>> GOOD MORNING, EVERYBODY. SO DESPITE THE HIGH RISK HIGH REWARD NATURE OF YOU, YOU ALL SIT IN THE BACK OF THE ROOM. --NO, I'M NOT KIDDING. YOUR AWARDS STATEMENT ARE NOT MADE YET. I'M ONLY TEASING ABOUT THE LAST PORTION. THANK YOU, I APPRECIATE IT. SO I HAVE NOW DETERMED THE ONLY PERK OF BEING THE PRINCIPAL DEPUTY DIRECTOR OF THE AGENCY, IS GETTING EVERYONE TO COME FORWARD. YOU MIGHT WONDER WHAT I DO? IT'S A VERY SIMPLE JOB DESCRIPTION. I DO EVERYTHING THE DIRECTOR DOESN'T WANT TO DO. AND IN THIS CASE, IT'S BECAUSE HE COULDN'T DO IT, FRANCIS IS IN THE U. K. THIS MORNING. OTHERWISE HE WOULD HAVE BEEN HERE WITH YOU. AND THEN AS THE ETHICS COUNSELOR FOR THE AGENCY, I TELL EVERYBODY WHAT THEY HAVE TO DO. NAHEY MY JOB. THOSE THAT ARE COMING IN LATE, MOVE TO THE FRONT OF THE AUDITORIUM SO WE CAN SEE YOU, YES YOU IN THE BACK, MOVE TO THE FRONT. COME ON. THIS IS A HIGH RISK, HIGH REWARD GROUP. COME ON. GHEE, OKAY. SO I WANT TO WELCOME YOU ALL TO THIS EVENT THIS IS NOW THE THIRD YEAR WE'RE DOING THIS AND IT'S MEANT TO BE A CELEBRATION OF ALL THE IPT GREATER CREDIBLE IDEAS AND ACCOMPLISHMENTS THAT THE GROUP HAS ALREADY MADE OR WILL MAKE IN THE FUTURE. MOST IMPORTANTLY FOR YOU, IT GIVES YOU AN OPPORTUNITY TO WORK WITH ONE ANOTHER. THE TRACK RECORD HAS BEEN PRETTY GOOD. WE TYPICALLY HAVE A NUMBER OF IMPORTANT COLLABORATIONS THAT EMERGE FROM GETTING YOU ALL TOGETHER IN THE SAME ROOM AT THE SAME TIME. MANY OF YOU HAVE NEVER MET ONE ANOTHER. YOU MAY KNOW EACH OTHER BY REPUTATION BUT IN MANY INSTANCES, NOT--AND SO IT'S REALLY AN IMPORTANT OPPORTUNITY AND OF COURSE THE BREADTH OF SCIENCE IS TYPICAL AND NOT TYPICAL OF THE NIH PORTFOLIO AND THAT'S A GOOD THIPG AND SELFISHLY, IT ALSO ALLOWS FOLKS AT NIH TO HEAR SOME REALLY GREAT SCIENCE. SO THAT'S A GOOD THING AS WELL. SO HOPEFULLY TO STATE THE OBVIOUS, BUT MAYBE IT'S NOT SO OBVIOUS TO THOSE WHO HAVE BEEN INVOLVE WIDE NIH FAR LONG PERIODS OF TIME, NIH IS COMMITTED TO HIGH RISK RESEARCH AND THE ATTRIBUTES OF THAT WHICH YOU'RE ALL PROPONENTS OF SHOULD BE SELF--EVIDENT AND THEY'RE LISTED ON THIS SLIDE. LATER THIS MONTH, NIH IS GOING TO BE RELEASING IT'S FIRST IN A VERY, VERY LONG TIME NIH WIDE STRATEGIC PLAN AND A VERY IMPORTANT AND CENTRAL THEME TO THE PLAN IS THE NOTION THAT WE HAVE TO ASSUME MORE RISK, THAT WE HAVE TO BE MORE CREATIVE THAT WE HAVE TO DO EVERYTHING WE CAN TO CAT. CATALYZE INNOVATIVE APPROACHES AND IT'S THE SORT OF THING THAT SITS ON HARD DRIVES BECAUSE WE DON'T BRINS THESE THINGS ANYMORE, MERCIFULLY, THEY USED TO HIT ON SHELVES BUT IF YOU DO HAVE A CHANCE TO SEE TYOU MIGHT WANT TO PURUSE IT BECAUSE I HOPE YOU WILL FIND THAT THE AGENCY REALLY IS TRYING TO MAKE A COMMITMENT TO THE TYPE OF WORK THAT WE KNOW IS IMPORTANT TO MOVE SCIENCE FORWARD, YOU KNOW IN A DRAMATIC FASHION. NOW, THE--IT GETS A LITTLE COMPLICATED BUT BECAUSE OF THE WAY NIH IS ORGANIZED BUT I'M GOING TO FRY --TRY AND WORK THROUGH THIS, BUT WE HAVE WITHIN THE OFFICE OF THE DIRECTOR AND IT'S SOMETHING CALLED DIP A KIPSI, BUT SOMEBODY DOES, IT'S DIVISION OF STRATEGIC PLANNING AND ACCORD NATION. CLOSE ENOUGH FOR GOVERNMENT WORK AND WITHIN THAT DIVISION IS THE OFFICE OF STRATEGIC ACCORD NATION AND THAT'S WHERE THE SO CALLED COMMON FUND IS MANAGED. AND WITHIN THAT COMMON FUND ARE A SERIES OF HIGH RISK, HIGH REWARD OPPORTUNITIES AND THEY INCLUDE AS LISTED HERE THE PIONEER AWARD, INNOVATOR AWARD, TRANSFORMATIVE RESEARCH AND INDEPENDENCE AWARD AND WE HAVE EXAMPLES OF ALL OF THOSE AWARDEES HERE TODAY. THERE--ALL OF THESE PROGRAMS IN COMMON ARE ALTERNATIVES TO THE TRADITIONAL RO-1. WHICH AS MANY OF YOU KNOW FOR YEARS HAS BEEN CONSIDERED THE GOLD STANDARD AND EVERY TIME I HEAR THAT PHRASE, I CRINGE BUT THAT IS IN FACT, THE SORT OF NOMENCLATURE THAT THAT HAS BEEN ADOPTED. ALL OF THESE AWARDS ARE REVIEWED IN DIFFERENT WAYS AND ARE FUNDED IN DIFFERENT WAYS, AGAIN ALL WITH THE GOAL OF ENHANCING INNOVATION AND INCENTIVIZING YOU AND ENABLING YOU TO BE RISKY. TO DO RISK TAKING. SO THE NIHDIRECTOR'S I PIONEER AWARD AND THEY HAVE TO BE BOLD TO ADDRESS MAJOR CHALLENGES AND IN THIS YEAR WE HAVE 13 NEW AWARDS. SO THESE ARE THE PICTURES OF ALL OF THE AWARDEES AND AT THIS POINT, I AM TOLD DO NOT READ THE INDIVIDUAL NAMES BUT IF ALL THE PIONEER AWARDEES COULD STAND AND BE RECOGNIZED, PLEASE? [ APPLAUSE ] WELL, OKAY. THE DIRECTOR'S NEW INVOCABULARYIVATOR AWARD BEGAN IN 2007, ABOUT 400 OF THESE HAVE BEEN MADE THUS FAR. THIS AWARD SUPPORTS INDIVIDUAL EARLY CAREER INVESTIGATIONS WHO ARE PROPOSING BOTH INNOVATIVE AND IMPACTFUL PROJECTS AND THIS YEAR WE HAVE 41 NEW AWARDS, SO IF THOSE OF YOU WHO ARE DISPLAYED ON THE NEXT SERIES OF SLIDES COULD PLEASE STAND, I KNOW YOU'RE OUT THERE. OKAY. WELCOME. THE NIH DIRECTOR'S TRANSFORMATIVE RESEARCH AWARD BEGAN IN 2009 ABOUT 125 OF THESE HAVE BEEN MADE. AGAIN THEY SUPPORT EXCEPTIONALLY INNOVATIVE UNCONVENTIONAL PARADIGM SHIFTING RESEARCH PROJECTS THAT ARE INHERENTLY RISKY AND UNTESTED AND MAY REQUIRE MULTIPRINCIPAL INVESTIGATORS AND BUDGETS. THIS YEAR WE AWARDED NINE AWARDS TO 13 INVESTIGATORS UNDERSHE MULTIPI DIMEN