST THAT [INDISCERNIBLE] TARGETING METASTISES BY NO. SO WE WANTED TO KNOW IF WE COULD TARGET NOS WITH CONVENTIONAL THERAPY. WE USED RADIATION MODEL. WE HAD MICE THAT HAD TUMOR-BEARING LEGS AND WE GAVE THEM TEN GRAY OF RADIATION AND WE TREATED THESE MICE EITHER WITH GAUN DEAN OR GAVE THEM L MAIN. IN THIS CASE, THIS IS AN IMMUNOCOMPETENT MICE AND THE ANIMALS THAT GOT L MAIN EXHIBITED TRAMATICALLY F-REDUCED TUMOR GROWTH WITH L MAIN — DRAMATICALLY-REDUCED TUMOR GROWTH WITH L MAIN. I NOS DID SIGNIFICANTLY REDUCE TUMOR GROWTH BUT IT THE TUMOR GROWTH DELAY INDUCED BY RADIATION BUT NOT AS EFFECTED AS L MAIN. WE REPEATED THESE IN MICE USING HE 29 CELLS. BASICALLY THERE WAS NO AUGMENTATION OF THE RADIATION INDUCED. THIS SUGGESTS TO US THAT T CELLS ARE REQUIRED FOR TARGETING NOS IN THIS SYSTEM. THANK YOU. OKAY. WE WENT ON AND LOOKED AT CYTOKINE EXPRESSION PRO ARE FILES IN THESE TUMORS AND THE TU UH MORES THAT GOT — TUMORS THAT GOT RADIATION ALONE OVERALL IMMUNOSUPPRESSES SIGNALING PROFILE BUT THE TUMORS THAT GOT TEN GRAY AND L MAIN EXPRESSED HIGHER LEVELS OF PROINFLAMMATORY CYTOTOXIC CYTOKINES. SO IL 10 WAS ONE CYTOKINE THAT REAL HI JUMPED OUT AT US BECAUSE IT WAS UP REGULATED EARLY AND THE MICE THAT GOT TEN GRAY AND IT WAS SUPPRESSED WHEN WE GAVE L MAIN, AND SO WE WANTED TO KNOW IF WE COULD TARGET IL 10 AND INCREASE RADIATION-INDUCED. WE WERE ABLE DO THAT WHICH IS SHOWN HERE. UM, SO WE WENT ON TO LOOK AT THE TUMORS OF THESE MICE AND WE ISOLATED LEUKOCYTES FROM THESE TUMORS AND FOUND THAT IN THE ANIMALS THAT GOT THE TEN RADIATION AND L MAIN, THEY HAD HIGHER NUMBER OF CDA POSITIVE T CELLS AND HIGHERER NUMBER OF ACTIVATED CD 8 POZ T CELLS AS WELL. — POSITIVE. THIS DATA SUGGESTS THAT IF WE TARGET NOS IN A RADIATION THERAPEUTIC MODEL, WE CAN REDUCE IMMUNOSUPPRESSANT CRY TO FINE, INCREASE PROINFLAMMATORY CYTOKINE AND INCREASE THE NUMBER AND ACTIVATION OF CD 8 POSITIVE CYTOLYTIC TUMOR CELLS TO IMPROVE OVERALL TUMOR RESPONSE TO RADIATION. WE HAVE ALSO LOOKED AT CD 80-TREATED MICE IN THIS SAME MODEL. CD 40, WE’LL INITIATE A CYTOKINE FORM OF PROINFLAMMATORY CYTOKINES AND WE SHOWED HERE THAT IF WE DID CD 40 DAY BEFORE RERADIATE THE TUMOR THAT WE CAN INCREASE THE TUMOR GROWTH DELAY WHEN COMPARED TO RADIATION ALONE. TR A AKIOLY HAS DEMONSTRATED ANTIBIOTICS CAN LIMIT THE CHEMO THERAPEUTIC EFFECTS OF OXLOFATTEN A AND SO WE EXAMINED WHETHER OR NOT ANTIBIOTICS HAD AN EFFECT IN TUMOR GROWTH DELAY AND SIMILARLY WE EXAMINED THAT WHEN WE GAVE MICE ANTIBIOTICS IT DID REDUCE. THESE HAVE OPENED DOOR TO NEW AREA WHERE WE CAN EXAMINE TARGETING NOS OR, YOU KNOW, TARGETING POLARIZATION OF THE IMMUNE SYSTEM TO IMPROVE THERAPEUTIC RESPONSE TO RADIATION. SIMILARLY, CHANG ALSO SHOWED THAT L MAIN POE TENUATED BETWEEN THERAPEUTIC EFFECT OF DOSAP A AXEL IN MICE — DOSAPAXEL IN MICE AND YOU CAN SEE THE SURVIVAL OF THESE ANIMALS IS EXTENDED HERE. THESE DATA SHOW THAT I NOS PROMOTED TRIPLE NEGATIVE DISEASE PROGRESSION, POSTULATES THAT IT’S MODULATED IN STEM CELLS METASTATIC POTENTIAL, AND AGAIN, THEY IDENTIFIED TWO NEW BIOMARKER PROTEINS THAT WERE HIGH IN THEIR NEGATIVE BREAST TUMORS WITH HIGH NOS TWO AND KNOCK DOWN THESE TEAMS REDUCED TUMOR GROWTH AND ME IT’S A SCHISM IN MICE ME IT’S A
SCHIMICE METATISI
SM — WE ARE ALSO INTERESTED IN LOOKING AT OTHER NOVEL COMPOUNDS THAT MAYBE CAN TARGET CANCER STEM CELLS AND TIPS, AND ONE OF OUR FELLOWS IN OUR LAB HAS MADE A SERIES OF ABSENT CONJUGATEED BREAST WHERE SHE SUBS VE KNOW ON TO ASPIRIN AND iPANEL WHICH RELEASES A REDUCED FORM OF NITROOXIDE. THESE COMPOUNDS REDUCE TENNISTY OF ACT-RIN. GIVE THEM TO [INDISCERNIBLE] ANIMALS, REDUCE TUMOR GROWTH IN THESE MICE. INTERESTINGLY, THESE DRUGS CAN INCREASE OXIDATED STRESS AND DNA DAMAGE, BUT THEY ALSO CAN TARGET STEM CELL BIOMARKERS AND THEY CAN REDUCE ANGIOGENESIS. ANOTHER COLLEAGUE OF OURS FROM BRAZIL, THEY ARE MAKING SOME DRUGS THAT WILL RELEASE HNL AS WELL. THESE DRUGS ARE ACTIVATED TO RELEASE