>> GOOD AFTERNOON. WE'LL GET STARTED. THANK YOU ALL FOR BRAVING THE ELEMENTS. MY GRANDMOTHER USED TO SAY THIS IS A DAY THAT'S GOOD FOR DUCKS. [LAUGHTER] THIS IS -- YOU KNOW, MAYBE YOU MAY BE REALLY -- MOST OF YOU ARE MUCH YOUNGER AND SO UH YOU KNOW THESE THINGS, BUT I, WHEN I WAS ONLY ABOUT A YEAR, TWO-AND-A-HALF YEARS AGO THAT I THOUGHT MITOCHONDRIA WERE THE WAY, THEY LOOKED THE WAY I WAS TAUGHT AND DID SOME ELECTRON MICROSCOPY AND SO FORTH, I THOUGHT THEY WERE NICE STABLE THINGS THAT JUST SAT THERE AND OF COURSE THAT'S NOT TRUE, AND SO THIS IS A PICTURE OF THE DANCE OF THE MITOCHONDRIA WHICH IS GOING ON IN EVERY LIVING CELL EXCEPT THE ENRICHMENT SITE THAT DOESN'T HAVE ANY MITOCHONDRIA. AND IN THE PROCESS OF THE DANCE, THEY GET LONGER, THEY MOVE, THEY FRAGMENT, THEY COME BACK TOGETHER AGAIN. AND THIS IS A GRAMMATIC EXAMPLE OF THE IMPACT OF DYNAMIC IMAGING ON STATIC BIOLOGY AND EVEN TO SOME EXTENT BIOCHEMISTRY A AS STATIC BIOLOGY. OKAY. WHAT? >> [INDISCERNIBLE]. >> I DON'T REMEMBER BUT IT LOOKS THE SAME IN ALL OF THEM. [LAUGHTER] SO TODAY'S TOPIC OF THE MITOCHONDRIA IS ANOTHER AS ALL THE EXAMPLES ARE PRESENTED BEAUTIFUL ILLUSTRATION OF THE BRIDGING BETWEEN BASIC SCIENCE. IN THIS CASE IT'S [INDISCERNIBLE] ON THIS SIDE OF THE BRIDGE AND HUMAN DISEASE, ANY DISEASE -- THAT'S NEW YORK ON THE OTHER SIDE AND THIS OF COURSE IS THE BROOK LYNN BRIDGE. THE WHOLE NAME OF THE GAME WHETHER IT'S BACK IN THE 19th CENTURY OF BRIDGE BUILDING OR IN THE PRESENT TIME IS REALLY TO LINK THE INCREDIBLE ADVANCES GOING ON IN MODERN BIOLOGY WITH A BETTER UNDERSTANDING OF CONCEPTUAL PHYSIOLOGY AND IT'S RELATIONSHIP TO DISEASE, AND SO THAT'S THE POINT. NOW NORMALLY WE PRESENT A LIVE PATIENT WHICH WE'VE NOT BEEN DOING TOO MUCH THIS YEAR BUT WILL AGAIN, AND THE REASON WE DO IT IS BECAUSE YOU FOLKS KEEP TELLING US THAT IT PUTS A HUMAN FACE ON A DISEASE, SO I ASSURE YOU THAT THERE'S EVERY INTENTION TO CONTINUE THIS, IT'S JUST THAT SOME OF THE TOPICS, LIKE EBOLA AND DANGI, TUBERCULOSIS DIDN'T QUITE FIT WITH HAVING PATIENTS SO WE'LL MOVE ON. OKAY. SO I THINK IT'S INTERESTING JUST VERY BRIEFLY TO REFLECT ON THE TRANSITIONS IN SCIENCE AS TECHNOLOGIES AND HOW THEY, IN TURN, THINKING ABOUT THE BIOLOGY AND VICE VERSA. SO MITOCHONDRIA ARE GOOD EXAMPLE THAT SOMETHING WE'RE SEEING BACK IN 1890s, AND IT WASN'T QUITE CLEAR WHAT THESE STRUCTURES WERE. THEY WERE DONE WITH PRIMITIVE STAINING TECHNIQUES BUT THEY'RE SITTING IN CELLS THAT WERE A MYSTERY. IN THE 20th CENTURY, OF COURSE, THOSE STRUCTURES BECAME ASSOCIATED WITH BIOCHEMISTRY, CLASSIC WORK FIRST ARE RELATING TO RESPIRATION AND THEN THE DISCOVERY OF THE CYTOCHROMES BY HUMAN AND THEN LIPMINUTES WORK ON UH HOW ENERGY IS COUPLED TO THE POTENTIAL. BUT ALL OF THIS WAS DONE ON HA KNOWLEDGE NATES AND FRACTIONS AND YOU COULDN'T QUITE PIN IT DOWN TO ORGANELLES UNTIL THE PERIOD OF WHERE ALBERT QUAD PILATET DO YOU KNOW AND SO FORTH, NO, VACOFF CREATED THE WHOLE FIELD OF SUB CELLAR FRACTIONATION WHICH INCREASINGLY PERMITTED MORE PURIFIED FRACTIONS. SO YOU REALLY COULD HAVE SOMETHING THAT WAS NEVER PURE MITOCHONDRIA BUT AT LEAST HIGHLY ENRICHED. THAT FORMED THE BASIS OF WHAT'S BG BEEN SHOWN BIOCHEMICALLY WITH SOMETHING STRUCTURALLY. IT WAS AROUND THIS TIME THAT THE FIRST ELECTRON MICROSCOPY EMERGED WHICH THEN GAVE A MUCH CLEARER PICTURE OF WHAT THE ORGANELLE ACTUALLY LOOKS LIKE. IN THE LATE 1950s, SINKVIT COINED THE TERM WHICH IS APURE BIOLOGY FOLKS THAT THE MITOCHONDRIA IS THE POWER HOUSE OF THE CELL. THEN WE ENTER A WHOLE NEW AREA WHERE IT'S DISCOVERED THAT MITOCHONDRIA HAVE RIBOSOMES, GENES, THE GENOME HAS BEEN SEQUENCED, THE INHERITANCE THROUGH THE FEMALE ESTABLISHED, AND THESE GENOMIC STRUCTURES HAVE BEEN DONE REMARKABLE THINGS TO TRACE THE ORIGIN WHICH WE WON'T -- I'M NOT GOING TO DISCUSS OR MENTION ANYTHING ABOUT TODAY, BUT WHERE DID MITOCHONDRIA COME FROM? ARE THEY REALLY BACTERIA THAT ULTIMATELY THEIR GENOME WAS INCORPORATED? THEN THREE AND FOUR WHICH ARE THE SUBJECT OF TODAY'S EXCITING ADVENTURES. THIS APPLICATION OF DYNAMIC TECHNOLOGY TO DISCOVER THINGS OF HOW THE MITOCHONDRIA DIVIDE BY BINARY FISSION, HOW THEY MOVE, HOW THEY FUSE, HOW THEY FRAGMENT, AND WHAT DOES THAT MEAN? HOW DO THEY TURN OVER? HOW DO THEY DODGE? A AND WHAT'S THE ROLL OF ALL OF THIS IN INHERITABLE AND ACQUIRED DISEASES? A HUGE NUMBER IN BOTH OF THOSE CATEGORIES ARE NOW SUBJECT TO STUDY AND MANY MITOCHONDRIA Y'ALL DISEASES HAVE BEEN
ESTABLISHED. ALL OF THIS MOVES IN DIRECTION OF CONCEPT, THE PARADIGM THAT IF YOU KNOW HOW IT HAPPENS IN DETAIL, MAYBE YOU CAN FIGURE OUT SOME WAY TO CONTROL IT, PREVENT IT, BY PASS IT, TO BE ITEM SIX FOR THE SECTION 20, I SUSPECT. WE'RE REALLY FORTUNATE AT NIH TO HAVE SEVERAL MANY INDIVIDUALS WHO WORK ON MITOCHONDRIA BIOLOGY AND WE'RE FORTUNATE TO HAVE TWO OUTSTANDING SUCH ONES TO SPEAKTOUS TODAY. THE FIRST SPEAKER IS GOING TO BE RICHARD YOULE WHO GRADUATED FROM ALBIAN COLLEGE IN MICHIGAN AND THEN GOD GOT A Ph.D. AT THE UNIVERSITY OF SOUTH CAROLINA. RICHARD CAME HERE IN THE SECTION OF NEUROCHEMISTRY AND HAS BEEN HERE SINCE 1977? HE'S CURRENTLY CHIEF OF THE BIOCHEMISTRY SECTION OF THE SURGICAL NEUROLOGY BRANCH OF NINDS AND WE PUT ON THE WEB SITE -- AND I'M SURE MOST OF YOU HAVE SEEN HIS ARTICLES A AND PARTICULARLY TWO EXTRAORDINARY REVIEW ARTICLES THAT BIGGERER CONCEPT PARTICULARLY FOR PEOPLE LIKE LIVER DOCTORS MAYBE LIKE SOME OF YOU WHO DON'T NORMALLY EAT AND BREATHE MITOCHONDRIA. OUR SECOND SPEAKER IS MICHAEL SACK WHO TOOK HIS H.D. AND Ph.D. IN SOUTH AFRICA. HE CAME HERE TO WASHINGTON UNIVERSITY IN ST. LOUIS TO BE TRAINED IN CARDIOLOGY AND THEN WENT BACK TO SOUTH AFRICA AND GOT A Ph.D. IN MOLECULAR BIOLOGY. HE IS THE NEW SPECIES OF CARDIOLOGISTS, THE MOLECULAR CARDIOLOGIST, UH BUT THIS IS NOT TRIVIAL BECAUSE AS YOU WILL LEARN, HIS STUDIES OF IN RELATIONSHIP TO MYOCARDIAL FUNCTION FAILURE AND DISEASE LINK VERY POWERFULLY TO THE ROLE OF MITOCHONDRIA AND IN DETAIL WHERE ARE WE IN TERMS OF UNDERSTANDING THESE PROCESSES. SORRY CHA SORRY -- SO, RICHARD, WOULD YOU PLEASE BEGIN? >> THANK YOU FOR PUTTING ME AND MY