SIONS. SO IF THOSE OF YOU WHO ARE ON THIS SLIDE COULD STAND AND BE RECOGNIZED PLEASE? [ APPLAUSE ] AND THEN FINALLY, WE HAVE THE NIH DIRECTOR'S EARLY INDEPENDENCE AWARD, THESE BEGAN IN 2010, WE MADE 72 OF THESE AWARDS. EUPHEMISTICALLY THESE ARE CALLED SKIP THE POST DOCK AWARDS SO THESE ARE FOR YOUNG PEOPLE WHO RECEIVED PH DZ AND ARE SUFFICIENTLY BOLD AND BRAVE AND STRONG TO JUST GO RIGHT INTO AN INDEPENDENT CAREER TRACK. AND IT IS WE ARE MAKING 16 NEW AWARDS THIS YEAR. SO FOR THOSE OF YOU ON THIS SLIDE, COULD YOU PLEASE STAND? [ APPLAUSE ] THANK YOU. SO JUST TO LET YOU KNOW THAT THE VARIOUS AWARDS EVEN THOUGH THEY ARE COORDINATED BY AND OVERSEEN BY THE FOLKS IN THE OFFICE OF STRATEGIC--WHICH--COORDNATION, THERE ARE ALSO MANY GIVEN BY STRAY JECTORYS THE NIH SO IT'S A PROGRAM THAT ALLOWS THE PARTNERSHIP TO BE LARGER THAN THE NIH COMMON FUND ALLOWS AND THIS IS TED PLACE WHERE THAT ALL HAPPENS. SO WITH THAT, I AM UNDER TIME AND UNDER BUDGET AND WANT TO, YOU KNOW ASSURE YOU THAT BOTH FRANCIS AND I ARE KEENLY INTERESTED IN WHAT YOU ARE DOING. DR. COLLINS SPENDS A FAIR AMOUNT OF TIME WITH DR. ANDERSON AND HIS STAFF WORKING THROUGH THESE PROGRAMS AND IN PARTICULAR, THEIR OUTCOMES SO--OH GOOD. OKAY. I THINK I AM SUPPOSED TO BE BACK LATER THIS AFTERNOON, LATE THIS AFTERNOON. THIS IS SUCH A CREATIVE INNOVATIVE GROUP. I WILL GIVE THEM A TEST. SO, WHY NOT HAVE A LITTLE FUN? I'M ALLOWED, RIGHT? OR IS THAT AGAINST THE LAW? THIS THING--OKAY, AT THE END OF THE DAY, SOMEBODY HAS TO TELL ME WHAT THAT IS. IT WILL BE NO PRIZE BECAUSE WE WOULD HAVE TO PUT THAT IN A FEDERAL REGISTER NOTICE AND AND WE HAVEN'T DONE THAT YET BUT TELL BE VERY COOL TO SEE IF ANY OF YOU CAN FIGURE OUT WHAT THAT IS. IN ANY EVENT MY E-MAIL ADDRESS IS THERE IF YOU HAVE ANY INTEREST IN CORRESPONDING WITH ME, I LOOK AT MY OWN E-MAIL, PRETTY MENIACAL ABOUT IT AND WITH THAT I AM TURN IT BACK TO WHOM? TO DR. ANDERSON. THANKS, EVERYBODY. >> THANK YOU, LARRY. GOOD MORNING, I'M JIM ANDERSON, DEPUTY DIRECTOR FOR COORDINATING PLANNING INITIATIVES AND SS. WE TRIED TO CALL IT DIVISION BUT WE CALL IT DPKISI THANK YOU FOR SHARING YOUR PROJECTS AND THE COMMON FUND. I WANT TO TELL YOU ABOUT WHY IT'S DIFFERENT AT NIH. IT'S DESIGNED TO STIMULATE INNOVATION AS LARRY SAID, WE TRY TO GET MORE IMPACT FROM WHAT WE FUND HERE AND THAT'S SAID WE HAVE TO HAVE A WAY TO MEASURE THAT SO THE SECOND THEN I WANT TO TALK ABOUT IS THE NEW WAY THAT WE ARE TRYING TO EXPERIMENT WITH TO ACCESS PUBLICATIONS THAT'S IN ANOTHER DIVISION, SO THOSE TWO THINGS. WHAT IS THE COMMON FUND? IT'S CURRENTLY ABOUT A LITTLE OVER HALF A BILLION DOLLAR APPROPRIATION FROM CONGRESS THAT STARTS IN THE OFFICE OF THE DIRECTOR SO IT DOESN'T START AT OFFICE OF THE INSTITUTE, IT'S NOT CATEGORICAL--FIVE TO 10 YEARS LONG SO WE LIKE TO THINK OFs BEING A WAY TO TAKE ON MAJOR ROAD BLOCKS BROADLY IN BIOMEDICAL RESEARCH. THERE'S CURRENTLY 29 PROGRAMS. WE CONSIDER THE ONES YOU'RE ALL PART OF TO BE ONE OF THOSE 29. SO I MENTION A COUPLE OTHER EXAMPLES. IT IS REALLY A TEAM SPORT, THE FUNDS START WITH THE OFTENIS OF THE DIRECTOR FRANCIS IS INTERESTED IN THE COMMON FUND AND OFF IN A MEETING IN LONDON TODAY SO CAN'T BE HERE BUT T'RE MANAGED BOO I AN OFFICE IN OUR DIVISION WITH THE INSTITUTE, SO IT'S REALLY A GROUP ACTIVITY. EVERY PROJECT HAS MANY OF THE INSTITUTES INVOLVED. WHY ARE THESE PROGRAMS DIFFERENT IN THE INSTITUTES DO CAPITALIZE ON EMERGING OPPORTUNITIES BUT SPECIFICALLY THE COMMON FUND ASKS, WHAT'S HOLDING BACK SCIENCE BROADLY AND IS THIS THE RIGHT TIME TO MAKE A CATALYTIC INVESTMENT. WHAT'S NEEDED IN THIS AREA RIGHT NOW THAT WOULD MOVE THINGS FORWARD QUICKLY SO THAT'S THE WAY WE APPROACH THE DESIGN OF THE PROGRAMS OF IDENTIFICATION OF PROMULGATE ECTOMYOSINS THAT WE FUND. WE THINK OF IT IN FIVE CATEGORIES, ONE TRANSFORMATIVE. WE DON'T SAY WE NEED TO DO THIS AND LET'S WORK TOWARDS IT FOR FIVE OR 10 YEARS. WE SAY WHAT IS IT WE CAN DO NOW THAT IF WE GET IT DONE, IT WILL CHANGE OR TRANSFORM THE WAY WE DO SCIENCE. THE PRODUCTS WE DELIVER, THE SCIENCE, THE DATA, THEY SYNERGIZE WITH WHAT THE ITES TRY AND DO, IT'S NOT SOMETHING THAT'S COMLITELY SEPARATE FROM OUR INSTITUTES MISSIONS, AGAIN IT'S CATALYTIC IN THAT WE CAN SAY HOW CAN WE LOWERLET ENERGY TO GET FROM HERE TO THERE IN THE DEFINED SCIENCE IN THE DEFINED PERIOD OF TIME. IT'S ALSO UNIQUE, WE DON'T JUST ADD FUNDS TO SOMETHING THAT'S GOING ON, WE SAY WHAT'S THE UNIQUE NEED THAT WE CAN ADDRESS WITH THIS SPECIAL BUCKET OF MONEY. SO YOU'RE AQUANTED WITH THE HIGH RISK-HIGH INVESTIGATOR AWARD WHICH IS INITIATED BUT MOST OF OUR FUNDS GO TO TOP DOWN WHERE WE IDENTIFY WHAT THE NEED IS, AND THEN ORGANIZE A VERY LARGE TEAM, MANY PIs, MANY INSTITUTIONS, IT'S OFTEN INTERNATIONALLY COORDINATED IN ORDER TO PRODUCE SOMETHING THAT WE ANTICIPATE WILL CHANGE THE WORLD. SO WHAT FITS THOSE CRITERIA, OFTEN IT'S THINGS LIKE TOOLS, DAT AND I'LL GIVE YOU A COUPLE EXAMPLES, IT'S REAGENTS, SOMETIMES IT'S KNOWLEDGE SOLUTIONS, UNDERSTANDING, EVERYTHING WE DO IS DESIGNED FROM THE BEGINNING TO BE TRANSITIONED AND HANDED TO THE COMMUNITY. IT'S