HNO BY FILES. HERE WE SHOW THAT WHEN WE INDUCE IF ONE STABILIZATION BY STER-NO. TREAT CELLS WITH COMPOUNDS WE CAN SUPPRESS NO-INDUCED STABILIZATION OF IF TEMP ONE. WE THINK THAT THESE COMPOUNDS MAY BE SUITABLE FOR ER-NEGATIVE PATIENTS THAT HAVE HIGH NOS TWO BECAUSE THEN GOING BACK TO OUR TABLE, THESE PATIENTS EXPRESSED HIGH LEVELS OF GLUTE MAID SYSTEM LIGATE — GLUTAMATE SYSTEM LIGATE, AND ALSO IF THESE DRUGS CAN TEMP AND MAYBE REDUCE ANGIOGENESIS WE ALSO KNOW IN OUR COHORTS THAT INCREASED VASCULARIZATION HAS BEEN IDENTIFIED IN TU UH MORES THAT HAVE HIGH NOS TWO. BAY SUCKLY, IN SHORT, USE DISINIUM COMPOUNDS MAY BE A SUITABLE DRUG FOR TREATING PATIENTS WITH THIS SIGNATURE. TO END THE TALK, I THINK THESE DATA HAVE SHOWN THAT WE CAN USE READ-OUTS INFLAMMATION TO FOLLOW CANCER PROGRESSION AND TREATMENT AND BASICALLY THESE STUDIES HAVE KIND OF STARTED FROM BED-SIDE TO BENCH AND BACK TO BEDSIDE BECAUSE CHAN SGSHGS, THEY’RE GOING TO BEGIN A CLINICAL TRIAL USING NOS INHIBITORS IN COMBINATION WITH CHEMOTHERAPY INCLUDING BOTH PAXIL AND IN PART, WE THINK THAT IT’S TARGETING CANCER STEM CELLS AND IT’S PROMOTING METASTISES OF CANCER CELLS AND NITRATION AND INCREASING MNP 9; RATIO MAY BE IMPORTANT METHOD IN THIS PROCESS. A LOT OF PEOPLE HELPED WITH ALL OF THIS WORK. STEFAN’S GROUP HERE AT NCI. CHANG COHORT. THEY HAVE EXAMINED TONS AND TONS OF PROTEIN EXPRESSIONS IN THESE TUMORS THAT WE CAN GO BACK AND LOOK P MECHANISTICALLY AT RELATIONSHIP WITH NOS TWO. WE HAVE ACTIVE COLLABORATIONS WITH DR. WELLCHILD AND TRANKIAR DWROSHGS EXAMINE RULES AS IMMUNE POLARIZATION IN RADIATION THERAPY. THANK ERNIE WHO DID ALL OF THE MASS SPECK I SHOWED YOU BECAUSE THAT WAS IMPORTANT. GROUP AT DUKE, SHE HAD THE A ALZHEIMER’S MODELING THAT WE USED. EVERYBODY IN DAVE’S LAB. WE HAD A LOT OF HIGH SCHOOL KIPS COME THROUGH THE LAB AND DID A LOT OF THE WORK I E SHOWED YOU TODAY. THANKFUL FOR THEM AND WISH THEM WELL IN THEIR FUTURES. THANK YOU AND I’LL TRY TO ANSWER ANY OF YOUR QUESTIONS. [APPLAUSE]>>[LOW AUDIO].>>WELL, AGAIN, BENNY SHOWED THAT A LOT OF DMC MARKERS, THOSE MARKERS ARE UP IN HER MOUSE MODEL AND I THINK SHE SHOWED THAT THEY WERE ALSO — I DON’T KNOW IF SHE SHOWED THEY WERE UP IN THE PATIENTS BUT I DO KNOW SHE SHOWED THEY WERE UP IN MOUSE MODEL AND SHE COULD SUPPRESS THEM WITH NOS INHIBITORS. TEMP ONE IS ALSO NT MARKER, SO THE IF TEMP IS HIGH AND CAN BE MODIFIED IN PRESENCE OF HIGH NO, HIGH NOS TWO OR DERIVED NO, THEN THAT MAY ALSO WITH PROMOTING EMC AS WELL. [APPLAUSE]>>DR. PACHER HAS ARRIVED. HE GOT HIS MEDICAL DEGREE IN EUROPE AND CAME TO U.S. HE’S ONE OF THE WORLD’S EXPERTS IN PHYSIOLOGY AND HE’S IN THE LABORATORY OF CARDIOVASCULAR. HE’S SECTION CHIEF HERE AT NIH. AT NIH TRIPLE A AT BEN BROOK, OFF CAMPUS. INTERPLAY OF OXIDATED STRESS, INFLAMMATION, LIPID AND CELL DEATH SIGNALING PATHWAYS DURING TISSUE — IMPLICATION F F FOR PHYSIOLOGY AND PATHOLOGY. PAUL.>>THANK YOU VERY MUCH. THANK YOUER VERY MUCH FOR COMING TO THIS SEMINAR. [LOW AUDIO]. OKAY. SORRY. SO TODAY I WOULD LIKE TO TALK TO YOU ABOUT OXIDATIVE STRESS OF IN VITRO SYSTEM HOW IMPORTANT TO UNDERSTAND HOW OXIDATIVE STRESS IS LINKED WITH INFLAMMATION [INDISCERNIBLE] PHOSPHATES. SO FIRST, THIS IS VARIOUS [INDISCERNIBLE] SHOULD BE IN THE CENTER OF MY PRESENTATION AND DIFFERENT PROJECTS [INDISCERNIBLE]. DURING ANY KIND OF TISSUE NIR — INJURY, YOU HAVE [INDISCERNIBLE] DIFFERENT SOURCES CONTRIBUTE LIKEMITOCHONDRIA [INDISCERNIBLE] — OTHERS. SOME EXAMPLES GENERATED [INDISCERNIBLE] INTERACT AS WITH NITRIC OXIDE [INDISCERNIBLE] INCLUDING [INDISCERNIBLE] NITROGEN SPECIES AS WELL. FINALLY, THIS OXYGEN NITROGEN GENERATION SPECIES [INDISCERNIBLE] OXIDATED DNA INJURY WHICH [INDISCERNIBLE] — MOST ABUNDANT NUCLEAR ENZYME. [INDISCERNIBLE] DNA CONSUMPTION, LOOK AT CONSUMPTION AND [INDISCERNIBLE]. WALKING NEXT EACH OTHER AND THEREFORE IT IS ALSO IMPORTANT [INDISCERNIBLE] TRANSCRIPTION PROCESS LIKE NUCLEAR [INDISCERNIBLE] INVOLVED