COLLEAGUE TOGETHER. THIS SHOULD BE FUN. HE TOOK LIKE MY FIRST TWO SLIDES. ANYWAY, SO IN A CELL, THERE'S NUCLEUS WITH NUCLEAR DNA AND NUMEROUS MEMBRANE-BOUND COMPARTMENTS AND ORGANELLES. ONE WHICH IS THE MITOCHONDRIA THAT HAS OUTER MEMBRANE AND INNER MEMBRANE. WHAT'S UNIQUE ABOUT MITOCHONDRIA IS THE OTHER ORGANELLES JUST GOT ITS OWN DNA, CIRCULAR DNA THAT WE MENTIONED, AND ENCODES 13 PROTEINS, TRNAs AND RNAs WITH 13 PROTEINS THAT GET INCORP. RATD INTO THE OXIDATED PHOSPHORYLATION MACHINERY. THIS IS THE OUTER MEMBRANE AND THIS IS THE ANYONE PER. THERE'S MANY DIFFERENT COMPONENTS MOST OF WHICH ENCODED IN THE NUCLEAR DNA. THIS IS A VERY INTERESTING AND COMPLEX EVOLUTIONARY RELATIONSHIP BETWEEN THE MITOCHONDRIA DNA AND NUCLEAR DNA AND A ALSO TRANSLATIONAL AND EXPRESSION RELATIONSHIP BETWEEN THE NUCLEAR GENES AND THE MITOCHONDRIA GENES. ALSO SINCE WE'RE TALKING ABOUT MEDICINE, I NOTE THAT THE [INDISCERNIBLE] DON'T [INDISCERNIBLE] MITOCHONDRIA TO THE FETUS EMBRYO AS THESE ALL COME FROM THE MOTHER AND THERE'S INTERESTING PROCESS DURING DEN DEN GENESIS CALLED PURIFICATION. THESE MITOCHONDRIA CAN ACCUMULATE MUTATIONS. HOW DO YOU KEEP THIS RIGOROUS OVER TEN THOUSAND YEARS? OOGOO GENESIS THERE WAS TWO PROCESSES SELECTED FOR PERFECT MITOCHONDRIA DNA SO HOPEFULLY MOST OF U.S. STARTED OUT PURE AND CLEAN. THERE ARE POER [INDISCERNIBLE] SYSTEM AND THOSE CHILDREN HAVE ARE DEVASTATING MATERNALLY INHERITED MITOCHONDRIA DNA MUTATION DISEASES AND I WON'T GO INTO THAT TODAY, BUT THERE CAN BEER RORS IN MITOCHONDRIA DNA ITSELF THAT WILL LEAD TO MUTATIONS IN SOME OF THESE COMPONENTS. SINCE MOST THERE ARE THOUSANDS OF PROTEINS. MITOCHONDRIA IN CALCIUM, IRON SULPHUR CLUSTERS. M METABOLICALLY IMPORTANT. HOW DO THESE GET INTO THIS COULD BELIEVE MEMBRANE VIOLATOR? WORK OVER DECADES FROM WALTER AND CLOUS AND MANY OTHERS I HAVE THREE POST DOCS IN MY LAB FROM DIFFERENT MEETING GROUPS AT ONE POINT. PROTEINS GET IMPORTED THROUGH THIS MEMBRANE, TO THE MATRIX. SOME MEMBRANE PROTEINS CAN BE IMBEDDED STOP ALONG THE WAY AND THERE CAN BE PROTEINS IN THE MEMBRANE SPACE. I'LL COME BACK TO THIS LATER. THIS IS DEPICTION YOU SEE IN ALL THE TEXTBOOKS AND THAT'S BASED ON THE EM IMAGES A AND THESE ARE THE CHRISTY, THE INNER MEMBRANE, O OUTER MEMBRANE. ERA OF EM TALKING ABOUT THAT TALKING AA BOUT THE KINDS OF MENTALITY AND WITH THE RECENT ADVENT THE BIG ADVANCES IN CON FOCAL MICROSCOPY OR LIGHT IMAGING IT'S MORE APPARENT TO ALL OF US THAT THEY'RE NOT NECESSARILY BEAN-SHAPED. THEY HAVE THIS HETEROGENOUS -- I'LL SHOW ANOTHER MOVIE, THESE ARE HELLA CELLS. THEY'RE QUITE MOBILE AND MOVE AROUND. THEY HAVE DIFFERENT MORE THOL JIS. SOME ARE ROUND, SOME TUBULAR. THIS INTERESTING NEW TOPIC PEOPLE THINK IT'S KIND OF CURIOUS BECAUSE WE USED TO THINK THEY LOOK LIKE THIS AND NOW WE THINK THIS. BUT IN FACT BEFORE EM WHEN THESE WERE DISCOVERED A HUNDRED YEARS AGO THEY USED LIGHT MICROSCOPY AND THEY KNEW THIS ALREADY AND IN FACT THAT'S EMBODIES IN THE NAME OF MITOCHONDRIA BECAUSE THIS IS GREEK FOR MITO. A HUNDRED YEARS AGO THEY KNEW THEY HAD THIS PLASTIC HETERO GENIUS MORPHOLOGY. WHAT THEY DIDN'T KNOW A HUNDRED YEARS AGO IS THAT SOME OF THE GENES THAT ARE REGULATING THIS MORPHOGENESIS HAVE BEEN IDENTIFIED. THESE ARE SOME OF THE PIONEERS THAT DISCOVERED THESE THREE TYPES OF LARGE GTP ACES. HE'S INVOLVED IN FISSION OF BOTH INNER MITOCHONDRIA MEMBRANE AND THEN THE OUTER MEMBRANE GETS DIVIDED. INFUSION, INVOLVED IN FUSING JUST OUTER MEMBRANE. OPRAH ONE THIS INVOLVED IN FUSION OF THE INNERxD MEMBRANE. FOCUS ON JE NET CALL LINKS TO MITOCHONDRIA IS THAT MUTATIONS -- SO INITIALLY YOU THINK WHY DO THEY BOTHER TO DO THIS? WHAT IS ALL THIS FISSION, FISSION? LET ME BACK UP A MINUTE. FORGOT TO SHOW THE MOVIE. NOTICE THESE COUPLE SPOTS DOWN HERE. SEE THIS TUBE Y'ALL IS GOING TO BREAK OFF IN A SECOND. SEE HOW BROKE OFF THERE, THAT'S A FISSION. THEY'RE BROKE IN HALF AGAIN. THIS ONE JUST BROKE OFF. WATCH THESE TWO ARE GOING TO FUSE. QUESTION IS WHY? IS THIS IMPORTANT FOR ANYTHING? AND IF SO, WHAT? THE REASON WE KNOW IT'S IMPORTANT IS THERE'S TWO FUSION GENES HAVE BEEN IDENTIFIED TO HAVE GENETIC LINKS IN NEUROGENETIC DISEASES. LOSS OF ONE ALLELE CAUSES MOST BLINDNESS. TWO GENES IN THE FUSION PATHWAY SHOWING THERE THIS IS AN ESSENTIAL PROCESS AND WHAT I'M GOING TALK ABOUT IS WHY. IT'S INTERESTING AND CURIOUS WHY THESE DIFFERENT CELL TYPE ACTIVITY OF TWO FUSION GENES AS IN MANY MITOCHONDRIAL DISEASES IS NOT KNOWN. IT DOES SHOW IMPORTANT OF THIS PROCESS. IT'S A LITTLE BIT HARD TO STUDY IF YOU JUST LOOK AT CELLS AND COUNT EVENTS, BUT A POST DOC DEVELOPED A VERY NICE QUANTITATIVE METHOD TO ASSESS THIS. HE DID IT SHORTLY AFTER THIS GROUND-BREAKING WORK HERE AT THE NIH BY GEORGE PATTERSON WHO DEVELOPED FERTILE ACTIVATABLE FORM OF GFP. POST DOC GOT IT FROM THEM WITHIN DAYS. BEING IN NIH, YOU SEE A PAPER AND NEXT DAY GO GET A CONSTRUCT. HE TARGETED THROUGH PATHWAY TO MAY TRICK COMPARTMENT AND THIS IS SA HELI CELL BEEN TRANSFECTED BY -- YOU CAN SEE THE MITOCHONDRIA THROUGHOUT THE WHOLE CELL BUT AS GEORGE AND JENNIFER SHOWED, IF YOU SHINE A LASER ON THIS, YOU CAN PHOTO ACTIVATE IT AND MAKE A VERY SMALL RAIZOR THROUGH TWO SPOTS AND WITHIN MILLISECONDS IT FILLS THE BOTTOM OF THAT TUBE. IT'D BE HARD TO SAY WHERE THE BEGINNING OF ONE IS AND WHERE THE END OF THE ANOTHER. THEN YOU CAN WATCH THIS OVER THE NEXT 30 MINUTES AND YOU CAN QUANTIFY THE FUSION RATE BY LOOKING AT THE [INDISCERNIBLE] THROUGHOUT THE WHOLE NETWORK. OVER 30 MINCE YOU SEE THE FUSION THERE, IT DILUTES IT OUT. HERE'S THE NEXT ONE. GENERALLY IN HELY CELLS WITHIN 30 MINUTES THIS IS THROUGHOUT THE ENTIRE CELL. OPRAH ONE, IF YOU DO RNA NINE TO KNOCK DOWN OPRAH ONE, YOU SEE OPRAH ONE RNI MUCH MORE PUNK FORM BUT WATCH THE FUSION PROCESS DUE OVER THE SAME TIME COURSE. THESE COMPLETELY E QUILL BRAT. THEY CAN'T FUSE AND GFP DOES NOT DILUTE OUT. TECHNIQUES TO QUANTIFY THIS PROCESS. VRP IS IN SAME FAMILY BUT INVOLVED IN FUSION PROCESS. IMPORTANT WORK WAS DONE HERE AT NIH AND OFFICIALLY BY ALEX VAN DER BEAK'S GROUP AT UCLA WHERE HE WAS ABLE TO LABEL AND WATCH IT