NOT I'M DOING MY RESEARCH HERE, IT'S HOW DOES THAT FIT INTO DATA THAT'S IN AN ACCESSIBLE DATABASE THAT PEOPLE CAN USE, WHETHER THEY NEED FROM IT, HOW TO DESIGN THE DATABASE. EVERYTHING WE DO IS DESIGNED TO PUSH IT OUT TO THE COMMUNITY OVER THE PERIOD OF THE FUNDING. SO WHAT HAS THIS COMMON FUND BEEN OFFERED TO THE COMMUNITY. WELL FIRST OF ALL FUNDS AND SUPPORT AND I'LL ENCOURAGE TO YOU CHECK OUR WEB SITE, AND YOU'LL SEE NEW PROGRAMS THAT DON'T HAVE FUNDING OPPORTUNITY THAT'S BEEN OUT YET BUT YOU CAN SEE WHERE IT'S BEEN HEADED AND MANY OF THE PROGRAMS WILL HAVE NEW FUNDING OPPORTUNITIES DURING THE PHASE OF THE OVERALL PROGRAM SO KEEP AN EYE ON THAT. WE THINK IT ALLOWS A VERY SPECIAL OPPORTUNITY FOR NETWORK AND COLLABORATION THAT CAN TAKE PLACE ACROSS INSTITUTES. SO MANY FOLKS WILL BE FUNDED BOY A SINGLE INSTITUTE OR TWO THROUGHOUT THEIR CAREER AND THAT'S WHERE THEY TEND TO KEEP THEIR THINKING, THE COMMON FUND IS REALLY VERY BROAD SO I HOPE YOU MEDE A LOT OF PEOPLE TODAY, THE YOUNGER FOLKS, YOU WILL KNOW THESE PEOPLE FOR THE REST OF YOUR CAREER, THIS IS AN IMPORTANT OPPORTUNITY TO NETWORK AND THEN I'LL GIVE YOU INTANGIBLE THINGS THAT THE PROJECT PRODUCES. SO IT WASN'T LONG AGO THAT PEOPLE STUDIED THE EPIGENOMIC MODIFICATIONS OF GENES OR A SET OF GENES AT A TIME AND IT WASN'T THE REFERENCE OF THE WHOLE GENOME SOW CAN START TO SEE THE PATTERN OF HOW MARKS WERE USED, USED IN DIFFERENT CELL TYPES, USED IN DIFFERENTIATION. SO THIS IS NOT SOMETHING AN INSTITUTE WOULD DO. IT'S NOT SOMETHING A SINGLE LAB WOULD DO. SO THIS IS ONE OF THOSE OBSTACLES TAKEN ON BY THE COMMON FUND WITH THIS PROGRAM AND IT SAID IF YOU WERE TO HAVE REFERENCE--REFERENCE GENOME WIDE EPIGENOMIC MARKING AND IT WAS ACCESSIBLE ON WEB SITES, PEOPLE COULD USE IT, THIS WAS AN AMAZING REFERENCE THAT EVERYONE CAN TAKE THEIR DATA TO TO TRY AND BED IT BETTER SO AS TYPICAL WITH OUR PROGRAMS, MULTIINITIATIVES AND MULTIPIs AND THIS ONE IS COORDINATED WITH INTERNATIONAL PARTNERS BUT TO GET THE WORK DONE, WE NEED CENTERS THAT DID THE MAPPING OF MARKS, WE NEED A CENTER THAT COORDINATED THE DATA AND TURNED IT INTO FORMAT THAT INVESTIGATORS COULD USE. THERE WAS A REALIZE THAT THERE REALLY WASN'T, THERE WAS A NEED TO DEVELOP MORE UNDERSTANDING AND TECHNOLOGY TO MEASURE THE MARKS ON A GENOME LEVEL AND THEN THEY WERE NOVEL MARKS THAT NEED TO BE DISCOVERED. SO SOME OF IT WAS DISCOVERY AND A LOT OF IT WAS SIMPLY PRODUCING A DATABASE OF GENOME WIDE INFORMATION THAT WOULD BE USEFUL TO MANY, MANY RESEARCHERS, SO IN IS A PRETTY ADVANCED PROGRAM AT THIS POINT. IT HAS ALMOST 3000 GENOME WIDE DATA SETS THAT HAS MANY OF THE HISTON MODIFICATIONS, DNA SENSITIVITY, MAPPING ACROSS THE WHOLE GENOMES AND MULTIPETRESSABLE CELL TYPES, SO CAN YOU SEE WHAT THIS WOULD DO. IT JUST TURNS THE LIGHT ON SO YOU CAN SEE HOW THE MARKS ARE USED THROUGHOUT THE GENOME AND THAT WAS SOMETHING THAT WAS NOT REALLY THERE, 10 TO 15A YEARS AGO. OKAY, A SECOND EXAMPLE OF A THEME OF HOW THE COMMON FUND PROGRAMS IDENTIFIED TARGET AND AN ORGANIZED DISSOLUTION WILL BE THE HUMAN MICROBIOME PROJECT. THIS IS VERY SIMILAR BECAUSE 10-15 PLUS YEARS AGO, PEOPLE WERE STUDYING A SINGLE OR FEW BUGS AT A TIME. BUT THERE WAS THE OPPORTUNITY WITH HIGH THROUGH PUT SEQUENCING AND COMPUTATION METHODS YOU COULD IDENTIFY THE BUGS THERE THAT IT WAS TIME TO SAY WHO'S THERE. OKAY? AS A REFERENCE. SO THIS PROGRAM TOOK ON A LIMITED NUMBER OF BODY SITES IN NORMAL PEOPLE AND ASKED WHO'S THERE. AND IT PRODUCED A MAPPING, DATABASE OF THE MICROBIOME, IN A VARIETY OF BODY SITES. SO IT WAS JUST A SIMPLE BUT THROUGH THAT IT CREATED DATA THAT COULD BE USED BROADLY AND IT STIMULATED THE MICROBIOME RESEARCH IMENSELLY. SO WE'RE IN A SECOND FACE OF THIS PROGRAM WHICH IS WE'RE ASKING, WHAT'RE WHAT ARE THEY DOING, OKAY? SO THIS IS NOT JUST THE METAGENOME, ALL THE GENES THAT'RE THERE, BUT IT'S ALSO LOOKING AT TRANSCRIPTOMICS, PROTEOMICS AND METABALOMICS IN THESE SITES WHERE THESE COLLECTIONS OF MIKE ON ORGANISMS AND US RESIDE AND THIS IS BEING DONE IN PRIMATES THREE CONDITIONS, IN PREGNANCY, AND PRETERM BIRTH, INFLAMMATORY B DISEASE AND PREDIABETES. THIS IS MOSTLY DATA BUT IT'S DATA THAT'S VERY BROAD AND VERY DEEP AND HAS HELPED PEOPLE THINK IN NEW WAYS ABOUT THE MICROBIOME. I WANT TO GIVE YOU THESE TWO EXAMPLES OF THE KIND OF PROGRAMS WE TAKE ON. OKAY SO WE CLAIM THIS IS AN INNOVATIVE SPACE, THAT THE AWARDS YOU HAVE THAT ARE DIFFERENT FROM RO-1S, ALLOWS TO TAKE RISKS, DO THINGS, ALLOW YOU TO HAVE INFLUENCEOT PUBLISHED COMMUNITY. WE WOULD LIKE TO SEE WHAT HAPPENED TO YOUR WORK IN 10 OR 20 YEARS DID IT RESULT IN A NEW TREATMENT FOR SOMETHING. WE OFTEN HAVE TO USE SHORT-TERM MEASURES LIKE BIBLIOMETRICS. THERE ARE PROBLEMS WITH MANY THAT WE USE NOW, FOR EXAMPLE, THE IMPACT FACTOSH HAS COME JUSTIFIABLY UNDER GREAT ATTACK IN THE MOST RECENT YEARS, YOU MAY KNOW AND HAVE SIGNED ON TO THE PETITION FROM DORA, FROM THE DECLARATION ON RESEARCH ASSESSMENT WHO WANTS TO KILL THE IMPACT FACTOR. THERE HAVE BEEN PUBLICATIONS IN THE VARIETY OF JOURNAS ABOUT HOW IMPACT FACTOR IS DRIVING