IN INFLAMMATION AND IT CAN ALSO INTERACT WITH MITOCHONDRIA AS WELL AS [INDISCERNIBLE] EITHER GO TO MITOCHONDRIA AND HELP WITH [INDISCERNIBLE] AND FINALLY, [INDISCERNIBLE] — ARE REACTION AND ATTRACTION OF INFLAMMATORY CELL [INDISCERNIBLE] OXYGEN AND NITROGEN [INDISCERNIBLE]. SO TODAY I WOULD LIKE TO SHOW US AN EXAMPLE OF [INDISCERNIBLE] INJURY AND WE’LL GO TO SOME OF THIS INTERACTION SO [INDISCERNIBLE] OXYGEN AND NITROGEN SPECIES. SOURCES ON THAT AS WELL [INDISCERNIBLE] NOT SURE WHAT IS HAPPENING AND WHY [INDISCERNIBLE] SO IMPORTANT [INDISCERNIBLE]. MIE TO CON DRI Y’ALL INFARCTIONS — MITOCHONDRIAL INFARCTIONS [INDISCERNIBLE]. FOR EXAMPLE IF YOU HAVE MITOCHONDRIAL INFARCTION DECISION INJURY [INDISCERNIBLE]. ACTIVATION INFLAMMATORY [INDISCERNIBLE] TRACK INFLAMMATORY PRODUCED BY OXYGEN AND NITROGEN SPECIES LEADS TO FURTHER INJURY. VERY IMPORTANT [INDISCERNIBLE] THEY DIE FROM MASSIVE TISSUE INJURY AS WELL AS DAMAGE JUST [INDISCERNIBLE] HAVE YOU HAVE NEXT GO HERE WHICH CAN BE DNA FRAGMENT, CAN BE LIGATIONS, MOST KNOWN OR EVEN CAN BE OXIDATIVE STRESS BY PRODUCTIVE LIPID SHOWN [INDISCERNIBLE]. ALSO USED FOR MARKER SO IN SOUTH AND MEASURE IT. THIS HAPPENS THROUGH SECONDARY COMING FROM REACTION BECAUSE THIS [INDISCERNIBLE] BIND TO CERTAIN RECEPTORS INFLAMMATORY RESPONSE. IT GOES BACK UP PATHWAY. ARE RELEASE OF THIS [INDISCERNIBLE] USUALLY [INDISCERNIBLE] BY OTHER [INDISCERNIBLE] WHICH DO NOT LEAVE SECONDARY INFLAMMATION. THIS IS AN EXAMPLE OF DAMAGE [INDISCERNIBLE] WHICH ACT ON DIFFERENT AREA RECEPTORS LIKE ONE OF THE MOST KNOWN IS PROTEIN ONE IT’S ACTIVATED THE SAME AS [INDISCERNIBLE] CR RECEPTORS ANTITOXIN BUT ATT CAN ALSO [INDISCERNIBLE] CYTOKINES ONE ALPHA AND DNA FRAGMENT CAST BEFORE US FOR EXAMPLE DURING [INDISCERNIBLE] RELATED MORE JOINTS BUT AFTER MUSCLE CONSUMPTION [INDISCERNIBLE] JOINTS WAS VERY BAD INFLAMMATION. FOR EXAMPLE, THIS CAN ALSO TRIGGER ACTIVATION INFLAMMATORY CELLS. GOING BACK TO ANOTHER, I WOULD LIKE TO TALK E ABOUT IMAGE TODAY AND JUST OUTLINE THE [INDISCERNIBLE] PROCESSES INTERACT. [INDISCERNIBLE]. WHAT’S HAPPENS FOR ONE HOUR CELLS [INDISCERNIBLE] — THIS PROCESS THERE’S MITOCHONDRIAL FUNCTION AND DYSFUNCTION AND HEPATOCYTE [INDISCERNIBLE]. IT STARTS TO RELIEVE VARIOUS [INDISCERNIBLE] SHOWS VARIOUS [INDISCERNIBLE] THEY ACTIVATE THE CELLS MODIFIED MICROPHAGES THAT SIT [INDISCERNIBLE] LEVER OR NORMAL AND ONCE NORMAL CELLS ACTIVATED, THEY CAN [INDISCERNIBLE] OXIDATIVE WHICH SHOW TO PRODUCE HYDROGEN PEROXIDE AS WELL AS NITRIC OXIDE AS WELL AS [INDISCERNIBLE] REACTION TO [INDISCERNIBLE]. ATTRACT NEUTROPHIL TO THE SITE OF THE INJURY. FIRST TWO HOURS, THERE ARE NO NEW TRI — NEUTROPHILS YET. BUT THERE IS ACTIVATION OF CELL. ATTRACTION IN THIS EDITION OCCURS AFTER SIX HOURS AND PICKS UP 24 HOURS. YOU SEE VERY NICE DEMARCATION I UH WILL SHOW YOU AND ALSO IMPORTANT BECAUSE YOU SEE INFLAMMATION WAS UP UH BUT IT WAS BOUND LIKE HE E — HE HEPATOCYTE. [INDISCERNIBLE]. AT THE BEGINNING YOU HAVE [INDISCERNIBLE] LATER YOU HAVE MOST DIFFERENT FAST LIKE MICRO STAGES COMING LIKE TISSUE REPAIR. [INDISCERNIBLE] THIS IS EXPOSED TO ONE HOUR SKIN AND TWOER OR TWO HOUR AND SIX AND 4-HOUR RETROFUSION — 24-HOUR RETROFUSION. [INDISCERNIBLE] MACROPHAGES ARE RELEASED ALSO CLINICALLY AND LIVER DISEASES SO YOU SEE BOTH [INDISCERNIBLE] PEAK SIX HOURS AND THEN 24 HOURS. THIS IS WHERE IT WAS BOUND BECAUSE [INDISCERNIBLE] MADE PROBLEM AROUND [INDISCERNIBLE]. WHERE ALSO THIS IS NUCLEAR ENZYME [INDISCERNIBLE] WHICH I SHOWED YOU ON THAT FIRST SLIDE OXIDATIVE DNA INJURY, THIS IS INCREASE [INDISCERNIBLE] DNA FRAGMENTATION, MARKER OF UPWARD [INDISCERNIBLE], YOU SEE TWO HOURS YOU DON’T SAY UPWARD TWO [INDISCERNIBLE] AND YOU’VE GOT TO REALLY MORE SIGNIFICANT 24 HOURS. [INDISCERNIBLE] IT’S SHIFTED. YOU SEE MICROSCOPE, FIVE THIS IS A NORMAL [INDISCERNIBLE] MITOCHONDRIAL, AND TWO HOURS AFTER [INDISCERNIBLE] IS THIS ONE MIE TOE CON DRI ARE Y’ALL DECREASED STRUCTURE, AND