IN REAL TIME. YOU CAN SEE NS IT'S RED GREEN DOTS. IT'S BIGGER, THERE'S A BREAKAGE IN OTHER DIVISION OF THAT. OTHER WORK BY NUMBER OF GROUPS SHOWS IT FORMS A SPIRAL AROUND THE MITOCHONDRIA DRI YEAH AND CAUSES FISSION. IF YOU KNOCK DOWN GRP OR KNOCK IT OUT, THE MITOCHONDRIA DRI YEAH BECOME HIGHLY INTERCONNECTED. THE OPPOSITE OF THAT ONE; ALL COME TOGETHER. ONE I WANTED TO UPDATE MOST RECENT ADVANCES COMING FROM LABS AT COLORADO AND UC DAVIS. THEY FOUND THIS INCREDIBLE PROCESS WHERE ER TUBULES WILL WRAP AROUND A. IT'S NOT WHEN WHY THIS HAPPENS. -- LATEST IMPORTANT TOP NICK THE FIELD FOR WHAT DOES THAT DO, HOW DOES THAT TRIGGER FISSION AND HOW DID THAT EVER COME ABOUT? ANYWAY WHAT ALL MY FOLKS NOW [INDISCERNIBLE] THIS IS NOT SO OBVIOUS. THEY CLEARLY HAVE [INDISCERNIBLE] MAINTAIN HEALTH. ONE THEORY IS THAT THERE'S DAMAGE OR THIS IS DEFICIENT IN ONE SUB SPENS OR PROTEIN AND THIS IS DEFICIENT IN ANOTHER. IF THEY USE THEY NEE MAY I COMPLIMENT ONE ANOTHER EETS ACTIVITY [INDISCERNIBLE]. FUSION IS STIMLATE BID TWO PROCESS: INCREASED ENERGY E DEMAND CAUSES AND ALSOçó
STRESSFULxD CONDITIONS. FUSION'S Q#áRQ) TO UNDERSTAND IN SOME WAYS. WE OBVIOUSLY IT'S UNDERSTAND YOU NEED TO DIVIDE MITOCHONDRIA DRI YEAH TO REPOPULATE MORE CELLS. ANY TIME YOU HAVE MORE PEOPLE ON THE [INDISCERNIBLE] YOU NEED MORE MITOCHONDRIA. WHAT I'LL COME BACK TO LATER IS THIS IDEA AS FISSION AS QUALITY CONTROL PROCESS. THIS IS FIRST NOTED. AFTER FUSION E EVENTS ONE DAUGHTER CELLS WOULD BECOME DEGRADED. LEADING TO HIS IDEAS AND SOME EXTENSIONS OF GOD -- GOT MITOCHONDRIA IF YOU EXERCISE MAYBE SOME FORWARD PROTEINS AND DAMAGE ACCUMULATION AND ACTIVITY OF THE MITOCHONDRIA -- AS WELL AS THIS BRIE WILL AGGRAVATE A FISSION EVENT ANYWHERE WILL BE SOME KIND OF AGGREGATION OF GOOD AND BAD. THERE'S EXAMPLE OF THIS IN WIDE SHAPE BACTERIA KNOWN TWO DAUGHTER CELLS OF BACTERIA DIVISION RO NOT EQUAL. ONE WILL BE MORE RIG LOUSE ROUSLY MAINTAINED FOR FUTURE GENERATIONS AND DEBRIS WILL BE [INDISCERNIBLE] ONE OTHER DAUGHTER CELLS. I'M GOING COME BACK TO THAT BECAUSE RECENTLY ME JE NE MECHANISMS HAVE BEEN WORKED OUT. FIRST I WANT TO GO TO ONE OF THE OTHER INTERESTING PROCESSES THAT OCCURS DURING FISSION AND IT OCCURRED DURING APOPTOSIS OF PROGRAM CELL DEATH. I THINK PROGRAM SAW REVERSIBLE PHASE BECAUSE SIGNAL COMMITMENT STAFF AND THEN IRREVERSIBLE PHASE. A LOT OF THINGS HAPPEN IN IRREVERSIBLE PHASE. THE DECISION SPOINT WHEN ONE OF THESE B CELL TWO MEMBERS TRANSLOCATES FROM CYTOCELL TO MITOCHONDRIA, CAUSES RELEASE OF CYTOCHROME C FROM MITOCHONDRIA AND THAT ACTIVATE CASP A TE. [INDISCERNIBLE]. I HAD A POST DOC AND MEDICAL STUDENT WHO MADE THIS INTERESTING FIND MAYBE TEN YEARS AGO THAT RELATES TO THIS COMMITMENT STEP. BEFORE BACK STRAP TRANSLOCATES YOU CAN REVERSE BUT AFTERWARDS YOU CAN'T. CELLS ARE DOOMED IN ANY CASE. WHAT THIS MEDICAL STUDENT USES GREEN PROTEINS AND LABELED THESE FIVE CELLS. TWO OF THEM HAD BEEN TRANSFECTED WITH RFP MITOCHONDRIA IMPORT. TWO CELLS HAVE GOT THE MITOCHONDRIA LABELED. BACKS IN CITE SOL OF ALL OF THEM. THIS IS WHAT IT LOOKS LIKE, VERY DIFFUSED. IF YOU DO APOPTOSIS WATCH WHAT HAPPENS OVER 30 MINUTES. LOOK AT THAT CHANGE IN THE BACK DISTRIBUTION. MITOCHONDRIA TRANSLOCATES TO THE MITOCHONDRIA, [INDISCERNIBLE] SPOTS ON MITOCHONDRIA AND THAT'S WHEN CITE CHROME C IS RELEASED FROM MITOCHONDRIA. HERE'S A BLOWUP OF ONE MITOCHONDRIA AND THE BACKS IN RED. THIS IS DONE BY EXPERT OF MIE CROSS PI. YOU CAN SEE OVER SECONDS THIS DOTS FORM ON MITOCHONDRIA AND THEY ASSEMBLE AT DECRYPTION SITES. YOU SEE THOSE TWO COME TOGETHER AND THEIR MOTHER KIND OF BREAKS OFF AND THERE'S BACK ON THE TIPS. HERE'S MORE STATIC IMAGES SHOWING THAT THIS BACK GOING FROM MITOCHONDRIA ASSEMBLES AT THE TIPS ON THE MITOCHONDRIA. YOU CAN SEE THIS BY ANY NOTE EM. [INDISCERNIBLE] MAJOR OF DR P P I SHOWED YOU. BACKS IS GOING TO THESE DRP SITES. AS I MENTIONED CITE CHROME C IS ARE RELEASED. HERE'S WHAT FIELD OF CELLS INDUCED. THESE ARE MITOCHONDRIA WHERE CITE CHROME C IS STILL IN MITOCHONDRIA. HERE'S A CELL WHERE IT'S COMPLETELY RELEASED. LOOK AT THESE THREE CELLS. THEY CLEARLY HAVE CITE CHROME C STILL IN MITOCHONDRIA, SOME RELEASED HERE, BUT YOU CAN SEE MITOCHONDRIA ARE FRAGMENTED. THAT'S WHAT I'M TALKING ABOUT CORRESPONDING TO TRANSLOCATION OF MITE FISSION OCCURS, MITOCHONDRIA FRAGMENTS, YOU'LL NEVER FIND A TUBULAR MITOCHONDRIA AT CITE CHROME C RELEASE. IF UH YOU INHIBIT, YOU PREVENT THAT FRAGMENTATION. MITOCHONDRIA WEB. LOOK AT THE BACKS TRANSLOCATES AND GREEN SPOTS AND YOU CAN SEE MANY AMONG TIPS AND CONSTRICTION SITES. EM OF BACKS ON ONE OF THESE MITOCHONDRIA THAT'S PREVENTED FROM DIVIDING BUT YOU SEE BACKS ASSEMBLING THESE LARGE SPOTS. WHAT DO THESE REPRESENT? WHAT'S THE NATURE OF THIS? THIS IS PROBABLY WHERE CITE CHROME C IS COMING OUT. HOW LONG SHALL I GO? A QUARTER TO FIVE? I WANT TO COME BACK TO THIS BECAUSE THIS IS THE CURRENT FOCUS OF MY LAB. DUOG MUTATED IN PARK INSON'S DISEASE HAVING LINKED TO THIS MITOCHONDRIA ONTOLOGY. THERE'S ONLY THREE KNOWN MONO GENIC AUTO SEW MALL [INDISCERNIBLE] PARKINSON'S DISEASE. THERE ARE DOMINANT CAUSES LIKE LARK TWO. THOSE ARE PROBABLY, YET ONE COPY OF A MUTANT GENE AND YOU GET THE DISEASE. A LOT OF THOSE THOUGHT TO BE GAIN FUNCTION MUTATIONS. FOLDS PROTEIN IS DOING SOMETHING BAD 'CUZ YOU GOT A GOOD COPY AND BAD COP PUPPY AND IF YOU GOT THE BAD COPY YOU'RE GOING GET THE DISEASE. THERE'S ONLY, THESE AWE AUTO SOW MALL RESE SIEVE. IN MOST OF THOSE CASES I CAN'T THINK OF ONE CASE WHERE PEOPLE FIGURED TOUT CAUSE OF DISEASE. THESE ARE LEEKLY LOSS OF FUNCTION MUTATIONS. DON'T HAVE PARKIN', YOU GET PARKINSON'SES. IF YOU DON'T HAVE PINK ONE YOU GET PARKINSON'S. THIS IS BEFORE WE WORKED ON IT. WINK ONE WAS KNOWN TO BE A KINASE AND IT HAD A MITOCHONDRIA TARGETING SEQUENCE AND THAT LED PEOPLE DISCOVERING MUTATED DV IN CELLS IT'S MITOCHONDRIA. THERE'S A THIRD ONE CALLED DJ ONE AND I DON'T KNOW WHERE IS DJ ONE IN CITE SOEM? WHAT DO THESE DO AND IS THERE ANY E RELATIONSHIP BETWEEN THEM? PEOPLE MAID KNOCKOUT MICE, NO PHENOTYPE. PEOPLE WITH MUTATIONS NEEDED THOSE FEE