PEOPLE'S CAREERS, POST DOCS, CHOICE OF TOPICS, TIMING OF PAPERS, IT'S HAVING AN ERESOURCESSIVE EFFECT ON THE WAY WE DO SCIENCE, TENURE AND PROMOTION. THE PROBLEM IS IT'S A JOURNAL METRIC, IT DOESN'T MEASURE THE PAPER'S INFLUENCE SO IT'S QUITE FLAWED AND IT'S HAVING AN ERESOURCESSIVE EFFECT PERHAPS EVEN ON REPRODUCIBILITY ISSUES. SO WHAT ARE SOME METRICS THAT WE DO USE. YOU COULD USE PUBLICATION COUNTS, HOW MANY PUBLICATIONS YOU PRODUCE, BUT THAT'S REALLY DEPENDENT VERY MUCH ON THE FIELD, BEHAVIORIAL SOCIAL SCIENCE IS TYPICALLY FEWER THAN MOLECULAR BIOLOGY OR CELL BIOLOGY. YOU CAN ALSO PUBLISH A PAPER THAT'S NEVER USED. SO THAT'S NOT VERY INFLUENTIAL, YOU MAY HAVE A LOT OF PAPERS BUT IF NO ONE READS THEM, THAT'S NOT PARTICULARLY WHAT WE WERE LOOKING FOR, THE IMPACT FACTOR AGAIN IS A JOURNAL LEVEL METRIC, DOESN'T SAY ANYTHING ABOUT THE INDIVIDUAL PAPER, ALTHOUGH, YES THERE'S A CORRELATION BECAUSE IT'S BASED ON THE JOURNAL. CAN YOU LOOK AT CITATION RATES, HOW OFTEN IS NIGH JOURNAL CITED? BUT THERE'S A CERTAIN LUSTER THAT'S ACQUIRED BI YOU CITING A PAPER IN SCIENCE OR NATURE TADDS A LUSTER TO YOUR OWN WORK, SO THAT'S--IS THAT FAIR? IS THAT REALLY TELLING YOU ABOUT THE IMPACT OF THE ORIGINAL PAPER, I'M NOT SURE. SO ONE OF THE OFFICES IN OUR DIVISION IS CHARGED WITH COMPUTATIONAL WAYS AND ONE THING THEY'VE DONE IS DEVELOP A NEW METRIC THAT WE'RE EXPLORING AND I WOULD LIKE TO YOU KNOW ABOUT. IT'S CALLED RELATIVE CITATION RATE. AND THIS ADDRESSES THE NEED THAT WE WANT TO KNOW THE INFLUENCE OF INDIVIDUAL ARTICLE NOT THE JOURNAL BUT THAT ARTICLE. BUT IT IS BASED ON THE ASSUMPTION THAT PEOPLE CITE ARTICLES BECAUSE THEY HAVE INFLUENCE, OKAY? SO WE HAVE TO ASSUME IT DOESN'T MEAN IS THE WORK IMPORTANT, IT JUST SAYS DOES THE ARTICLE HAS IT INFLUENCED OTHER INVESTIGATORS AND THEN WHAT IT DOES IS IT USES THE CO CITATION NETWORK FOR THE ARTICLE AS A DENOMINATOR SO LET ME QUICKLY GO THROUGH THAT. IF WE'RE INTERESTED IN THE RED ARTICLE, THAT'S THE LAST PAPER YOU WROTE, WELL, LET'S MAKE IT FIVE YEARS AGO SO WE CAN ACQUIRE CITATIONS, THAT ARTICLE CITED OTHER ARTICLES AND IF YOU LOOK AT 200,000 NIH PUBLICATIONS, THE AVERAGE IS 30, AN ARTICLE WILL CITE 30 OTHER ARTICLES, THAT'S A STATIC NUMBER NUMBER, THAT CHANGES AFTER PUBLICATION, SO THAT'S NOT A GOOD REFERENCE SO YOU CAN LOOK AT THE PEOPLE CITING YOUR ARTICLE, THAT ACUES OVER TIME SO THAT'S MORE VALUNE BUT WHAT WE FOUND TO BE THE MOST USEFUL IS TO LOOK AT CO CITATION NETWORK. SO AS PEOPLE SITE YOUR ARTICLE THEY ALSO CITE OTHER ARTICLES AND THIS ACCUMULATES OVER TIME. THEY CITED THESE PAPERS BECAUSE THEY HAD SOMETHING TO DO WITH THEIR ARGUMENT WHEN THEY CITED YOU. SO WHAT WE DO IS TAKE THE CITATION RATE FOR YOUR ARTICLE AND NORMALIZE IT TO THE EXPECTED CITATION RATE, OF THE CO CITATION NETWORK AND THIS IS A BIG COMPUTATIONAL THING TO DO EACH TIME FOR EACH ARTICLE, BUT IT'S ACTUALLY PRETTY SIMPLE. AND WHAT DO YOU LEARN FROM THEM? IN ORDERED FOR THE MATHEMATICS, I WANTED TO REFER YOU TO THIS ARTICLE, IT'S A FREE PUBLICATION POSTING YOU WILL FIND ON THE COLD SPRING HARBOR BIOMATHEMATICAL WEB SITE WHERE WE GO FLEW AND GIVE LOT LIST OF EXAMPLES OF IT AND IF YOU WANT TO USE IT, WE'VE MADE THIS AVAILABLE AT iCITE. YOU CAN ENTER THE PUBMED I.D. NUMBERS, OF A PARTICULAR PAPER, ALL OF YOUR PAPERS, PORTFOLIO PAPERS, HUNDREDS OF PAPERS THOUSANDS OF PAPERS AND IT WILL TURN OUT THE RCRs AND GIVE YOU DATA ABOUT CITATION RATES AND GRAPHS SO I ENCOURAGE YOU TO PLAY WITH WITH IT. WHEN I LOOK AT IT, I SAY OH, I KNOW WHY, THAT PAPER OF MINE HAS THAT NUMBER AND THIS HAS THAT NUMBER, YOU KNOW I HAD TO GET THE POST DOC OUT OF THE LAB SO THAT'S NOT SUCH A GOOD PAPER IN TERMS OF HOW FAR IT INFLUENCED THE COMMUNITY SORE THAT'S REALLY FUNDAMENTAL. I CAN SEE WHY THAT'S BEING CITED. SO I ENCOURAGE YOU TO HAVE FUN AND GO AND USE THAT CITE. SO I WILL GIVE YOU EXAMPLES OF NIH COMMON FUND USING THIS METRIC. FIRST, THIS SHOWS YOU ALL--THESE ARE RESULT FROM ALL PUBLICATIONS THAT CAME FROM NIH FUNDING FROM 2003-2010 AND EVERY DOT IS A PUBLICATION AND THESE ARE BINNED BY THE WENTILE NUMBER FOR THE JOURNALS AND THESE ARE THE NEW ENGLAND JOURNAL OF MEDICINE, CELL SCIENCE UP IN HERE AND THESE ARE LOWER IMPACT JOURNALS AND OF COURSE, THIS IS THE MEAN AND THE RANGE OF THE IMPACT FACTORS FOR THE DIFFERENT WENTILES SO THEY FALL IN A NICE TIGHTLY GROUP BUT IF YOU LOOK AT INDIVIDUAL PAPERS THAT ARE OCCURRING OR ARE PUBLISHED IN KNEES JOURNALS SO AGAIN BY WENTILES BUT HERE WE'RE LOOKING AT RELATIVE CITATION RATES, YOU CAN SEE THE INFLUENCE OF INDIVIDUAL PAPERS IN THESE JOURNALS IS QUITE BROAD SO YOU CAN HAVE A PAPER AND A LOW IMPACT JOURNAL THAT HAS TREMENDOUS INFLUENCE ON ITS FIELD COMPARED TO SOMETHING IN CELL, OCCASIONALLY. THERE IS A RELATIONSHIP OBVIOUSLY BECAUSE THE IMPACT FACTOR IS AN AVERAGE OF ALL THE PAPERS BUT IF YOU LOOK AT INDIVIDUAL PAPERS, IF YOU CAN PICK OUT THEIR SPECIFIC INFLUENCE ON THE FIELD, SO LET'S LOOK AT COMMON FUND PROGRAMS. FIRST OF ALL YOUR PROGRAMS. THE DP-ONE AND TWOs THAT'S THE PIONEERS AND THE NEW INNOVATORS AND ALL THESE ARE