YOU ALSO SEE AT TWO HOURS DECREASE OF MITOCHONDRIAL COMPLEX ONE ACTIVITY AND YOU ALSO SEE INCREASE OF [INDISCERNIBLE] OXIDATION SO THIS FAMILY SHOWN HERE EACH IS A MARKER OF [INDISCERNIBLE] EDITION BUT THIS IS MORE STABLE MORE RELIABLE MARKER WHICH CAN BE EFFECTIVE [INDISCERNIBLE]. RELIABLE. ALSO INCREASE IN CARBON [INDISCERNIBLE] AT TWO HOURS FROM MITOCHONDRIA AND ALSO [INDISCERNIBLE] WHICH IS A MARKER OF NITRATION IN THERE ALSO IN PART [INDISCERNIBLE] GENERATION. SO THIS MARKER GO UP AND YOU SEE MITOCHONDRIA DYSFUNCTION, BUT THEREFORE ACTUALLY BY SIX, 24 HOURS MITOCHONDRIA DYSFUNCTION 24 HOURS AND IF YOU SEE LIPID OXIDATION THEY START TO GO UP VERY SIGNIFICANTLY AND THEREFORE GP 91 [INDISCERNIBLE] THIS IS ISOFORM, THIS IS NEXT TWO [INDISCERNIBLE] YOU FEEL TWO HOURS ACTIVE [INDISCERNIBLE] MITOCHONDRIA PART AND THEN LIPID GO UP. WHY SO IMPORTANT TO SEPARATE FROM IN VIVO EXPERIMENTS FROM IN VIVO? BECAUSE FOR EXAMPLE IF YOU TAKE TISSUE AND MOST OF THE PREVIOUS STUDIES FROM WHICH DRUG DEVELOPMENT AND PRACTICAL FIELD THIS FUSION ISOACTIVITY SHOE PREPARATION, ISOHEART, THIS SUBJECT HIGH [INDISCERNIBLE] JUST TOOK NITROGEN SOLUTION AND PROFUSED AND THEREFORE USED HIGH OXYGEN CONTENT WITH SOLUTION WHICH [INDISCERNIBLE] OXYGEN AND PART OF CO 2. THEN THEY INDUCED FOR AND BUT THEN THERE’S MORE [INDISCERNIBLE] OXYGEN/NITROGEN SPECIES HAPPENED RIGHT AFTER THE INTRODUCTION OF THIS OXYGEN SOLUTION AND IT LASTED FOR VARIABLE IF FOR SECONDS, BRIEF PERIOD. AS YOU SEE HERE IN VIVO, THE SITUATION WAS TOTALLY DIFFERENT AND THEY INDUCED [INDISCERNIBLE] INFINITELY AND THOSE ARE ARE EXTREMELY IMPORTANT IN INJURY OR IN REPAIR. ONE MORE REVEES STEM CELL HYPE S SO YOU SEE MARKED INCREASE AS BEEN IN THE EXPRESSION 24 HOURS [INDISCERNIBLE] SO THIS IS HIGH MODIFICATION YOU SEE IN HEPATOCYTES AND YOU SEE INFLAMMATORY CELLS AND THEY ARE ATTACHED TO [INDISCERNIBLE]. AND THIS H IS MARKER FOR [INDISCERNIBLE] FOR DNA INJURY, DG. YOU SEE HERE THIS IS HIGH MODIFICATION MARK IN THE CUP SPINNING IN NUCLEAR [INDISCERNIBLE] NO INJURY MARKERS SO THIS DNA DEP DEP E HEPATOCYTES. GO BACK TO TIME COURSE OF TWO HOURS WHERE NITRO GOING UP BUT THIS IS COMING TO OXYGEN/NITROGEN SPECIES IN MITOCHONDRIA, YOU SEE [INDISCERNIBLE] LARGER, THIS IS MORE MODIFICATION SO YOU SEE PART OF MODIFICATION YOU SEE THIS IS HEPATOCYTE, THIS IS [INDISCERNIBL [INDISCERNIBLE] — THEN INJURED MY WORKING TO TISSUES. HERE YOU CAN DAMAGED [INDISCERNIBLE] IN BLACK. DAMAGE IN TISSUE TO HELP US DECIDE INJURY. INFLAMMATION AND AGAIN THIS IS VERY IMPORTANT TO DISTINGUISH COLOR INFORMATION AND INFLAMMATORY REACTION WHICH WE CAN MEASURE CYTOKINES OR [INDISCERNIBLE]. FOR EXAMPLE YOU HAVE INFLAMMATION YOU CAN HAVE JUST MICROOXIDATION AND INCREASE IN [INDISCERNIBLE] INFLAMMATORY MARKERS. YOU BUY SINGLE HUMAN CELLS. DIFFERENT CELL NEEDS TO PASS BAD BRAIN BARRIER. IN THIS CASE, YOU SEE THAT THERE’S LOVELY INCREASE OF [INDISCERNIBLE], TWO HOURS. THIS IS COMING MOSTLY FROM ACTIVATED [INDISCERNIBLE] MICROSTAGES AND VERY IMPORTANT WORK, BUT LATER, IT HAS BEEN GOING BACK AND [INDISCERNIBLE] ALPHA AND THIS IS APPLICATION EXPRERGS PRESSED IN TISSUE. — EXPRESSED IN TISSUE. [INDISCERNIBLE] AGAIN, TWO HOURS, THEN THREE, INFLAMMATORY [INDISCERNIBLE]; SIX HOURS [INDISCERNIBLE], AND THEN 24 HOURS YOU HAVE HUGE NUMBER, BUT THIS PART ON NEUTROPHIL [INDISCERNIBLE]. SUMMARIZING AS TO WHAT HAPPENS DURING THIS PROCESS [INDISCERNIBLE]. SO YOU HAVE INJURY WHICH IS FOR EXAMPLE [INDISCERNIBLE] LIVER SURGERY OR LIVER TRANSPLANTATION AND THEN PUT BACK THIS LIVER [INDISCERNIBLE]. OXYGEN TO THE BLOOD WAS COMING AND YOU HAVE TISSUE INJURY AND WHERE THIS DAMAGE [INDISCERNIBLE] — ACTIVATED SICKLE CELLS EXPRESSED [INDISCERNIBLE] — NITROGEN — N NEUTROPHIL — AND FINALLY [INDISCERNIBLE]. ATTACHED TO DAMAGED [INDISCERNIBLE] AND THEN [INDISCERNIBLE] TISSUE OCTOBER — — LATER SECONDARY REACTION AND MIE TO CON DRI Y’ALL REACTION
TOCHONDRIAL