KNOWTOPY, IN OTHER WORDS, PARKINSON'S BUT MICE KNOCK OUT PINK ONE, NO PHENOTYPE. LIKE MITOCHONDRIA DISEASE YOU CAN IMAGINE MITOCHONDRIA DIFFERENT SPECIES ARE IMPORTANT IN DIFFERENT TISSUES. LIKE BUMBLEBEES AREN'T SUPPOSED TO BE ABLE TO FLY, THEY'RE FLIGHT MUSCLES ARE VERY AEROBIC. EAGLES HAVE MUSCLES IN THEIR EYES. FLIES HAVE MUSCLES. DID EM ON FLIES AND MITOCHONDRIA WERE SWOLLEN. HERE'S A LINK OF THIS PROTEIN TO MIKE KOOND THIS ONE'S BEEN LOCALIZED. COUPLE OTHER LABS STEM FROM THAT, THEY DID THE SAME THING WITH PINK ONE AND FEE KNOW COPIES THE PARKIN' IN THE FIVE. THEY DID EPI STAY SIS. THE QUESTION IS, WHAT IS THE PATH WAY? DO THEY FUNCTION TOGETHER AND WHAT DO THEY DO? I'M JUS GOING TO SUMMARIZE A BUNCH OF WORK FROM OUR LAB AND OTHER LABS OVER THE LAST FIVE YEARS. COULD WE HAVE FOUND THEY FUNCTION TOGETHER? I'LL COME BACK TOGETHER HOW THIS MAY BE RELATED TO PARKINSON'S. IF YOU HAVE A CELL AND ONE OF THOSE MITOCHONDRIA GETS DAMAGED, WHAT HAPPENS IS THIS KINASE PING ONE ACCUMULATES JUST ON THE DAMAGED ONE. HOW CAN YOU IMAGINE THAT WOULD HAPPEN? HOW DOES PING ONE KNOW? MITOCHONDRIA IMPORT SEQUENCE. I'LL SHOW YOU THIS IN A MINUTE. PINK ONE'S A KINASE. WHAT IT DOES IS THROUGH ITS KINASE FUKS IT RECRUITS PARKIN' TO THE MITOCHONDRIA AND
ACTIVATExD PARKIN'S IN A OFF STATE. PING ONE ACTIVATES IT, RECRUITS IT TO MITOCHONDRIA SURFACE [INDISCERNIBLE] OUTER MEMBRANE. THESE CHANGE STIMULATE [INDISCERNIBLE] AND ELIMINATION SUGGESTING THAT IF MITOCHONDRIA GETS DAMAGE AND ELIMINATE MAYBE THIS IS A QUALITY CONTROL PATHWAY. THIS IS IMPORTANT BECAUSE IF THIS IS TRUE AND IF IT'S ALL THAT THEY'RE DOING IT SUGGESTSÑi AN IDEOLOGY OF PARKINSON'S. I'LL GIVE YOU EVIDENCE FOR IT. ONE OF MY STUDENTS DID THIS, SURGERY DEPARTMENT, MIDDLE OF MEDICAL SCHOOL. WE'VE BEEN WATCHING OTHERS AND THOUGHT WE MIGHT TRY OUR OLD TRICK. WHY DON'T YOU TAKE PARKIN' USE GFP TO THAT AND SEE WHAT HAPPENS. YOU CAN'T SEE IT WELL BUT MAYBE THIS IS WORTH TURNING THESE LIGHTS OUT. CHANGE THE PROGRAM. CAN'T DO THAT. IT'LL BECOME VERY CLEAR. TWO CELLS, MITOCHONDRIA STAINED IN RED, GFP FUSED IN PARKIN' AND IT'S DEFINITELY IN CYTOSOL. -- THEN, THEY'RE THOUGHT LET'S ADD HERBICIDE. SAW SOME VERY STRANGE THINGS HAPPENING. PARAQUAD, PLAYING AROUND WITH THOSE THINGS AND FOUND A VERY SIMPLE NON-TOXIC REVERSIBLE STEP CALLED ADD UNCOUPLER. REMEMBER IN SECOND SLIDE OX FOS GENERATE ARES [INDISCERNIBLE] ACROSS INTERMITOCHONDRIA MEMBRANE. HOW MUCH TIME HAVE I GOT? I GOT TIME? UNCOUPLE REVERSES THAT PROTON GRADIENT. THERE'S UNCOUPER WE USE A LOT. HE ADD THIS CCCP AND WATCH WHAT HAPPENED TOSS THE GREEN PARKIN' AFTER HE TRIED MANY THINGS AND NOTHING WOULD CHANGE, HE NOW SEES ROBUST TRANSLOCATION TO THE MITOCHONDRIA. THAT'S WHY WE REMENTIONED IN THAT REVIEW SLIDE WE KNOCKED OUT PINK ONE AND THAT PREVENT BONE TO MITOCHONDRIA. THIS LINKS THESE TWO GENE PRODUCTS LOCATED IN SAME PATHWAY. PINK ONE RECRUITS -- -- PATIENT MUTATIONS IN PARKIN' AND PING ONE REE DEUCED OR COMPLETELY BLOCKED TRANCE ACTIVITY. WHAT DOES THIS MEAN FOR NORMAL HUMAN BIOLOGY. WE LOOKED A AT CELL TYPES. REMEMBER I WAS TALKING ABOUT MUTATIONS THAT CAUSE TOOTH DISEASE? DAVID 1 AND 2. MADE MICE EMBRYONIC FIBERGLASS AND GENEROUSLY E SHARED THEM. LOOKED AT MITOCHONDRIA MEMBRANE POTENTIAL. UNCOUPLERS, UNCOUPLED MITOCHONDRIA MEMBRANE POTENTIAL. DOUBLE KNOCKOUT HAS ABOUT 10% OF MITOCHONDRIA ARE UNCOUPLED. NONE UNCOUPLED HERE OR HERE. WHAT ABOUT A GE NATE MODEL? -- -- 98% OF THE CELLS LOOK LIKE THIS, SAME HERE, SAME HERE, BUT IN DOUBLE KNOCKOUT MOST BY FAR 90% LOOK LIKE THIS WITH THESE SPOTS OF PARKIN'. WHAT DO THEY REPRESENT? SAME TYPESETUP END IN ONE, TWO, DOUBLE KNOCKOUT CELLS EXPRESSING GFP PARKIN' ALSO [INDISCERNIBLE] MEASURE MEMBRANE POTENTIAL. HERE'S CYTOSOEM C. UH YOU SEE OFF THE BAT THAT THERE'S PARKIN' NOT IN ALL THE MITOCHONDRIA. LOOK AT THESE TWO, LOOK ON THE RIGHT SIDE ARE, ONE OF THEM HAS LOST MEMBRANE POTENTIAL AND ONE HAS NOT. IF YOU LOOK HERE WHERE THE PARKIN' IS, PARKIN''S ON THE OTHER ONE. THERE'S MITOCHONDRIA LOST NUMBER POTENTIAL. SEEMS PARKIN' CAN SOMEHOW IDENTIFY THE ONE DAMAGED OUT OF 50. HOW DOES IT DO THAT? A LOT OF PAPERS HAVE COME OUT AND A LOT OF DIFFERENT UNCOUPERLERS HAVE BEEN SHOWN. EVEN UNFOLDED PROTEINS WITHOUT ANY UNCOUPLING IS MITOCHONDRIA MEMBRANE CAN CAUSE [INDISCERNIBLE] WHAT'S THE COMMON DENOMINATOR? PING ONE ACCUMULATION ON THE OTHER MITOCHONDRIA MEMBRANE. HOW WOULD THAT WORK? YOU KNOW HOW SOME DO POST DOCS, MENTORS? I SUGGEST SOMETHING AND THEN THEY OF COURSE SAY THAT'S CRAZY. I'M THINKING KINASES ARE TURNED ON AND OFF. GUYS AND GALS ARE REALLY GOOD AT WESTERN BLOTTING AND THEY WANT TO DO THAT. WHAT ARE THE CHANCES THAT'S GOING WORK WITH A KINASE? THEYTH DID ANYWAY. THEY BLOT FOR PINK ONE. THIS IS ENDOGENOUS HEE LA CELLS. [INDISCERNIBLE] PING ONE ANTIBODIES DON'T WORK. ADD CCCP. THIS CAUSES PARKIN' TRANSPORTATION AND YOU SEE [INDISCERNIBLE] BAND RIGHT AT THE RIGHT EXACT PINK ONE SO WE TALK TO MARK COOKSON AND SHARE RNAI FOR PINK ONE. ARE RNA PINK 1 COMPLETELY RUBBED IT OUT. THE REASON YOU CAN'T SEE IT IS BECAUSE WELL WHY CAN'T YOU SEE IT THERE AND WHY CAN YOU SEE IT HERE? A CCCP IS EASILY WASHED OUT. IF YOU WASH IT OUT FOR TWO OR FIVE MINUTES, VERY RAPIDLY DISAPPEARS. PING ONES RAPIDLY TURN OVER. WE FOUND ONE CALLED PAROL, KNOCK IT OUT, IT PREVENTS THIS DEGRADATION. CCCP [INDISCERNIBLE] GOES AWAY, UPPER BAN -- LOWER BAND COMES BACK AGAIN. [INDISCERNIBLE] -- -- THIS PUT TOGETHER WITH A LOT OF DATA I DON'T HAVE TIME TO GO INTO LEADS TO THIS MODEL. MITOCHONDRIA TARGETING SEQUENCE -- REMEMBER UH I SHOWED YOU TOM COMPLEX. THIS IS CLEAVE ON IMPORTED PROTEIN BY MPP. THAT'S WHAT THIS IS. WHEN IT GETS IMPORTED IT GETS CLIPPED BY MPP THEN I'M LIKE ANY OTHER PROTEIN KNOWN IN THE MAMMALIAN MEMBRANE ANYWAY. SUB STRAIGHTS KNOWN IN YEAST BUT NOT IN MAMMALS; THIS IS THE FIRST ONE. IT CLINGS RIGHT ABOUT THERE IN THE MEMBRANE SPANNING DOMAIN OF PINK ONE. THEN I HAD VERY CLEVER JAPANESE POST DOC AND WE KNEW PEOPLE SHOWN EARLIER YOU COUNT SEE PICK ONE BUT IF YOU ADDÑi PROTOOWE SOEM -- [INDISCERNIBLE] IT'S 52 