PUBLISHED IN THE BRACKETS AND ARE BLOWN UP HERE BUT LET'S LOOK AT NUMBERS HERE. WE'RE COMPARING THIS TO ALL RO-1S DURING THIS PERIOD OF TIME AND WHAT YOU CAN SEE THAT THE MEDIAN CITATION PER YEAR FOR THE COMMON FUND HIGH RISK PROGRAMS IS AT LEAST 50% HIGHER THAN STANDARD RO-ONES. THE MEAN AND MEDIAN RCRs ARE ALSO HIGHER THAN THE RO-1S. ANOTHER EXAMPLE. HOW ABOUT THE EPIGENOMICS PROGRAM THAT I SHOWED YOU THAT PROVIDED A REFERENCE EPIGENOMES FOR THE COMMUNITY AND ALLOWED WORK TO GO FASTER IN THE SPECIFIC AREAS. WELL, HERE ARE THE GRAPHS AGAIN, THESE ARE THE COMMON EPIGENOME PAPERS. AND THESE ARE PUBMED THAT WAS JUST SEARCHED WITH EPIGENOMICS AND YOU CAN SEE THAT THE MEAN CITATIONS FOR YEAR FOR THE COMMON FUND PAPERS ARE OVER TWICE THE MEAN FOR ALL OF THE OTHER PAPERS THAT ARE STUDYING EPIGENOMICS AND IF YOU LOOK AT THE MEAN RCR, IT'S OVER TWICE AS HIGH. SO THIS IS--THE VALUE OF AN RCR IS A LITTLE HARD TO GET YOUR MIND AROUND BECAUSE IT'S NOT A LINEAR SALE, BUT IF YOU CONVERT THIS FOR ALL NIH PUBLICATIONS, THE COMMON FUND EPIGENOMICS PROGRAMS ARE IN THE TOP 15 PERCENTILE VERSES 40th PERCENTILE AVERAGE FOR THE NONCOMMON FUND. SO IT'S REALLY CLEAR THAT ON AN ARTICLE LEVEL BASIS, THE COMMON FUND PUBLICATIONS ARE MUCH MORE INFLUENTIAL TO THE COMMUNITY IN TERMS OF USE WITH THE CITE. SO I'LL END IT HERE WITH THE NUMBERS FROM THE MICROBIOME, THE COMMON FUND FUNDED MICROBIOME PAPERS VERSES ALL PUBLICATIONS OF PUBMED THAT USE HUMAN AND MICROBIOME. AND THE YOU CAN SEE THAT THE PROGRAM IS OVER TWICE AS HIGH AND THE MEAN RCR IS HIGHER AND YOU CAN CONVERT THAT SOMETHING INTO SOMETHING TO VERBALIZE THE PERCENTILE OF ALL PUBS, THE COMMON SUPPORTED FUND ARE IN THE TOP 5 PERCENTILE OF PAPERS VERSES 25th PERCENTILE FOR THE NON. SO THIS SHOWS IT'S NOT 50, IT IS 25. SO IT SHOWS THE INFLUENCE AND THE INTEREST OF THE COMMUNITY IN MICROBIOME OBVIOUSLY VERSES OTHER WORK BUT IT SHOWS THAT THE COMMON FUND APPROACH TO SUPPORTING THIS RESEARCH RESULTS IN MUCH MORE INFLUENTIAL WORK. SO I THINK THIS IS VALIDATION THAT WHAT WE TRY AND DO IN THE COMMON FUND IN TERMS OF CHOOSING OUR PROBLEMS, DESIGNING PROGRAMS TO OVERCOME, AND THEN ALSO, THE KIND OF SUPPORT AND FLEXIBILITY THAT WE GIVE YOU FOLKS TO DO YOUR HIGH RISK WORK. SO I WANT TO THANK YOU FOR BEING PART OF THIS ACTIVITY. AND I'LL HAND IT OVER TO THE NEXT SPEAKER IS THAT ROBBIE? [ APPLAUSE ] >> THANKS VERY MUCH LARRY WHEREVER YOU ARE AND THANK YOU JIM FOR THOSE REMARKS, SO WE'LL BEGIN OUR FIRST SCIENTIFIC SESSION THAT WILL BE MODERATED BY DR. RICHARD CONROY, HE WAS A DIVISION DIRECTOR OF NATIONAL INTUITY OF BIOENGINEERING AND BIOMEDICINE, THANKS. >> I'M ALSO PART OF THE HIGH RISK HIGH REWARD RESEARCH GROUP AT NIH AS WELL THAT HELPS MONITOR THESE PROGRAMS SO I WOULD LIKE TO BEGIN BY WELCOMING OUR FIRST SPEAKER THIS MORNING, LIHONG WANG WHO WILL TALK ABOUT COMPRESSED ULTRA FAST PHOTOGRAPHY AND EACH SPEAKIER WILL HAVE THREE MINUTES A COUPLE ENDS WITH QUESTIONS AND THERE'S A TRAP DOOR UNDER THE PODIUM IF THEY GO PAST 20 MINUTES SO IF THEY DISAPPEAR DON'T BE SURPRISED. >> LET ME FIRST THANK THE COMMON FUND FOR WORKING AS THE DP-ONE MECHANISM WHICH ALLOWS US TO USE THE UNBRIDLED FUNDS TO EXPLORE HIGH RISK, HIGH PAYOFF DIRECTIONS, THE COMPRESSED ULTRA FAST CAMERA I'LL BE TALKING ABOUT IS A DIRECT PRODUCT OF MY DP-ONE AWARD, WE BUILT THE--TO REST CAMERA IN THE WORLD WHICH ALLOWS US TO CAPTURE LIGHT PROPAGATION, AS WE KNOW FROM THE SPECIAL THEORY OF RELATIVITY. LIGHT PROPAGATION IS AT THE HIGHEST POSSIBLE SPEED OF ANY MATTER OR ABOUT ENERGY, YOU CAN'T JUST GO BEYOND THAT. SO THERE ARE A FEW EXAMPLES I WILL EXPLORE IN MORE DETAIL. OVER A HELPED YEARS AGO, PEOPLE WANTED TO IMAGE FASTER PHENOMENA THAT COULD NOT BE PERCEIVED BOY OUR EYE, LEELAND STAND FARRED, THE FOUNDER OF STANFORD, HAD AN INTEREST IN STUDYING THE GATES OF HORSES, HE TOOK THE POSITION ON A POPULAR DEBATE FOR A WHILE THAT 4 FEET WERE OFF THE GROUND WHILE GALLOPING. HE COMMISSIONED A POPULAR PHOTOGRAPH TO DEVELOP A MOVIE EMPLOY YOU CAN SEE HE USED 24 HIRES AND WHEN THE HORSE PASSES BY THE WIRES WILL BE TRIPPED TO TRIGGER EACH OF THE CAMERAS SO IT'S REALLY CREATIVE AT A TIME OF COURSE AND ALLOWS US TO CAPTURE THE MOTION OF THE HORSE. THERE ARE SNAPSHOTS, STILL SHOTS TRIGGERED AT DIFFERENT TIME POINTS THAT WAS ASSEMBLED INTO THE MOVIE, YOU CAN SEE RIGHT HERE SO THE DEBATE WAS ABOUT WHETHER THE 4 FEET WERE OFF THE GROUND AT THE SAME TIME WHILE IN THE TROTTING POSITION, THIS IS A MOVIE AT A GALLOP POSITION. SO CAN YOU LOOK AT THIS PHASE OF THE CALOP, ALL 4 FEET WERE OFF THE GROUNDMENT IF YOU LOOK AT OLDER PAINTINGS YOU CAN GOOGLE THAT SOME OF THE PAINTERS DREW THIS POSITION SHOWING ALL 4 FEET WERE OFF THE GROUND. SO HAD A HUNDRED YEAR OLD TECHNOLOGY, NOW WE'RE MOVING TO MODERN TECHNOLOGY, OF COURSE THE RATE IS MUCH FASTER, WE CAN USE IT NOW USING OUR SMART PHONE NOW DAYS BUT YOU CAN USE A HIGHER RATE AS USING CCDN, AND CMOS. IT HURTS AS YOU CAN TELL. SO CAN YOU GET MILLIONS OF FRAMES PER SECOND BUT WITH A LIMITED NUMBER OF TOTAL FRAMES PER MOVIE, MAYBE UP TO 10 