REACTION — MITOCHONDRIAL [INDISCERNIBLE]. WHAT’S HATCHED IF UH YOU STOP TO PAUSE [INDISCERNIBLE] WHERE FOR EXAMPLE [INDISCERNIBLE] NEXT TO THIS OR [INDISCERNIBLE] MITOCHONDRIA — I’VE CHANGED COUPLE AND UPDATED BUT I CAN SEND YOU ALL THIS PRESENTATION OR SEND YOU MOST OF THE SLIDES FROM THE PRESENTATION BUT THERE ARE A COUPLE NUANCE AND THERE IS A STAFF ORGANIZATION. SOME OF THE FIRST [INDISCERNIBLE] HAPPENS OXYGEN DOWN WHICH ARE POSSIBLE TO TARGET TO MITOCHONDRIAL AND ACTUALLY NOW THIS IS VERY SPECIAL [INDISCERNIBLE] TO WHERE YOU BASICALLY ALMOST [INDISCERNIBLE] EVERYTHING IN [INDISCERNIBLE] ORGANIZED. FOR EXAMPLE [INDISCERNIBLE] — FOR EXAMPLE USE COUPLE COMPOUNDS AND TRY TO DETERMINE WHAT IS [INDISCERNIBLE] THIS PROCESS AND BASICALLY IF YOU DID MIE TO CON DRI ARE YEAH TARGET AROUND THIS CORE — MITOCHONDRIA TARGET AROUND THIS CORE, THEY ARE VERY PROTECTIVE AND THEY CAN STOP ALMOST ALL THIS PROCESS BECAUSE THEY CAN STOP THE INITIAL HE HEPATOCYTE; HOWEVER, IF YOU START TO LEAD THEM UP TO SIGS THEY EXTREMELY LOSE EFFECTIVENESS. THEY ARE NOT VERY USEFUL BECAUSE USUALLY WHEN YOU HAVE [INDISCERNIBLE] MITOCHONDRIAL INFARCTION WILL STOP FOR CERTAIN PERIOD OF TIME SO YOU CANNOT [INDISCERNIBLE] THIS PATIENT. THIS IS VERY IMPORTANT BECAUSE MITOCHONDRIA ARE IMPORTANT [INDISCERNIBLE] INFLAMMATION AND TISSUE INJURIES [INDISCERNIBLE] BY INFLAMMATORY CELLS. THAT IS WORST THAN OTHERS [INDISCERNIBLE] DISTINCTION BETWEEN EXTINCTION AND [INDISCERNIBLE] — MOBILE SPIKE LONG-TIME PREPARATION OF THE HEART IN CLINICAL TRIALS BECAUSE THEY HAVE NO EFFECT IN THE SECONDARY PHASE. COUPLE OTHER EXAMPLES. WHAT HAPPENS IF YOU [INDISCERNIBLE] INDUCE [INDISCERNIBLE] YOU ACTUALLY INDUCE RESTRICTION AND [INDISCERNIBLE] AND MORE INJURY. INDUCE INDUCE LIPCYTE [INDISCERNIBLE]. SIMILAR TO MITOCHONDRIA, IT IS BECAUSE THEY ARE MOSTLY EFFECTIVE IN STAGES. [INDISCERNIBLE] — INJURIES BECAUSE ACTIVE OXYGEN PUSHES SUB SUBDIVISION [INDISCERNIBLE]. PROBLEM IS PRODUCTION OF LIPID BENEFITS THAN [INDISCERNIBLE] — INFLAMMATORY PROCESS BY THEMSELVES [INDISCERNIBLE] LIPID ARE AVAILABLE. AN EXAMPLE OF ACTIVITY, THERE ARE LOTS OF DIFFERENT [INDISCERNIBLE] THAT SOME PEOPLE FIND THEM. NITROGEN SPECIES, OXI NITRATES [INDISCERNIBLE] MORE PROTECTIVE THAN [INDISCERNIBLE] PERFUSION INJURY, BUT EASILY OTHER PROBLEMS THAT THEY ARE [INDISCERNIBLE] VERY DIFFICULT TO SEFRP THESE THIS COMPOUND — SYMPATHIZE THIS COMPOUND [INDISCERNIBLE] USUALLY BLACK OR GREEN [INDISCERNIBLE] SOME IN CLINICAL DEVELOPMENT. VERY DIFFERENT DRUGS AND PRECISION INJURY, AGAIN, THE SOONER YOU GIVE, MORE EFFECTIVE THEY ARE. I’M SURE YOU HAVE HEARD ABOUT OXYGEN DURING THE COURSE OF THIS PRESENTATIONS EARLIER AND OXYGEN [INDISCERNIBLE] VERY IMPORTANT ROLE IN ACTIVE OXYGEN NITROGEN SPECIES AND THE REASON WHY I’M ALSO MENTIONING BECAUSE WHEN E TALK ABOUT [INDISCERNIBLE] VERY STRONG INTERACTIONS WITH TYPE OXYGEN LIKE THE. OXYGEN LIKE THIS IS PERFECTED BUT HERE AGAIN THINK ABOUT GENERATION WITH WHAT’S COST AROUND HIM [INDISCERNIBLE] — IS VERY BAD IN THIS BUT TOSSED AROUND IN HERE TOO FOR EXAMPLE WE WERE A GENERATION AND [INDISCERNIBLE], SO [INDISCERNIBLE] ACCORDING TO THIS IT CAN ADVISORY AFFECT THE WHOLE SYSTEM. NOW WE TALK ABOUT [INDISCERNIBLE] SIGNALING AND THE REST OF HOW INTERACT WITH OXIDATIVE STRESS AND [INDISCERNIBLE] LIPID [INDISCERNIBLE]. INTRODUCTION I’M SURE YOU ALREADY KNOW OR HEARD ABOUT MARK WARNER WHICH HAS [INDISCERNIBLE] BUT ALSO [INDISCERNIBLE]. [INDISCERNIBLE] AFFECT MOSTLY THIS COMPOUND THC AS POSSIBLE. THIS HAS TWO RECEPTORS; ONE CD 1 MADE IN THE BRAIN SO IT INDUCES [INDISCERNIBLE] EFFECT BUT ALSO INCREASES [INDISCERNIBLE]. THIS WAS LEFT FOR THE DEVELOPMENT OF [INDISCERNIBLE]. WHICH MARKUP HAS BEEN LARGE NUMBER OF COUNTRIES EUROPE MORE THAN 40 COUNTRIES, BUT THEY INTRODUCE [INDISCERNIBLE] BUT THEY RAN IT BACK THROUGH ENTIRE [INDISCERNIBLE] COMPLICATION AND WERE EFFECTIVE IN [INDISCERNIBLE]. CD 1 RECEPTOR WAS MOST ABUNDANT