KD. GROUP IN LONG LONDON SEQUENCED THIS. -- -- PHYSICALLY SHOWS MU TAS THAT STABLE WAY CLEAR, IMPORTED, CLIP, CLIP, THERE BY THE PROTOOWE SOEM. THAT SOME TOTAL GENERATES THIS. LOW STATE OF PING ONE. YOU MAY NOT ALL KNOW BUT IF YOU UNCOUPLE MITOCHONDRIA YOU BLOCK IMPORT THROUGH THE TEN PATHWAY BUT NOT THE CON PATHWAY. THIS IS PRENTD BY UNCOUPLING. IT'S WELL-KNOWN BY IMPORT PEOPLE. SO IF YOU BLOCK IMPORT OF PINK ONE IT'S NEVER ACCESS TO PAROL AND IT'S SHOWN AT VERY GOOD BUY E YO DEKE BIO KE CHEMIST. THIS IS BOUND TO A TIME COMPLEX -- TO PREVENT IMPORT HERE AND FROM THERE KINASE FUNCTION RECRUITS BACK INTO THE MITOCHONDRIA. THIS EXPLAINS REALLY INTERESTING HOW NATURE FIGURED THIS OUT. HOW COULD PINPOINT ACCUMULATE JUST ON DAMAGED MITOCHONDRIA JUST BECAUSE HEALTHY ARE IMPORTING IT AND DEGRADING IT AND IMPORT HAS SOME KIND OF SENSOR FOR DAMAGE AND THAT ALLOWS IT TO CUMULATE ON OUTER MEMBRANE AND RECRUIT PAROL. WHAT'S PINK ONE SUB STRAIGHT. MOST EXCITING THING E WE PUBLISHED PAPER LAST YEAR ALONG WITH A A LOT OF OTHER PEOPLE. AT LEAST IT'S ARE REPRODUCEABLE. UH I WON'T GO INTO THE WHOLE STORY I'LL CUT TO THE CHASE. PINK ONE HAD BEEN SHOWN BY THIS GROUP -- THIS IS CALLED A GROUP OF MARKET DUNDEE. PEOPLE SAY WHY DOES PING ONE PHOSPHORYLATE? FIRST SEMESTER ANSWER WOULD BE MAYBE PHOSPHORYLATES PARKIN'. PAPER SAID IT DID AND WE COUNT REPEAT ANY OF THOSE DATA. THIS PAPER IN 2012 SAID PINK ONE PHOSPHORYLATES PARKIN' AND THIS IS IS THE PAPER WE COULD REPRODUCE. MUTATED THAT OUT HERE IN C TERM NOUSZ. SOME PROTEINS HAVE UBIQUITOUS. THESE GUYS SAY IT'S UBIQUITOUS AT 1065. WE DID IT IN THIS WHOLE E DOMAIN. PING ONE STILL REE KRUTED PARKIN'S MITOCHONDRIA SO E WE KNEW THERE HAD TO BE ANOTHER SUB STRAIGHT. A YEAR AGO LAB DID MAS SPECK STUDY. SHE'D BEEN TRYING FOR FOUR YEARS TO FIND SUB STRAIGHT. BASICALLY I'M PAIRING MITOCHONDRIA, WE KNEW IT SHOULD BE UT NT OUTER MITOCHONDRIA. NEVER COULD FIGURE OIT ANYTHING OUT. IN THE LAST YEAR, CRISPER TECHNOLOGY CAME OUT AND SO WE KNOCKED OUT PINK ONE IN UC CELLS. KNOCKED OUT PING ONE AND ORDERED PCCC SO BOTH. SHE IDENTIFIED SUB STRAIGHT AND AS SOON AS WE SAW IT WE KNEW IT WAS THE RIGHT THING BECAUSE E SHE FOUND UBIQUITOUS IN ITSELF. PINK ONE PHOSPHORYLATES YOU BIK TON. THESE GUYS CONFIRMS PINK ONE PHOSPHORYLATES IN UBIQUITOUS NIGHT FRAME. [INDISCERNIBLE] MIX -- -- PAPER IN APRIL THESE PEOPLE HAD SAME IDEA AND THEY PUBLISHED ANOTHER PAPER WITHIN A MONTH OF OURS. THEN THE JAP PAN GROUP HAD A PAPER THE SAME MONTH. THERE'S TWO OTHER REALLY BIG STUDIES HAVE COME OUT ALL IN THE SAME YEAR. ALL SHOWING THESE BOTH ARE POSSIBLE BUT CLEARLY FOX FOUR YOU BIK TON IS SUFFICIENT. THAT LEADS TO AN INTERESTING MODEL AI BOUT HOW TO WORKS. WE'VE BEEN WORKING ON THIS FOR MANY YEARS. THERE'S A PROCESS OF TURN OVER. WHEN PING ONE ACCUMULATES AFTER DAMAGE, IT CAN PHOSPHORYLATE THESE CHAINS AND THEN THESE CHAINS BECOME RECEPTORS FOR PARKIN'S AND PARKIN' BINDS TO THESE CHAINS AND IT ACTIVATES. IN PARKIN'S IN EACH YOU BIK TON LIE FACE. THE PARK INS PUTS CHAINS ON THE [INDISCERNIBLE] --. -- -- THIS BINDS AND ACTIVATES PARKIN'S AND PARKIN'S YOU BIK TATS -- -- GENERATE MORE PHOSPHATE YOU BIK TON AND THAT DRIVES. ONCE YOU GET THIS THING STARTS THE FEEDBACK AMPLIFICATION. LAST THING IF I'VE GOT TIME IS HOW DO THESE CHANGES SIGNAL ITOTHOGY? ONE PIECE OF EVIDENCE COMES FROM JAPAN AFTER WE FOUND THIS PATHWAY A LEADER LOOKED AT PARKIN'. THERE'S AN ADAPTER LIKE P 62 PRODUCTS NOBODY TO BIND YOU BIK TON BUT I ALSO HAVE A LURE DOMAIN BINDS TO LC 3. B 62 SO THOUGHT TO BE A LINK BETWEEN PROTEINS AND AUTO FAN. WE KNOCKOUT ALL FIVE KNOWN ADAPTERS AND THIS IS WHAT WE FOUND. I'M GOING TO BACK UP FOR A SECOND. HOW DO UH YOU MEASURE MIETHE TOSCOPY? ALL THESE LITTLE GREEN SPECKLE LS IN THE CELLS, THAT'S MITOCHONDRIA DNA. ADD AN UNCOUPLER FOR THREE HOURS, LITTLE CLUMPING. BY 24 HOURS THE GREEN STAIN'S ALMOST GONE. WE LOCKED OUT HEG 5 AND YOU'LL SEE THE GREEN DOTS. YOU SEE IT COMES UP QUITE A BIT THREE HOURS, MORE CLUMPED AT 24 HOURS BUT THEY DON'T DISAPPEAR. THEN WE KNOCK OUT ALL FIVE OF THESE ADAPTERS AND LOOKED AT THIS HERE. WE CALL IT THE FIVE TAIL AND YOU SEE THIS GREEN SPECKLES, AND HERE IT'S NOT AS CLUMPED HERE, DEFINITELY NOT DISAPPEARING, SO YOU NEED SOME OF THESE
ATDAPTERS. YOU NEED SOME OF THESE. I'M GOING WRAP UP HERE JUST KIND OF SUMMARIZE WHAT'S INTERESTING ABOUT THIS STEP IS THERE'S A NEW MECHANISM OF UH KINASE REGULATIONS THAT IMPORT CONDITIONAL DEGRADATION AND THERE'S A NEW MECHANISM REVEALED. SIMILAR TO A SUB STRAIGHT ACTIVATION OF AN ENZYME. BIG QUESTIONS ARE BOTH THE ABSENCE OF THIS PATHWAY EXIST CAUSING PARKINSON'S? THAT'S QUITE IMPORTANT. WHY WOULD SUCH A PATHWAY BE COLLECTED FOR CERTAIN TYPES OF NEURONS? NO CLUE. THERE ARE A LOT [INDISCERNIBLE] PATH TREAT MITE CONDITION DISEASES OR PARKINSON'S DISEASE? E REMINDER MITOCHONDRIA MORPHOGENESIS AND DYNAMICS -- -- MITOCHONDRIA APOPTOSIS -- -- I TRIED TO MENTION ALL MY POSTBACS A ALONG THE WAY. I'LL SHOW A PICK OF THEM. THIS IS MY GROUP UH NOW. ONE GRADUATE OPPORTUNITY RIGHT THERE AND THESE ARE THE FORMER POST DOCS I'VE HAD THAT HAVE CONTRIBUTED TO THE SLIDES I SHOWED. THANK YOU. [APPLAUSE] >> TIME FOR A FEW QUESTIONS. >> NICE TALK. WHEN YOU POLARIZE MITOCHONDRIA, LONG TERM POLAR TIEIZATION HOW DO UH YOU DO THAT CANNOT KEEP THESE GUYS POLARIZED FOR LONG? THE THING IS EVEN IF YOU TRY TO CREATE ZERO CELLS, IT'S NOT MITOCHONDRIA DNA COMPLETELY BIND IS DIMINISHED. DO YOU THINK THERE'S SELECT MECHANISM TO GET A BIT OF THE BAD MITOCHONDRIA DNA AND CELL KEEPS THEM FULL OF HEALTHY DNA? JUST A SPECULATION, DON'T HAVE ANYTHING IN MIND. >> IF YOU HAD VERY VOLUNTARIES YOU MITOCHONDRIA DNA MUTATION -- WE COLLABORATED WITH -- I'M BLANKING ON THE GUY'S NAME -- COMPLEX ONE MUTATIONS, PARKIN' WILL SELECTIVELY SHIFT TO HETERO MRAZ MY FROM MUTANT TO WILD TYPE. FOR OTHER ONES WE HAVE NOT SEEN ANY PURIFICATION. WE JUST DID A BIG STUDY WHERE WE LOOKED AT MUTATOR MOUSE. IT'S GOT MUTATION IN PRELIMINARY SCAN AND IT'S A MUTATION THAT SO THESE MICE ACCUMULATE A BIG VARIETY OF MITOCHONDRIA MUTATIONS, AND IF THESE