FRAMES. AND IF YOU WANT TO HAVE CONTINUOUS STREAMING OR HUNDREDS OF FRAMES SOPHISTICATED SO THEN YOU HAVE TO SLOW DOWN THE ARRAY TO KILLA HERTZ RANGE. SO THE MAIN LIMITING FACTORS ARE CHIP STORAGE AND THEIR READ OUT. THE ULTRA FAST CAMERA WE HAVE TO UNDERSTAND THE PRINCIPLE OF THE STREET CAMERA. THIS IS A ONE DIMENSION ULTRA FAST IMAGE AND GERONTOLOGYSTS VISE, AND LET'S JUST ASSUME FOR ILISTRATION PURPOSES WE HAVE FOUR MICROPULSES. WE SPACED, DISTRIBUTED IN BOTH SPACE, LET'S CALL THIS X-AXIS AND TIME. SO YOU SHOOT YOUR BEAM TOWARD THIS STREET CAMERA, THE FIRST ELEMENT IS A SLIT, IT LIMITS THE SCOPE OF THE Y-DIMENSION. THEY USE A COUPLE LENSES TO FOCUS THIS SLIP ON TO THIS PHOTOELECTRODE AS A CATHODE, DUE TO EINSTEIN'S PHOTOELECTRIC EFFECT, PHOTONS ARE CONVERTED INTEREST ELECTRONS. THEN WE HAVE THE MESH ELECTRON THEY PULL TO THE RIGHT. AS THE ELECTRONS ADD TO ANOTHER PAIR OF ELECTRODES, THE ELECTRONS WILL BE PULLED UP AND THE VOLTAGE WILL BE RASMED AS FAST AS POSSIBLE VERY MUCH IN THE SCOPE, DEPENDING ON THE TIME OF ARRIVAL THEY WILL BE PULLED DIFFERENTLY, ON THIS MICROCHANNEL PLATE, WE CONVERT THE ELECTRONS, WE AMPLIFY THE ELECTRONS SO WE GET SUFFICIENT SIGNAL TO NOISE RATIO WE WORK WITH AND THEN WE USE THIS PHOSPHOR SCREEN TO CONVERT ELECTRONS BACK AND THEN AT THE END, THE STANDARD CCD OR CMOS TO CAPTURE IMAGE. WHAT WE GET IS THE Y-AXIS OR VERTICAL AXIS IN TIME, AND THE SPACE AXIS DOESN'T CHANGE. SO YOU'RE ABLE TO GET A ONE HF D ULTRA FAST IMAGE. BECAUSE YOUR CCD DOESN'T RESPOND TO LIGHT PROPAGATION, DOES NOT GET YOU A PICO SECOND OR CENT O SECOND RESOLUTION BUT YOU CAN INTEGRATE EVERYTHING INTO TIME AND SPACE, AND IT ALLOWS YOU TO FORM AN IMAGE, YOU CAN LATER ON PROCESS THE NIH, AND YOU'RE CONVERTING WIDE POSITION BACK INTO TIME AGAIN BY GOING THROUGH THIS IMAGE. BUT TO GET A TWO D IMAGE YOU REALLY HAVE TO SCAN. SO THE GOOD NEWS IS YOU CAN HAVE A REALLY HIGH RATE, UP TO TRILLION ONE D IMAGES PER SECOND EMPLOYED IT'S ONLY A ONE DIMENSIONAL IMAGE, IF YOU SCAN AT REPEAT EFFECTS, YOU CAN GET A TWO D IMAGE, BUT IT TAKES MANY, MANY SHOTS TO FORM THE IMAGE. SO SCAN SUGGEST IS REQUIRED ALONG THE VERTICAL AXIS AND IT MUSET BE HIGHLY COMPETITIVE IF YOU HAVE DISCREPANCY BETWEEN SHOTS THEN YOU WON'T GET A SMOOTH MOVIE. SO HOW DO WE FORM OUR SINGLE SHOT, COMPRESSED ULTRA FAST IMAGE, AND WE'RE ABLE TO GET ONE BILLION FRAMES PER SECOND AS A RESULT. WE WILL START WITH OBJECT, WITH THE LIGHT INTENSITY WHICH IS A 3D DISTRIBUTION, IT'S INTENSITY EYE VERSES X, Y, AND T, AND WE WILL GO THROUGH A SERIES OF OPTIC COMPONENTS TO FORM THORS LIGHT INTENSITY FROM THE IMAGE OF THE LIGHT INTENSITY ON THE D& D, DIGITAL MICROARRAY DEVICE THAT WE USE IN THE PROJECTOR, WE HAVE A PSEUDORANDOM ENCODING PATTERN, IT'S ONES AND ZEROS. IT WILL ENCODE YOUR LIGHT INTENSITY, WE WILL ROUTE THE BEAM INTO THE ENTRANCE OF THE STREET CAMERA, UNLIKE THE USE OF THE STREET CAMERA HERE WE WILL WIDEN THE SLIT, BECAUSE NOW WE WANT TO ADD WHITE DIMENSION, SPACIAL DIMENSION, NOT JUST FOR TIME BUT ALSO FOR SPACE. SO THIS UNCONVENTIONAL USE ALLOWS US TO GET WIDE RESOLUTION. THEN YOU GO THROUGH THE STANDARD PROCESS, THERE'S THIS, HEARING BECAUSE YOU'RE RAMPING THE VOLTAGE OF THE VERTICAL ELECTRODES. THEN YOU HAVE THE CCD OR C-MOUSE INTERNATIONAL CLASSIFICATION GRIGS OVER TIME. AND IN THE END YOU GET IN EQUATION, THE CCD WILL REPORT ENERGY PER PIXEL SO IT'S A FUNCTION VERSES X AND Y THAT YOU GO THROUGH A SERIES OF OPERATORS, ENCODING, E-FOR SHARING, T-FOR TIME INTEGRATION, IF YOU CAN INVERT THIS EQUATION YOU CAN FORM A SERIES OF IMAGES WHICH WILL GIVE YOU A MOVIE. WITHOUT LOOKING AT DETAILS OF THIS DEVICE, LET'S LOOK AT THE PHYSICS BEHIND THIS CAMERA. AGAIN WE START WITH OBJECT, YOU HAVE I WHICH IS A FUNCTION OF X, Y, AND T, TIME VARIANT. WE 50 GO THROUGH THE CODING PROCESS, THROUGH THE DNV, Y OU--DMD, YOU GO THROUGH THE PIXELS AND GO THROUGH THE SHARING OPERATION OF THE STREAK CAMERA. THE YOU TILT THE DATA ESSENTIALLY, THEN YOU HAVE TO DO TIME INTEGRATION, THROUGH THE CCD CAMERA AND IN THE END YOU GET ENERGY MATRIX, E, SO THE ENERGY MATRIX AND A TAIL HERE EVEN THOUGH IT'S MUCH TALLER THAN THE ORIGINAL CUBE. WE CAN FLATTEN THE ORIGINAL E, AND YOU HAVE M-ELEMENTS FOR A NUMBER OF MEASUREMENTS, WE CAN FLAT AN EYE, TO A COLUMN LECTURE WITHIN ELEMENTS, SO HAVE YOU A TOTAL OF N ELEMENTS FOR YOUR OBJECT. THEN WE USE O TO REPRESENT ALL THE O UPONERATORS INVOLVED AND IT'S NBY N MATRIX. THE I IS 3D. ENERGY E IS TWO D. SO IN GENERAL M IS MUCH LESS THAN N, THIS IS A--THIS IS UNDER SAMPLED PROBLEM SO GENERALLY DON'T HAVE A SOLUTION TO ITS PROBLEM. HOWEVER WE HAVE PRIOR KNOWLEDGE. IN MOST CASES WE ARE DEALING WITH A SPARSE MOVIE, SO IT'S THE STANDARD SNAPSHOT IS COMPRESSIBLE WHICH WE DO THAT ALL THE TIME AND A STANDARD MOVIE IS MORE COMPRESSIBLE BECAUSE OFTEN TIMES THERE'S TEMPORAL CORRELATION EMPLOYING WHAT WE DO HERE FOR EXAMPLE IN THIS CASE, THERE'S ZERO GRADIENT, IN A LARGE PATCH OF SPACE IN THIS PHOTOGRAPH AND IT MEANS WE DESCROANT A LOT OF ZEROS AND WE CAN LIMIT THE NUMBER OF NONZERO ELEMENTS, USUALLY IF