GENE COUPLE RECEPTOR IN THE BRAIN SO IT IS IN EVERY SINGLE SYNOPSIS. [INDISCERNIBLE] IS THIS CONDITION HAVE THIS EFFECT. OTHER COMPOUND [INDISCERNIBLE] WAS THOUGHT FOR MANY YEARS THAT IT IS COMPLETELY AS [INDISCERNIBLE] EFFECT. [INDISCERNIBLE]. MIXTURE OF COMPOUND [INDISCERNIBLE] HAS PROVED IN MANY COUNTRIES CERTAIN CONDITIONS AND CERTAIN BENEFICIAL EFFECTS. MORE INTEREST IN THIS [INDISCERNIBLE] NEITHER CD 1, NOO DMOOETER IN — NEITHER IN THE OTHER ONE WHICH IS ANTI-INFLAMMATORY. NEITHER SHOWN BENEFICIAL EFFECTS OF INFLAMMATION AND USUALLY [INDISCERNIBLE] THIS IS ALSO VERY POWERFUL ANTI-INFLAMMATORY [INDISCERNIBLE] MIGRATION [INDISCERNIBLE] VERY PROTECTIVE IN PROFUSION INJURY AND RECENTLY IT IS APPROVED BY FDA IN CHILDREN WITH EPILEPSY AND ALSO FOR SVRL SEVERAL INDICATIONS. [INDISCERNIBLE] MIXTURE OF [INDISCERNIBLE] WITH THC. AGAIN THE MOST INTERESTING THOUGHT HERE IS NOT THAT YOU HAVE LIGAND BUT YOU HAVE ANTIGEN IN THE SYSTEM [INDISCERNIBLE] VERY THE TISSUE YOU HAVE [INDISCERNIBLE] DEGREE OF CB 1 AND CB 2 RECEPTORS AND THIS PLAY IMPORTANT WORK AND THIS ALSO VERY IMPORTANT [INDISCERNIBLE] ACTIVE OXYGEN SPECIES GENERATION AND REST OF [INDISCERNIBLE] OXYGENS. SO [INDISCERNIBLE] AND ALSO SECOND IS [INDISCERNIBLE]. CD 1 AND CD 2 RECEPTORS. MORE ALIVE THAN SUPPOSED TO BE TWO RECEPTORS AND ALSO MORE IMPORTANTLY [INDISCERNIBLE] PROXIMITY [INDISCERNIBLE] ARE HIGHER SO CAN BE VERY SIGNIFICANT SOURCE OF [INDISCERNIBLE]. [INDISCERNIBLE] EXHIBIT BY FDH AND TWO AG BY MAGL [INDISCERNIBLE]. SO I WILL TALK BRIEFLY ABOUT THESE TWO [INDISCERNIBLE]. BASICALLY NOW THEY ARE TWOS, TWO [INDISCERNIBLE] ENZYME [INDISCERNIBLE] 2 AG 11. I WANT TO TALK PRIMARILY ABOUT THIS ONE BECAUSE THIS IS [INDISCERNIBLE] TIE IN TISSUES CAN BE SIGNIFICANT SOURCE OF [INDISCERNIBLE]. CD 1 RECEPTORS IN [INDISCERNIBLE] CARDIOVASCULAR EFFECT OF [INDISCERNIBLE] MYOCARDIAL INFARCTIONS [INDISCERNIBLE] YOU KNOW WHY I CALL IT THE SPLICE AND K 2 ARE DIFFERENT NAMES FOR [INDISCERNIBLE] USUALLY MIXTURE OF FIVE, SIX [INDISCERNIBLE] BUT THEY ARE ABOUT ROUGHLY ON THE BLACK MARKET AND FOR AS YOU CAN BUY [INDISCERNIBLE] AND USE POUND TAKING [INDISCERNIBLE] VERY PROFOUND [INDISCERNIBLE]. ALSO STUDY INFLAMMATORY CELLS CD 1 RECEPTOR MICROPHAGES FOR EXAMPLE CANNOT [INDISCERNIBLE] DIABETES AND DIFFERENT OTHER COMPLICATION. SO OTHER RECEPTOR IS CB 2 RECEPTOR WHICH HAS NO DETRIMENTAL EFFECT IN CELL AND USUALLY CB 2 RECEPTOR ACTIVATION LEADS TO DECREASE OF [INDISCERNIBLE] SINGLE CELL, LOCATION AND MIGRATION DECREASE [INDISCERNIBLE] BENEFICIAL [INDISCERNIBLE] INJURY [INDISCERNIBLE] INJURY. NOW GOING BACK TO THIS [INDISCERNIBLE] TALK LITTLE BIT WHAT IS THE ROLE OF HOW [INDISCERNIBLE] ALSO INTERACTS WITH [INDISCERNIBLE] BENEFITS OF CD 9 AND CORPS TWO. [INDISCERNIBLE] — DURING THE [INDISCERNIBLE] BY DURING THE HEPATOCYTES FIRST OF ALL AND [INDISCERNIBLE] ITSELF. [INDISCERNIBLE] INJURY, FROM THIS ONE CONFUSION CAN BE ANTIGEN GENERATED BUT TO PUT TISSUES ALL [INDISCERNIBLE] RECEPTORS FROM FURTHER INJURY OR [INDISCERNIBLE] INJURY. ALSO THIS QUESTION, VARIOUS MICE AS I MENTIONED ONE JUST GOING BACK SO ONE [INDISCERNIBLE] MAGL [INDISCERNIBLE]. SO [INDISCERNIBLE] RECEPTORS IN [INDISCERNIBLE] CELLS FOR WHICH I DESCRIBE MICROPHAGES AND HE PA — HEPATOCYTES [INDISCERNIBLE]. THIS KIND OF PRECISION MAKES [INDISCERNIBLE] LARGELY DECREASED BY IT [INDISCERNIBLE]. YOU ALSO INCREASE AT THREE HOURS [INDISCERNIBLE] MARKERS AS I MENTIONED OXIDATE, THEN LOOKS TWO AND GOES UP [INDISCERNIBLE] 24 HOURS DECREASE. ALSO I DON’T WANT TO GO IN THIS [INDISCERNIBLE] IMPORTANT CONCLUSION OF THESE DIFFERENT [INDISCERNIBLE] CB TWO RECEPTORS. CONCLUSION WAS RECEPTOR WAS FOR [INDISCERNIBLE] RECEPTORS [INDISCERNIBLE]. YOU SEE THAT EXAMPLE [INDISCERNIBLE] FUSIONS [INDISCERNIBLE] WHICH IS DECREASED BY [INDISCERNIBLE] BECAUSE LEVER IT IS VERY IMPORTANT [INDISCERNIBLE] PROCESS. TWO AG GOES TO [INDISCERNIBLE] AGAINST INFLAMMATION BUT YOU ALSO