MICE DON'T GO AS LONG, THEY HAVE SHORTENED LIFE SPAN, THEY LOOK OLD, BUT THERE'S PHENOTYPE. WE TOOK THOSE MICE WHERE WE KNOW THE CAUSE OF DISEASE. WE KNOW THE IT WAS A MITOCHONDRIA DISEASE AND WE CROSSED THEM WITH PARKIN' KNOCKOUT TO LOOK AT THE ROLL OF THE ENDOGENOUS PARKIN' IN THE MUTATOR MOUSE AND WE SAW SYNTHETIC PHENOTYPE, MEANING THEY DID WORSE IN THE ABSENCE OF PARKIN'. WE CAN RESCUE THAT WITH ALDOPA. WE DID SOME A REALLY HEAVY DEEP SEQUENCING WITH MITOCHONDRIA DNA. WE HAVE GOOD EVIDENCE THAT PARKIN' -- AND NOT JUST PARKIN' -- RESCUES THOSE MICE TO CERTAIN EXTENT, BUT WE DID THE DEEP CONVINCING OF MITOCHONDRIA DNA THAT DOES NOT RESCUE SEQUENCE. SO I THINK -- I'M JUST KIND OF GO WITH THE FLOW, I WOULD HAVE THOUGHT WHAT YOU THOUGHT AND THAT'S WHY WE DID EXPERIMENT. MAYBE IT'S PARKIN' IS RESCUING DOWNSTREAM OF DNA DAMAGE AT PROTEIN LEVEL AT THE PROTEIN AGGREGATION LEVEL TO COMPENSATE FOR DAMAGE PROTEINS OR MUTANT PROTEINS IN RESCUING THEM PERHAPS BY SOME KIND OF SELECTIVE SEGREGATION. IF YOU DON'T HAVE THAT PROCESS, THE MUTATIONS LEAD THE TO MORE GUMMING UP. >> [LOW AUDIO]. >> BASED ON PINK ONE. YOU'RE RIGHT IT CAN'T REALLY TELL WHERE THAT DNA IS. UNLESS IT'S ONE OF THE BIG THAT DON'T DIFFUSE WELL. 30 MINUTES IT'S EVER WHERE. DOESN'T PURIFY. BY AND LARGE AT THE DNA LEVEL, DOWNSTREAM PROBABLY. >> [LOW AUDIO]. >> WONDERFUL OVERVIEW OF THIS MOLECULAR CHARACTERIZATION OF MITOCHONDRIA FUNCTIONS -- DIFFERENT TREK NOW AND I'M GOING TO ACQUIRE CHANGES IN MITOCHONDRIA -- OVERLOAD OR FEEDING AND FASTING AND THE AFFECTS ON HUMAN DISEASES. I'M GOING TO TRY AND KEEP THESE -- ESSENTIALLY I'M GOING TO TALK ABOUT CALORIC LOAD, MITOCHONDR MITOCHONDRIA DEASSET LACE AND REGULATION OF [INDISCERNIBLE]. RESPECT TO SPECIFIC DISEASE THE ROLE OF FASTING AND LIVERTOX SISTIES DUE TO TYLENOL. I'LL GO BACK TO ONE SPECIAL FIELD OF LIVER DISEASE. THEN LOOK AT CERTAIN BIOLOGY MORE GLOBAL UHLY IN QUALITY CONTROL AND TALK ABOUT NEW STUDY IN THE LAB WHICH IS UNPUBLISHED LOOKING AT ROLE OF FASTING ON MITOCHONDRIA QUALITY CONTROL IMMUNE PROGRAM CALLED DNR FEE THREE INFLAMSON. LITTLE BACKGROUND HERE. I I THINK WE ALL KNOW THAT NUTRIENT AND OBESITY ARE CREATING INCREASING DISEASE BURDENS AND WE JUST LOOK AT THE U.S. FROM 1980-2008 THERE'S BEEN A HUGE INCREASE IN THIS PROCESS. WE STUDIED FROM MINNESOTA BUT SOME WERE SUGGESTING THAT 30% OF EXCESS DEATH TO CANTER, HEART DISEASE, ETC ARE IS DUE TO OBESITY AND THERE'S A HUGE FINANCIAL BURDEN ON EVERY SINGLE STATE BASED ON THAT. THIS IS JUST AN EXAMPLE OF HOW NUTRIENT OVERLOAD CAN AFFECT INTRACELLAR PROGRAMS AND PATHWAYS AND THIS IS LOOKING AT GROWTH. SO I'VE EXCESS AMINO ACIDS INCREASING SUB STRAIGHT. I'M NOT GOING TALK ABOUT THE GUYS GOLATION BUT INCREASE GLUCOSE CAN CAUSE -- [INDISCERNIBLE] -- E EITHER GLUCOSE OR FATTY ACID CAN GENERATE A METABOLIC [INDISCERNIBLE] COA. -- GET DOWN TO MITOCHONDRIA DRI YEAH AND BE K BE USED TO -- THEN ACTIVATE GENES INVOLVING GROWTH AND AMINO ACIDS TO IMPORT [INDISCERNIBLE]. SO TOGETHER THESE METABOLITES CAN TRY PROCESSES IN THE CELL. THE HISTONE ACETYLATION BRINGS UP TOPIC OF LIE SCENE ACETYLATION. SINCE THE FIRST HISTONES WERE DISCOVERED TO BE ASSOCIATE IN 1958 AND IN 1997 THE MOST KNOWN HISTONE PROTEIN ASITLATION WAS FOUND IN 53. BEFORE 2006 OR IN THE LAST DECADE THERE ARE ONLY ABOUT 19 PROTEINS KNOWN TO BE ACETYLATED AND THEN THIS IS SORT OF ALIGNED WITH INCREASED MAS SPECTROSCOPY WHERE THEY FOUND 196 PROTEINS AND NOW THERE'S EXCESS OF TWO THOUSAND ACETYLATED PROTEINS. MOST INTERESTING THING ABOUT THIS STUDY 2006, EXCUSE ME, WAS THEY NOTICED THE IT WAS A DIFFERENT IN WHICH PROTEINS AND WHICH RESIDENCE DEUCE FOR ACETYLATED DEPENDING ON THE FATAL FASTED STATE. THIS WAS IN THE LIVER. THERE WAS A HIGH ENRICHMENT OF ACETYLATED PROTEIN IN MITOCHONDRIA. THIS IS BLOT FROM THE LIVER FROM THE KIM PAPER IN 2006, AND THEY SHOW E THAT COME PROTEINS, WHICH IS AN ANTIBODY TO ACETYLATED LAS TEEN. THIS IS A STUDY FROM OUR LAB SHOWING IF YOU LOOK AT A CHARRED DIET, HIGH FAT DIET PORTION IN THE LIVER INCREASED IN RESPONSE. WITH RESPECT TO THIS REGULATION OF ASITLATION, THERE COULD BE SOME [INDISCERNIBLE] PRO SAYING, BUT THERE'S ALSO AN ACTIVE ENZYME SYSTEM AND TRANSFER ACETYLATEÑi PROTEINS AND
RESIDUES AND DEER SETLATIONS AREÑi NAD DEPENDENT WE MAKE THEM TO PRODUCE SUB STRAIGHTS [INDISCERNIBLE] INTERESTINGLY INHIBITS ENZYME. SEVEN ENZYMES DISSTRICT FROM HISTONE DEASSET LACES WHICH ARE ZINK DEPENDENT. -- -- THIS JUST SHOWS THE GROUPS ON THE EXXON MIEN OF LIE SCENE LYSINE RESIDUE. THIS LYSINE RESIDUE -- -- GROUP CREATES BROMO DOE XHAN FACILITATES@Qx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s WILD TYPE CERT THREE AND THE FLAG TAGGED OVEREXPRESSION OF ALDH 2 WITH SAG THE NEP 2 I YOU SEE INCREASED BINDING AND IF YOU USE MUTANT YOU RETAIN ACETYLATION OF ALDH 2 YOU GET LESS BINDING SUGGESTING THAT THIS ASITLATION OF ALDH 2 COULD BE MODULATING THE BINDING OF THIS; THEREFORE PLAY A ROLE IN TOXICITY. WE THEN USE MASS SPEC -- -- COULD BE MODULATED BY THIS MODIFICATION. YOU GET A 42 CHANGE IN SPECTRA WHEN THE LIE SCENES ARE SPECULATED AND WE FIND TWO FINDS. LYSINE AND LYSINE 77. WHEN WE MUTATED THOSE TO EITHER A GLUTAMINE WHICH MIMICS ACETYLATION ACETYLATION OR ARGUMEAN. MIMIC ACETYLATION YOU GET LESS BINDING. S A INHIBITOR -- -- LIVER INJURIES. THAT WAS AN EXAMPLE OFlp FASTING AND DISEASE PROCESS THAT'S MODIFIED BY ACETYLATION WITH DEPOSITING OF SPECIFIC PROTEIN INVOLVED IN THE METABOLISM OF TYLENOL. I'M NOW GOING TO LOOKING MORE THL/+GH ALL THE CERTAIN VILES OF MITOCHONDRIA xDCONTROL. AT FIST I'M GOING TO INTRODUCE CONCEPT OF INFLAMMASOME. A MULTIPROTEINi] INTERCELLULAR COMPLEX THAT ALSO SENSES PATHOGEN OR DAMAGE ASSOCIATED MOLECULAR PACKAGE WHICH COULD BE INTERCELLULAR WHICH ACTIVATES TEST BASE ONE. NUMEROUS DIFFERENT INFLAM PROGRAMS. A FEATURE IS THE NOD-LIKE RECEPTOR AND THE SECOND PROTEIN INVOLVED IS THE ADAPTER OF APOPTOSIS DECK LINE PROTEIN HIRE ASC. UNLESS YOU SEE NRP WITH ASC DOMAIN IN TEST PHASE THAT FORM THIS MULTIPROTEIN COMPLEX WHICH ENABLE AED ACTIVATION OF CASH PHASE