YOU USE THE RIGHT SPACE TO REPRESENT I WHICH IS A VECTOR, WE WILL USE IT FOR DIFFERENCE OR WAVELET SPACE, AND THEN YOU USE I, IT MAY HAVE NONZERO ELEMENTS BUT IF YOU USE THE RIGHT SPACE, X HAS A NUMBER OF NONZERO ELEMENTS, WOOY CALL THAT S-PARSE. IN GENERAL IT IS MUCH LESS THAN N, NOW THERE'S HOPE TO SOLVE THAT EQUATION. COMPRESSED SENSING IS A NEW THEORY WE FOUND A LOT OF APPLICATIONS BUT IN MOST APPLICATIONS PEOPLE TRY TO MAKE A CHEAPER DEVICE, BUT HERE WE TRY NOT TO USE THE THEORY TO DEVELOP A CHEAPER DEVICE BUT INSTEAD WE WANT TO START WITH A RELATIVELY EXPENSIVE DEVICE AND PUSH THE CAPABILITY OF THE DEVICE AND SEE WHAT WE CAN DO WITH IT, WHAT IS COMPRESS SENSING. LET'S SAY WE START WITH THE SIMPLE UNKNOWNS WE WANT TO SOLVE. X-ONE AND TWO, SO IN THIS TWO, CAN WE LIVE WITH THE SINGLE MEASUREMENT WHILE IN GENERAL ONE MEASUREMENT IS GOING TO YIELD MANY POSSIBLE SOLUTIONS IN THIS TWO D PLOT, ANYTHING ALONG THIS LINE CAN BE A POSSIBLE SOLUTION. SO WE HAVE AN NUMBER OF SOLUTIONS. HOWEVER WE HAVE SOME KNOWLEDGE, WE ONLY HAVE ONE VOXEL AT A TIME--IF YOU DO A LOG OF N MEASUREMENTS, YOU HAVE ONE MORE MEASUREMENT IN THIS CASE, YOU CAN FIND A UNIQUE SOLUTION BUT OF COURSE BY THEN, YOU DON'T REALLY WIN. IN AT THIS TIME END YOU STILL HAVE--YOU NEED ONE PLUS ONE MEASUREMENTS, THAT'S TWO. IF YOU HAVE TWO MEASUREMENTS YOU CAN FIND A UNIQUE SOLUTION ANYWAY. BUT WHERE YOU WIN IS--AND IS LARGE, IN GENERAL, SAND N VOXELS WITH BE THERE AT A TIME, S-LOG-IN MEASUREMENTS ARE REQUIRED TO YIELD A UNIQUE SOLUTION. FOR THE SMALL S AND SUFFICIENTLY LGE N, THIS IS WHY WE CAN TOLERATE THIS AND STILL FIND A UNIQUE SOLUTION. USING THIS THEORY WE'RE ABLE TO WATCH A LIGHT PULSE PROPAGATING IN SPACE WITH A SINGLE SHOT, SO WE'RE NOW REPEATING THE LASER PULSE AT ALL. WE CAN FIRE A LASER PULSE, WE CAN WATCH IT, WE CAN CAPTURE A MOVIE AT 100 BILLION FRAMES PER SECOND, AND NOW OF COURSE, THESE ARE THE SLOWEST SLOW MOTION PICTURES YOU'VE EVER SEEN BECAUSE WE WE CAN SLOW DOWN THE MOVIES CONTINUE BILLION TIMES IN ORDER FOR US TO WATCH. TIS IS A LIGHT PULSE GETTING REFLECTED BY A MIRROR. THE LIGHT PULSE ENTERS INTO A DIFFERENT MEDIA SO YOU'RE SEEING LIGHT REFRACTIONS WITH A SINGLE PULSE, THE TWO PULSES ARE HAVING A RACE IN TWO DIFFERENT MEDIA, SOME OF YOU MIGHT HAVE HEARD FLAME MICROSCOPY WHICH STANDS FOR FLUORESCENCE LIFETIME IMAGING AND THIS IS THE FASTEST SINGLE SHOT FLAME IMAGING, YOU HAVE TO SCAN, EITHER IN TIME OR SPACE SO THAT TAKES A LOT OF TIME, A LOT OF REPEATED SHOTS, HERE IT'S A SINGLE SHOT, THE GROWN SHADOW IS THE EXCITATION SO THE CAMERA IS COLED WITH THE TWO COLOR IN THIS CAMERA NOW, YOU SEE THE GREEN SHOT FOR EXCITATION AND THE RED SHOT IS FOR FLUORESCENCE SUBMISSION, THE FLUORESCENCE SUBMISSION LASTS NANO SECONDS BECAUSE WE'RE RESOLVING PICK O SECONDS SO NANO SECONDS FEELS LIKE AN ETERNITY SO CAN YOU GET THIS VERY EASILY BY FITTING THE U. K. CURVE. WELL WE DON'T FLY CONCORD ANYMORE FOR SUPER JET PLANES, YOU WILL CAUSE SOMETHNG CALLED THE SUPER SONIC MACH CALL, NOW WE CAN OBSERVE A PHOTONIC MACH CONE. IT IS SPEED OF LIGHT IN THE MATERIAL, CUP DO THAT, YOU DON'T VIOLATE EINSTEIN'S SPECIAL THEORY OF RELATIVITY IN THAT CASE, YOU CAN SEE THE CONE HERE WHICH RESEMBLES THE SONIC COUNTERPART VERY WELL. THE TIME OF FLIGHT CAN ALSO BE CONVERT INTOED DISTANCE FOR IMAGING PURPOSES LIKE SONAR OR RADAR, YOU GET SPEED, SPACE, YOU GET THE ACTUAL POSITION. WE CAN USE THIS TO IMAGE THROUGH THE SCATTER MEDIUM LIKE A BIOLOGICAL TISSUE, SO THIS IS A TEST, WE HAVE A MOVING TARGET BEHIND THIS SCATTER AND SCREEN, AND WE USE OUR CAMERA TO FIRE THE PULSE AND WE MEASURE THE ROUND TRIP TIME AND WE SY THIS MOVING TARGET THROUGH THIS SCATTER MEDIA, CAN YOU ALREADY IMAGINE SOME OF THE APPLICATIONS IN BIOLOGICAL TISSUE. WHERE ARE WE GOING FROM HERE? ARE OUR CAMERA IS VERY GENERIC. IT CAN BE COUPLED WITH MICRO SCOPE FOR BIOLOGICAL STUDY SAYS, BIOLOGICAL REACTIONS, CHEMICAL REACTIONS. WE ALSO COUPLED THIS WITH THE HUBBLE TELESCOPE SO CAN YOU GET THE BEST RESOLUTION SO CAN YOU POTENTIALLY DISCOVER NEW PHENOMENA IN OUTER SPACE. AS WE SPEAK, WE'VE INVISSED OUR OWN CAMERA BY ONE HELPED TIMES PER SPEED NOW WE'RE TALKING 10 TRILLION FRAMES PER SECOND, THIS IS THE OLD CAMERA, THIS IS OUR NEW CAMERA, NOTICE THE SPACIAL SCALE HERE, THIS IS 10-MILLIMETERS, THIS IS 1 MILLIMETER, WE ARE ABLE TO RESOLVE LIGHT PROPAGATION AGAINST SINGLE SHOT, MUCH BETTER IN SPACE AND TIME. ADAM COHENS LAB REPORTED SOMETHING VERY EXCITING LAST YEAR AND THEY SHOWED THE FIRST IMAGES OF ACTION POTENTIAL PROPAGATION IN A SINGLE NEURON. BUT HE HAS TO REALLY IMAGE A LOT OF FRAMES IN ORDER TO GET THE RIGHT FRAME RATE. IN THE END HIS TECHNOLOGY IS LIMITED TO 1.1-MILLIMETERS PER SECOND, .1-METERS PER SECOND, ACTUALLY POTENTIAL SPEED AS WE KNOW THE SPEED CAN BE AS HIGH AS ONE OR 50-METERS PER SECOND. HE HAD TO INTERPOLATE HIS MANY FRAMES, HUNDREDS OF FRAMES IN ORDER TO INCREASE THE IMMUNE SYSTEMMAGE AND RATE FROM ONE KILL HERTZ TO 50 KILL HERTZ. HE HAD TROUBLE REPEATING TECHNIQUE OVER MANY NEURONS. SO WHEN YOU DEAL WITH A NETWORK, YOU