GET [INDISCERNIBLE] — [INDISCERNIBLE] WHICH IS A [INDISCERNIBLE] PROFUSION. [INDISCERNIBL [INDISCERNIBLE] DOING THE [INDISCERNIBLE] DECREASE. AGAIN [INDISCERNIBLE] LIVER INJURY OR THIS IS INDUCED BY ANTITOXIN [INDISCERNIBLE] INFLAMMATION [INDISCERNIBLE] DECREASE OF THE INJURY, LIVER ENZYME [INDISCERNIBLE]. THIS H IS ANOTHER [INDISCERNIBLE]. HERE COMES TO WHETHER YOU HAVE ENOUGH LIVER INJURY OR INDUCE [INDISCERNIBLE] MARKING HEPATOCYTE AND YOU HAVE [INDISCERNIBLE] YOU ALSO HAVE [INDISCERNIBLE] POSITION [INDISCERNIBLE], YOU ALSO LOOK AT [INDISCERNIBLE] HAVELESS HAVE LESS [INDISCERNIBLE] TAG CAN CREATE ACTIVATION OF [INDISCERNIBLE] CELLS AND [INDISCERNIBLE] AND ATTACH TO — [INDISCERNIBLE]. AS I MENTIONED YOU, THIS IS UPDATE OF ANOTHER. FOR EXAMPLE, [INDISCERNIBLE] ET 2 IS PROTECTIVE [INDISCERNIBLE] GENERATION SO TAKING FOR EXAMPLE COX TWO [INDISCERNIBLE] FOR WHICH YOU CAN BUY OVER THE COUNTER AND USE FOR [INDISCERNIBLE] IS NO GOOD BECAUSE THIS IS [INDISCERNIBLE] GENERATION. COX TWO WOULD BE SHOWN TO ANY KIDNEY FUNCTION. FOR EXAMPLE, BECAUSE [INDISCERNIBLE] COX [INDISCERNIBLE] KEEP INJURY AND [INDISCERNIBLE] MAINTAINED BY NITRIC OXIDE BUT IF YOU HAVE KIDNEY [INDISCERNIBLE] NITRIC OXIDE SUSTAINS IN PART [INDISCERNIBLE]. SO IF YOU TAKE ALL OF TOXIN HIB TORS [INDISCERNIBLE] — INHIBITORS [INDISCERNIBLE] — TO KEEP NORMAL PERSON. IT IS KNOWN ONE OF FIVE EFFECT OF THIS [INDISCERNIBLE] INFLAMMATORY DRUG INDUCED [INDISCERNIBLE] AGAIN GOING BACK TO HEARTS AND [INDISCERNIBLE], THE REASON WHY I MENTION BECAUSE NOW INHIBITORS OF OTHER ENZYME [INDISCERNIBLE] WHICH I MENTION THIS WAS VERY [INDISCERNIBLE] AND FOR CERTAIN [INDISCERNIBLE] BECAUSE IF YOU GIVE [INDISCERNIBLE] GET IT. BUT BY THIS [INDISCERNIBLE] WHICH WOULD [INDISCERNIBLE] HAVE MUCH E EFFECT AND MAYBE YOU CAN ADD DEN — BENEFICIAL [INDISCERNIBLE] — ARE PERFECTED. IT HAPPENS WITH THIS CLINICAL TRIAL THAT LAST YEAR BY FRENCH COMPANY [INDISCERNIBLE] SO [INDISCERNIBLE] SOME OF THEM HAD DIVISIONS, INFLAMMATION, EVEN [INDISCERNIBLE] DIET AND THEN THEY BACK TO TRY TO MAKE [INDISCERNIBLE] INFLAMMATION BY LOOKS OF DIFFERENT [INDISCERNIBLE] AND SO GOING ENDOCRINE IN THE HEART [INDISCERNIBLE] INFLAMMATORY EFFECT, [INDISCERNIBLE] ANTI ANTI-INFLAMMATORY [INDISCERNIBLE] — EXPRESSED IN CELL. IF YOU HAVE MYOCARDIAL INFARCTION [INDISCERNIBLE] FROM INCREASED DEFINITION BUT IF YOU [INDISCERNIBLE] ANYTHING BENEFICIAL. [INDISCERNIBLE] IS BAD [INDISCERNIBLE] IN ADDITION TO THAT AS I MENTIONED, CAN EXERT METHOD ACTS AND FOLLOWING [INDISCERNIBLE] MUSCLE SPASM TURN INTO MYOCARDIAL INFARCTION. VERY COMPLICATED AND THIS IS [INDISCERNIBLE]. [INDISCERNIBLE] KNOCKOUT MICE [INDISCERNIBLE] SIGNIFICANTLY HIGHER SO DIFFERENT TISSUES [INDISCERNIBLE] SEVERAL IN THE HEART [INDISCERNIBLE]. INITIALLY [INDISCERNIBLE] EXPECT IT WOULD BE [INDISCERNIBLE]. USE [INDISCERNIBLE] DRUG AND WHAT WE FOUND IN THIS KNOCKOUT MICE [INDISCERNIBLE] INCREASE, [INDISCERNIBLE] TO THE BLOOD, AND [INDISCERNIBLE] RICH LIPID OXIDATION [INDISCERNIBLE] MYOCARDIAL AND SURPRISINGLY IN THE [INDISCERNIBLE] NOT ASSOCIATE WITH INFLAMMATION [INDISCERNIBLE] INFILTRATION. AGAIN, THIS IS LAST THING THAT WHEN YOU TARGET SOMETHING YOU HAVE TO TRY TO THINK ABOUT THE [INDISCERNIBLE] YOU TARGET SOMETHING IN THE BRAIN YOU HAVE TO THINK ABOUT HEART AND OTHER ORGANS. FOR EXAMPLE, LIKE LESSONS LEARNED FROM THIS CLINICAL TRIALS [INDISCERNIBLE]. FOR EXAMPLE, CELEBREX WHICH WAS THE MOST WIDELY-USED [INDISCERNIBLE] STARTED TO COME CAUSING BLOCKS AND LATER [INDISCERNIBLE] EVERYTHING YOU CAN BUY OVERCOUNTER [INDISCERNIBLE] PATIENT WITH HEART [INDISCERNIBLE] — AT RISK FOR MYOCARDIAL INFARCTION IN FAMILY. SUMMARIZING [INDISCERNIBLE] THAT OXIDATIVE STRESS INDUCES SYMPTOMS OF [INDISCERNIBLE] WHICH IS LIPIDS SIFTER WHICH CAN PROFOUNDLY AFFECT INFLAMMATION TO [INDISCERNIBLE] RECEPTORS WHICH IS [INDISCERNIBLE] AND USUALLY [INDISCERNIBLE] IT CAN ALSO LEAD TO BLIND AND [INDISCERNIBL [INDISCERNIBLE] MARKEDLY INCREASED [INDISCERNIBLE] TISSUE AND IMPORTANT CONTACT DEPENDENT E EFFECT. FINALLY, I WOULD LIKE TO JUST SHOW US AN EXAMPLE BRIEFLY THAT [INDISCERNIBLE] STRESS CAN BE BENEFICIAL AND ACTUALLY MIMIC COMPACT [INDISCERNIBLE] WHICH IS JUST AN EFFECT REACTION TO AN INJURY AND [INDISCERNIBLE] EMOTION WITH COX TWO [INDISCERNIBLE] SO IF YOU HAVE INJURY YOU CAN’T TELL [INDISCERNIBLE] BUT [INDISCERNIBLE] CONDITION [INDISCERNIBLE]. SO THIS IS RELATIVELY SIMPLE [INDISCERNIBLE] — JUST [INDISCERNIBLE] YOU SEE [INDISCERNIBLE] GENERATING BY IN THE MOUSE THREE. [INDISCERNIBLE] THERE IS MARKED INCREASE GENERATION AND IF THE INJURY [INDISCERNIBLE] GENERATION [INDISCERNIBLE]. THIS IS JUST AN EXAMPLE THINGS DIFFERENT [INDISCERNIBLE], AND YOU SEE HERE [INDISCERNIBLE] — SO THIS IS THE THING ACTUALLY [INDISCERNIBLE] NORMALIZE EVERYTHING LIKE BLOOD PRESSURE AND HIGH BLOOD GLUCOSE COMPOUND. THIS PATIENT STARTED TO DIE. WE LOOK AT CONFUSION WAS THAT PHOSPHATE NORMALLIZATION [INDISCERNIBLE] IS NEVER GOOD. [INDISCERNIBLE] AND YOU MAY INTERFERE WITH THIS TWO AND INDUCE MORE DAMAGE THAN BENEFIT. FINALLY, IS JUST IT IS VERY IMPORTANT TO US TO THINK ABOUT THAT COMPLEX. SO YOU MAY HAVE [INDISCERNIBLE] TRY TO WHAT’S HAPPENED [INDISCERNIBLE] OTHER SYSTEMS AND THIS IS VERY IMPORTANT BECAUSE LIKE MOST OF THE DRUGS ACTUALLY MEASURE [INDISCERNIBLE] BECAUSE IT TURNS OUT THAT THEY HAVE TO FIGHT EFFECT IN OTHER SYSTEMS [INDISCERNIBLE] LIKE CARDIOVASCULAR. [INDISCERNIBLE] FINALLY I WOULD LIKE TO THANK YOU FOR LISTENING AND COMING TO THIS PRESENTATION AND OUR SCIENTIFIC DIRECTOR, MY MENTOR [INDISCERNIBLE] CONTRIBUTED TO SOME OF THIS. THANK YOU VERY MUCH. [APPLAUSE]>>ANY QUESTIONS?>>TERMINAL CANCER PATIENT [LOW AUDIO].>>[INDISCERNIBLE] BENEFITS OF [INDISCERNIBLE] — THERE WERE SOME STUDIES [INDISCERNIBLE] AND IN THE FIRST STUDIES [INDISCERNIBLE] WHICH IS ONE OF THE [INDISCERNIBLE] IN BRAIN TUMORS WHICH IS VERY MALIGNANT AND ALMOST IMPOSSIBLE TO CURE AND THE PROBLEM IS [INDISCERNIBLE] BE SURE THEY INJECT THOSE [INDISCERNIBLE] IN THESE TUMORS AND THEY SHRUNK BUT THIS WAS MANY, MANY YEARS AGO [INDISCERNIBLE]. FOR EXAMPLE TUMOR WHICH IS BAY SUCKLY NOTHING BUT IN CASE OF [INDISCERNIBLE] WHICH I MENTIONED [INDISCERNIBLE] WHICH HAS NO EFFECT ON [INDISCERNIBLE] RECEPTORS. BUT IN THAT CASE [INDISCERNIBLE] STUDIES THEY ARE SHORING [INDISCERNIBLE] BUT THIS IS OBVIOUSLY MORE PROMISING AND THIS WAS [INDISCERNIBLE] — IN CERTAIN PATIENTS WHO [INDISCERNIBLE] AND DISEASE AND Ph.D. VERY LOW [INDISCERNIBLE] LEVELS INDUCED TO [INDISCERNIBLE]. IF THEY ARE RESPONDING TO THIS [INDISCERNIBLE] RESPONDING [INDISCERNIBLE] — AGAIN, IT’S COMPETITION EVEN IN CANCER PATIENTS WHO [INDISCERNIBLE] THEY ARE REACTING [INDISCERNIBLE] BUT I MEAN I THINK MOST STUDIES GOES ON AND MAJOR PROGRAM [INDISCERNIBLE] THAT YOU CAN BUY MOST ANYTHING BUT YOU HAVE NO IDEA WHAT EFFECT [INDISCERNIBLE] AND THIS MAKES DIFFERENCE BECAUSE NOW CHECK THE TRAM LIKE [INDISCERNIBLE] AND ALSO THE OTHER THING THAT — AND AGAIN I’M JUST [INDISCERNIBLE] MOUSE THAT CAUSES [INDISCERNIBLE] IMPOSSIBLE TO DO CLINICAL TRIALS BECAUSE EVERYBODY THAT GOES THROUGH [INDISCERNIBLE] YOU CAN BUY [INDISCERNIBLE] YOU HAVE NO IDEA WHAT IS THERE. THERE ARE FIVE HUNDRED [INDISCERNIBLE] WAS BECAUSE OF THE DISEASE [INDISCERNIBLE] THERE WAS JUST Ph.D. ON THE [INDISCERNIBLE] BECAUSE OF THE HIVE BUT THERE WAS NO REAL IMPROVEMENT. [INDISCERNIBLE] TEMPERATURE YOU WILL GET THE [INDISCERNIBLE] BUT LOOK AT DIFFERENT [INDISCERNIBLE] BUT YOU CAN GET [INDISCERNIBLE] CARCINOGEN, EVERYTHING, [INDISCERNIBLE] HEAVY METALS AND SO GOING THERE UH YOU CAN GO [INDISCERNIBLE]. SO THIS IS VERY COMPLICATED AND THINK DEFINITELY HAS TO BE [INDISCERNIBLE] BECAUSE OTHERWISE IT’S JUST A HUGE MESS AND EVENTUALLY JUST EXPLORE THEM REALLY DO NOTHING.>>[LOW AUDIO].>>YEAH, EXACTLY.>>[LOW AUDIO]. [APPLAUSE]>>THANK YOU VERY MUCH.

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