FOR EVE OF THE PRONE IR 1 AND IL 18 WHICH YOU SEE CYTOKINES THAT CASCADE. MOST RECOGNIZED AS A COMPONENT OF STERILE INFORMATION WHICH OCCURS WITH ES THROE SCOPE, DT A ASTHMA AND WITH AGING WE GET INCREASED CELL INFLAMMATION. IT'S REGULATE AT NUMEROUS LEVELS BOTH AFTER TRANSCRIPT AND POST TRANSLATION. TRANCE CAN SCRIPT LEVEL IS FIND PRIMING OF CHROMOSOME AND THAT CAN BE MIMICKED IN A MODEL SYSTEM USING POLLY SACK RIDE WHICH USES AND ACTIVATES IN KAPPA B AND ACTIVATES P 13 PROTEIN POWER WHEN BETA TRAN SKRICHTS THAT GENERATE THE PROTEINS THEN YOU GET ACTIVATION WHICH IS WHEN THIS CONTEXT COMES TOGETHER, SMALL PROTEIN COMPLEX TO DRIVE THE DISEASE. THIS CAN BE SIMILAR RA RAR TO$ ATP. INTERESTINGLY AND WHY WE GOT INTERESTED IN THIS IS THAT ONE OF THE DISEASES ASSOCIATED WITH MOLECULAR PATENT THAT CAN DRIVE NRC P SPSHGS DAMAGE TO MITOCHONDRIA. THERE WAS A WONDERFUL REVIEW BY MARTIN IN 2012 WHICH DEMONSTRATED NUMEROUS EXTRA CELLAR STRESSES POTASSIUM INTO THE CELLS LOSE MITOCHONDRIA POTENTIAL AND FOR SOME YOU COULD GET RELEASE. AT THE SAME TIME OTHER PEOPLE HAVE SHOWN AND SUGGESTED BECAUSE OF THE [INDISCERNIBLE] HYPOTHESIS OF MITOCHONDRIA BEAN BACTERIA DNA DIFFERENT TO MUTATE DNA IN THAT UH YOU HAVE THESE UNMETLATED EDT POE MOTIFS. -- -- THIS WAS WHAT MADE OF INFLAMMASOME AND INTERESTINGLY I PREVIOUSLY DISCUSSED THE FACT THAT [INDISCERNIBLE] COULD BE ACTIVATE BID FASTING AND OTHER SUPPRESSED BY OVERFEEDING. INTEREST STUDY CAME OUT FROM NIH SUGGESTING THAT ALL STAY TO LOW CONSTRICTION CAN PROVE ASTHMA. RHEUMATOID ARTHRITIS HAS ALSO BEEN SHOENL TO HAVE LEGISLATION FLARES IF UNDER GO FASTING. RECOGNITION THAT MITOCHONDRIA INTEGRITY OR LACK OF COULD CYTOSOEM, WE WANTED TO PURSUE FURTHER. JUST IN TERMS OF SEARCH THAT CONTROL MITOCHONDRIA INTEGRITY, ONE OF THE NUCLEUS IN CERT ONE CAN IMPROVE MITOCHONDRIA AND CHANGE INTEGRITY ABOUT DRIVING OR ACTIVATING TRANSCRIPTION FACTOR CALLED PCG ALPHA AND CERT THREE IN LOW NUTRIENT SPACE -- FAST KG AFFECT MANY DIFFERENT PATHWAYS BETWEEN OTHER INCREASED METABOLISM BUT ALSO INCREASED [INDISCERNIBLE] ANTIOXIDANT PROGRAMS THAT ACTIVATE MOTIF, ARE REDUCES MITOCHONDRIA TRANSITIONS -- AND IT'S SUGGESTED TO PLAY A ROLE IN PROMOTING MIE TOFIGY. HYPOTHESIS CAN MITOCHONDRIA DESCRIPTION ARES REGULATE IN RP 3 INFLAMMASOME. IS DEPENDENT ON MITOCHONDRIA HOMEOSTASIS. THE FIRST THINGxD THIS IS IN THE LAB B LOOKED AT MOUSE WHERE HE FASTED THEM FOR 48 HOURS AND LOOKED AT NEW MACROPHAGES AND DEMONSTRATED THERE WAS A ROBUST DESTRUCTION AND SIMILAR RELEASE OF ALPHA. SUGGESTING THERE COULD BE AN ASSOCIATED EVENT. WE THEN USED HUMAN Ph.D. ONE MACRO FAJS. MICROPHAGES. DEMONSTRATED THAT IT WAS THE DEMREGS OF CERT THREE THAT COULD ACTIVATE IO 1 BETA AND RELEASE OF [INDISCERNIBLE] ALL THESEñr CELLS. THEN LOOKED AT OVEREXPRESSION OF CERT THREE AND SHOWED HE COULD DECREASE MYOONE BAY BETA
RELEASE. HE REPEATED HIS FASTING STUDIES COMPARING WILD TOP AND KNOCKOUT ANIMAL SHOWING IN FIFTH SECT THERE WAS NO DIFFERENCE IN THE FASTER STATE THERE WAS A MUCH MORE ROBUST PRODUCTION IN IO 1 BETA RELEASE IN WILD TYPE MOUSE COMPARED TO THE SNOKOUT MOUSE. THIS WAS REP TATED AT THE LEVEL OF IO ONE B PROTEIN SHOWING IT WAS HIGHER IN KNOCKOUT AND LESS DURING THE WILD TYPE. HE REPLICATED THIS LOOKING AT BONE MARROW MACRO -- MICROPHAGES. THE STORIES HAVE BEEN MORE UNCOMPLEX BECAUSE EFFECT ON FEEDING AND PRIMING AND ACTIVATIONS. WE REDUCE THE TALK SOMEWHAT AND I'M NOT GOING TO SHOW THAT DATA, NEVERTHELESS WITH RESPECT TO THE MITOCHONDRIA WE'VE DISCUSSED THE BENEFICIAL EFFECTS ON MITOCHONDRIA AND OTHERS SHOW THAT IF YOU KNOCK OUT CERT THREE IN THP 1 CELLS YOU GET INCREASE OF MITOCHONDRIA REACT VANT SPECIES REPLICATED. HE LOOKED AT MITOCHONDRIA RELEASE INTO CITE SOL. WHEN YOU TAKE WILD TYPE AND ADD LPS AS A PRIME AND ATP YOU GET VERY MODEST RELEASE OF MITE CHROMES BUT IF YOU USE THE CERT THREE KNOCKOUT MOUSE WHICH ACTIVATED DEACTIVATION COMPONENT YOU SEE THIS HUGE INGREECE DMROOES MITOCHONDRIA ARE RELEASE INTO CITE SOL SUGGESTING THAT THE LACK OF MITOCHONDRIA INTEGRITY CAUSES DISRUPTION OF CERT THREE COULD PLAY A ROLE IN THIS PROCESS. PREVIOUS POST DOC IN THE LAB WAS LOOKING AT PROTEINS THAT COUNTER REGULATED CERT THREE AND IDENTIFIED A COMPONENT OF THE MITOCHONDRIA TRANCE FACE PROGRAM. NOT COMPLETE ENZYME SYSTEM BUT IT DOES PLAY A ROLE. INTERESTINGLY AS I'VE SHOWN YOU PREVIOUSLY FROM ERIC'S LAB WITH FASTING CERT THREE GOES UP AND GCM FIVE FROM WEBSTERS IS PROFOUNDLY REDUCED FROM FASTER TYPE COMPARED TO REGULAR STATE. THEY WERE TRANS REGULATED. THEY DO SHOW COUNTER REGULATORY EFFECTS. IF YOU KNOCK OUT CERT THREE YOU GET INCREASED MITOCHONDRIA PROTEIN ACETYLATION AND THIS IS LOST WHEN YOU KNOCK OUT BOTH PROTEINS TOGETHER. BRAD WEBSTER SHOWN IF YOU KNOCK OUT [INDISCERNIBLE] -- INCREASING CERT THREE YOU SEEM TO HAVE INCREASED A ACCUMULATION OF LT 33 AND P 632 -- -- IF YOU KNOCK OUT CERT THREE YOU LOSE THIS EFFECT. SUGGESTING THESE PROTEINS COURT REGULATORY EFFECT. JAVIER LOOKED TO SEE WHAT HAPPENS IN KNOCK DOWN MACRO MICROPHAGES -- -- GCN 35 SITS A NUCLEAR -- -- MITOCHONDRIA ENRICHED AND SEEMS TO BE A COMPONENT OF MITOCHONDRIA SL TANS FACE MACHINERY. WHEN YOU EXPRESS ALONE YOU
CAN'Tq CHANGE REGULATION. PROBABLY A REGULAR E EFFECT IN THAT PROGRAM OR E I JUST USE IT E AS A COUNTER REGULATORY FEE. IF YOU LOOK AT SPI SES DOWN IN THE KNOCK UHDOWN AND BY ADDING CALCIUM TO THE CELL TO LOOK AT RESISTANCE TO TRANSITIONS TO GCN 5 FLOCK DOWN ARE MUCH MORE ROBUST. SO WE GOT INTO THIS FIELD WAS OF HUMAN STUDIES SHOWING THAT FASTING COULD -- -- ASTHMA AND RHEUMATOID ARTHRITIS WE WANTED TO SEE IF THIS INFLAM ZONE WAS INDEED REGULATED IN HUMAN SUBJECTS. WE'RE STILL LOOKING FORKER A FEW VOLUNTEERS TO FINISH THIS ONE OFF. WE HAVE PROTOCOL AT THE MOMENT TO ACTUALLY SDU ST UH DI DISEASE BUT THIS IS NO NORMAL CONTROLS. IS E WE HAD THE INDIVIDUALS COME TO THE CLINICAL CENTER, SENT THEM ON THEIR WAY AND ALL THEY COULD DO WAS DRINK WATER. WE DID BLOOD TESTS, FATTY ACIDS, BOTH HORMONES JUST CHECK IF THEY WERE FASTING OR. THEY HAD ANOTHER FIXED MEAL AND WE REA ASSESSED THE TRUE BLOOD AGAIN IN ONE AND THREE HOURS SO WE CAN COMPARE CHROMOSOME ACTIVATION AND MITOCHONDRIA FUNCTION AT THESE THREE TIME POINTS. I GUESS THE TIME'S MUCH BETTER THAN I THOUGHT. THESE ARE THE DATA AT LEAST ON IO 1 BETA RELEASE AND YOU SEE THAT EVEN WITHOUT THE EDITION OF LPS TRULY IN THE BASIL STATE WHEN YOU -- -- THE FASTER HUMAN SUBJECTS BOTH [INDISCERNIBLE] -- IF YOU LOOK AT CELLS WITH EITHER POINT 1 OR LBS, YOU SEE THIS IS ASSOCIATED WITH INCREASED MITOCHONDRIA RACTANT SPECIES. WE'VE ADDED DATA USING STIR THREE AGONIST SUGGEST THAT WE CAN REVERSE THIS EFFECT. IN CONCLUSION, [INDISCERNIBLE] BY FASTING, BETA IN THE NUTRIENT-LEVEL DATA. DATA SUGGEST FASTING IN MITOCHONDRIA PROGRAM MODIFY MITE CONDITION DENSE IN THIS ACTIVITY AND OPERATIONAL IN HEALTHY HUMAN SUB JEKTS WND WE'RE GOING LOCK AT GENETICS NEXT. FINAL SCHEMATIC, JEKYLL AND HYDE. MY FIRST STORY SUGGEST THAT ACETYLATION IN BENEFICIAL. -- -- THE FLIP SIDE IS I'M FASTING ON NUTRIENT DEFICIENT INCREASE IN CERT BY W DECREASE -- -- ARE PROTECTION FROM DISEASES WITH MITOCHONDRIA INTEGRITY CAN EXACERBATE. END OFF I JUST WANTED TO GIVE WHO DID THE WORK .S WEBSTER IN MICROSCOPY. DATA DONE BY [INDISCERNIBLE] HELP FROM JESSICA. THANK YOU VERY MUCH. [APPLAUSE] >> THANK YOU VERY MUCH, MICHAEL. YES. SURE. >> [LOW AUDIO]. >> WANTED TO KNOW IF IT WAS RETINOL WHAT? THE BLINDNESS IS CALLED DOA, DOMINANT OPTIC ATROPHY, LOSS OF CELLS. QUESTION FOR MICHAEL L. PRETTY INTERESTING THAT INFLAMMASOME IN LRP 3 IS INDUCING THE RELEASE OF MITOCHONDRIA DNA. >> YES. THE RELEASE OF THE DNA ACTIVATES IS THOUGHT TO BE A DAMAGE ASSOCIATED PATENT WHICH THEN ACTIVATES COLISTING OF ALL THESE PROTEINS TO GENERATE THE INFLAMMASOME. >> BUT YOU DON'T SEE IT UNLESS YOU ADD ATP? I THOUGHT THE ATB WAS ACTIVATING IT BECAUSE THERE WERE [LOW AUDIO]. WHAT WERE YOU MEASURING? WHAT WAS ON THE Y AXIS? >> IO 1 BETA RELEASE. >> YOU HAVE TO ADD ATP TO GET IO 1 BETA? TO THE MEDIA? J TO THE MEDIA. >> TO ACTIVATE INFLAMMASOMES. ISN'T THERE MRET PLENTY TO ACTIVATE THE INFLAMMASOMES? >> I GUESS IT PROMOTES INFUSED POTASSIUM. WE USE ANOTHER TRIGGER CAN SHOW THE SAME -- >> I SEE SO IT'S NOT REALLY ABOUT THE ATP. >> ATP IS ALSO -- >> THANK YOU. [CROSS TALK]. >> AT P P IS ALSO A ADAPT. >> OTHER QUESTION. >> HAS ANYBODY MEASURED [INDISCERNIBLE] AFTER TYLENOL? >> THE PRO HIB TORS -- I DO KNOW TO ACTIVATE THE ALD HIDE -- -- ENZYME ITSELF IS BEEN SHOWN FOR MANY YEARS TO BE SUPPRESSED BY HIGHER DOSE OF TYLENOL. >> ANOTHER QUESTION. [LOW AUDIO] INFLAMMATION TO ALL THE OTHER -- -- >> RIGHT, SO WE DON'T. WE DEMONSTRATE THAT THIS ONE -- FIRST OF ALL ALD HIDE HIGH DROJ FACE IS IMPORTANT IN THE BREAKDOWN OF THE REACTING ME TAB LIFE OF ASSET MINUTE FIN BECAUSE WE KNOCK DOWN DH 2 WE LOSE PARTICULAR EFFECT. -- -- OTHER PROTEINS THAT COULD BEÑi TARGETS FOR OTHER -- --
THAT THNGD HAVE ANçó EFFECT ON. I DON'T THINK IT'S THE EXCUSE OF, UM, BUT IT IS IMPORTANT -- -- >> [LOW AUDIO]. >> THE PROBLEM IS THAT SOME OF THE DAMAGE ARE SO POWERFUL THAT AND [INDISCERNIBLE] BETTER WAY TO LOOK AT THE DATA BECAUSE THAT'S WHY YOU LOSE THE EFFECT OF TIMING BECAUSE HIGH AFP IS DAMAGING TO THE CELL. >> SO YOU THINK THAT THE TYLENOL PACKAGE ALSO SAY DON'T EAT ON AN EMPTY STOMACH? >> THERE ARE A LOT OF CASE REPORTS OF PEOPLE DURING RELIGIOUS FASTING WHO THEN TAKE TYLENOL AND GET TOXICITY, AND AS I SAID THAT BIG STUDY OUT OF PITTSBURGH SUGGEST USING LOWER DOSE BUT -- -- HAVEN'T BEEN FIGURED OUT. WE'RE A SOCIETY OF OVERA ABUNDANCE -- -- USUALLY PATIENT WITH APPEND SOOITS -- -- >> I THINK MOST PEOPLE WITH SEVERE CALORIC RESTRICTION VOLUNTARILY THEY ALL HAVE HEADACHES AND THEY PROBABLY POP A FUTILE NOLS AND IT ACCUMULATES. POSSIBLE. >> THERE ARE PEOPLE WHO ARGUE IF YOU VACCINATE IN MORNING AND MAX VACCINATE AT NIGHT YOU'LL GET DIFFERENT. >> WE DISCUSSED THAT AND TALKED TO RICHARD ABOUT IT, DEFINITELY, BUT SO THE INFLAM ZONE PLAYS A A ROLE IN THE VAC SOOENG AND THEREFORE CHANGE VACCINE RESPONSE. THE QUESTION IS, SHULD WE AFTER THE ASTHMA STUDY SHOULD WE BE DOING A STUDY OF OK GENERAL NAR YANS? YOU HAVE POOR RESPONSE. >> FACT YOU DON'T EAT FOR 12 HOURS AND THE MORNING YOU -- [CROSS TALK]. >> THE PEOPLE WERE 24. >> WOULD 12? >> I WOULDN'T DO 48. BUT NO SO I WOULD UH SPECULATE THAT YOU MIGHT HAVE A BETTER RESPONSE -- >> AT NIGHT. [CROSS TALK]. >> XABTED TO ACETYLATION LOOK AT IT THERE THE CLOCK PLAYS A ROLE WHEN YOU GET A CHANGE. SO THAT COULD BE ADDING ANOTHER DIMENSION WHICH WE HAVEN'T LOOKED A AT [INDISCERNIBLE] BUT I WOULD SPECULATE YOU COULD AUGMENT -- >> [LOW AUDIO]. >> TALK INTO THE -- >> >> [LOW AUDIO]. >> WAIT A MINUTE, WAIT A MINUTE. JEFFREY COX HAD A PAPER IN NATURE 16 MONTHS AGO SAYING PARKIN'S TRANSLOCATES TIEF NEUROYUM, IS IS THAT IT? MICROBACTERIUM, BUT HE SAID IT'S SORT OF LIKE YOU CAN'T REALLY DOES IT BECAUSE IT'S SOME KIND OF TYPE THREE E SECRETION SYSTEM AND MOST ONES WE CAN USE IN NORMAL LABORATORY ARE BLUNTED. HE HAS A P 3 STA FACILITY AND HE'S DWOTS THE FULL BLOWN TB AND IT TRANS LOCATE THERE IS AND NOT IN OTHERS. I THINK UH THE WORLD IS WATCHING WHICH WHETHER THERE'S A LINK BETWEEN MIE KOT GI AND ZENOTHAGY. IT'S A HOST AGAINST BACTERIA WHERE YOU USE THE AUTO FAGA-SONE. YOU BIK TIEIZATION ARE INVOLVED. I GOT SCREENING FOR E 3 LIE GAZE INVOLVED IN THOSE PATHWAYS. IT'S CLEARLY E 3 LIE GAZE. >> [LOW AUDIO]. >> WAIT A MINUTE, IF PEOPLE -- >> CROSS TALK]. >> INTERCELLULAR BACTERIA, APOLT GI IS GREAT WAY -- IS THAT BACTERI BACTERIUM TELL KG CELLS TO DO THIS? >> THERE'S SO MANY DIFFERENT PATHOGENS, PROBABLY 50 DIFFERENT WAYS, IT'S A KALT AND MOUTS GAME. WHATEVER THEY'RE DOING WE'RE COUNTERACTING THAT OR WE WOULDN'T BE HERE. I KNOW VIRUSS ARE ALSO VERY COMPLICATEDLY INVOLVED IN THESE PATHWAYS, AND SOME OF THEM STIMULATE AUTO PATH GI AND THEY'RE USINGS IT BUT MAYBE SOMETIMES WE USE AUTO CELL FAJ AGAINST THEM. IT'S PROBABLY ALL OF THEM ABOVE. J [LOW AUDIO]. >> YES. ALMOST EVERYTHING. 33,000 TO 13. ALL THE MIELT CON GENES ARE EITHER PRNAs OR IN OX FOX COMPLEX IMBEDDED IN MEMBRANE. >> THANK YOU VERY MUCH. THIS HAS BEEN QUITE AN EYE OPENER. [APPLAUSE]