CAN'T USE THE ALEGORITHMS. WE ARE--OUR NEXT STEP IS TO USE OUR CAMERA IF THIS TYPE OF PROVINCE TO IMAGE THIS OVER A LARGE NETWORK WITH A SINGLE LASER SHOT. I NODE TO THANK NIH FOR FUNDING THE COMMON FUND, CREDIT GOING TO MY LAB MEMBERS AND FOR MORE INFORMATION PLEASE VISIT OUR WEB SITE. THANK YOU VERY MUCH. [ APPLAUSE ] >> WE HAVE TIME FOR ONE OR TWO QUESTIONS FOR LIHONG. SO THE QUESTION IS HOW MANY FOCUSED ON TANS WE HAVE. WE ESTIMATED THE NUMBER, YOU HAVE TO HAVE THOUSANDS OF PHOTONS TO GET AS GOOD AS THEY ARE TO WORK WITH SO IT'S THE NUMBER OF RECEIVED PHOTONS THEY NEED. >> ANY OTHER QUESTIONS? ALSO GIVE A QUICK PLUG, WE HAVE LIHONG HERE ALL WEEK. HE'S TALKING ABOUT THE INVESTIGATOR MEETING LATER IN THE WEEK AND HE WILL TALK ABOUT COMPLIMENTARY WORK HE'S BEEN DOING AND DOING IMAGING IN THE BRAIN AS WELL AS PART OF THAT PROGRAM AND THIS IS SOMETHING WE'RE ALWAYS VERY KEEN TO SEE IS DISSEMINATION OF THESE IDEAS INTO DIFFERENT AREAS OF NIH RESEARCH. SO WITH THAT, PLEASE JOIN ME AGAIN IN TANKING LIHONG FOR HIS TALK AND I WOULD LIKE TO--[ APPLAUSE ] --INVITE THE SECOND SPEAKER IN THE SESSION MING HAM HAMMOND FROM UC BERKELEY AND SHE WILL TALK ABOUT ILMULEINATING BACTERIAL SIGNALING AND RNA-BASED BIOSENSORS. >> I WOULD LIKE TO THANK YOU FOR INSIGHTING ME TO THE MEETING. TODAY I WOULD LIKE TO TALK ABOUT THE WORK IN MY LAB. WE HAVE DEVELOPED TOOLS FOR LIFE CELL IMAGING AND SIGNALING PROCESSES IN BACTERIA BUT FOR TODAY I THOUGHT I WOULD FOCUS JUST ON ONE STORY WHICH CONCERNS THIS PARTICULAR QUESTION WHICH IS HOW DOES A BACTERIA ACTUALLY PERCEIVE THE SURFACE THAT IT'S IN CONTACT WITH, RIGHT? SO MOST OF THE BACTERIA IN THE BIOSPEAR ARE LIVING ON SURFACES AND WE KNOW THAT THIS QUESTION IS IMPORTANT BIOMEDICALLY FOR MANY REASONS, RIGHT? SO WHEN WE TALK ABOUT THIS, WE ARE TURNOVERRED WITH THE FACT THAT BACTERIA MAY WANT TO INTERACT WITH THE SURFACES AND ADHERE IN SUCH A MANNER THAT IT WOULD BE DIFFICULT TO STERILIZE MEDICAL EQUIPMENT. FOR THE SAME REASONS WE ALSO KNOW THAT BACTERIA ARE IN CONTACT WITH BIOTIC SURFACES SCH AS THE INTESTINAL LINING OF THE GUT, WHERE THEY CAN PROPAGATE AND FINALLY, WE HAD MENTION ALREADY OF THE HUMAN MICRO BIOME PROJECT. IN THAT CASE WE KNOW THAT THE ACTION OF A MICROBIAL COMMUNITY IS THROUGH ORGANISMS WORKING IN SYNERGY POTENTIALLY WITH ONE ANOTHER AND THAT INVOLVES BACTERIA CONTACTING EACH OTHER. AND SO THE TAKE HOME MESSAGE FOR TODAY, IS TO TELL YOU ABOUT SOME OF OUR WORK WHICH HAS REALLY HIGHLIGHTED THIS NEW SIGNALING MOLECULE THAT WAS ONLY DISCOVERED IN 2012. AS A SECOND MESSENGER ACTUALLY FOR HOW--FOR SURFACE SENSING BY BACTERIA AND IN PARTICULAR, THIS SECOND MESSENGER IS REGULATES THE FORMATION OF TYPE FOUR PILLI, THAT ARE SPECIALIZED TO INTERACT WITH DIFFERENT SURFACES AND I WILL GIVE YOU EXAMPLES OF HOW WE FOUND THIS SIGNALING INVOLVED IN DIFFERENT PROCESSES. SO THIS IS A BIT OF A COMPLICATED SLIDE BUT I WANT TO EXPLAIN WHAT THE SIBLING MESSENGER MEANS. SO THE SECOND MESSENGER SIGNALING MEDIATING THE FORMATION OF BIOFILM SYSTEM VIA THIS COMPOUND, RELATED COMPOUND CALLED CYCLIC DI-GMP. AND IN SECOND MESSENGER SIGNALING IS THAT YOU HAVE A PRIMARY SIGNALING ENVIRONMENTAL CUE THAT IS PERCEIVED ON THE CELL SURFACE BY PROTEINS THAT HAVE A SENSORY DOMAIN, THAT COMMUNICATE THEN TO AN ENZYME ACTIVE SITE DOMAIN THAT EITHER ACTIVATE THE SYNTHESIS OF THE SOLUBLE SIGNAL OR IN SOME CASES, THE SIGNAL ACTUALLY INHIBITS THE PRODUCTION OF THE SIGNAL, OR ACTIVATES FOSTER NURSED FOCUSED ON DIASTER ACES THAT DEGRADE THE SIGNAL. THE SOLUBLE FACTOR THEN CAN PROPAGATE THE PRIMARY STIMULI BY BYPASSING TO VARIOUS MOLECULAR EFFECTORS, THESE COULD BE BINDING PROTEINS, TRANSCRIPTION FACTORS OR RNAs DONE AS RIBOSWITCHES AND AS A MENTION THE DISCIPLINARY I-GMP SIGNALING IS UPREGULATION WHICH ITSELF IS AN INTERESTING PROCESS. SO IN 2012, THIS RELATED SECOND MESSENGER CYCLIC AG WAS FIRST DISCOVERED BY JOHN MC LLANOs GROUP WHO FOUND THAT THIS ENZYME DNCB WAS RESPONSIBLE FOR PRODUCING CYCLIC AMP-GMP IN CHOLERA, AND IT EFFECTED THE EFFECT TO COLONIZE THE INTESTINES OF MICE AND THEY HAD ASSOCIATED DN CV ALSO. JUST THIS YEAR WE PUBLISHED THIS INTERROGATORY NO. IN A COMPLETELY DIFFERENT ORGANISM, AND THE EFFECTOR AS YOU CAN SEE HERE, WE ALSO GOT THIS CRYSTAL STRUCTURE OF THIS RNA COLLABORATION WITH P A TEL, SERVEs AS A BEAUTIFUL RNA THAT BINDS AND CRATELES AT ITS HEART THE SIGNALING MOLECULE AND IT IN FACT FOLDS AROUND THIS SIGNALING MOLECULE AND THIS COMBINATION CHANGE EFFECTS THE MESS ERCHER RNAs THAT CARRY A COPY OF THIS SWITCH AND THE PRIEF UNTRANSLATED REGION SO THIS SMALL MOLECULE RNA INTERACTION PROPAGATES DIRECTLY INTO EFFECTS THE EXPRESSION OF THE DOWN STREAM GENES. NOW IN THIS ORGANISM, THE SELF-REDUCEINS THAT WE FIRST DISCOVERED THE RIBOSWITCH IS AN ENVIRONMENTAL BACTERIA, AND HERE IT GOES TO SHOW THAT THE SIGNALING AND BROADLY APPLIED BY BACTERIA. IN PARTICULAR IN GIVING--YOU O FACTOR, THE ASSOCIATION TO A SURFACE IS NOT FOCUSED ON A HOST CELL BUT IN FACT TO A MINERAL OXIDE SURFACE, SO ONE REASON WHY GEOBACTERIAER IS AN EXAMPLE OF A CURRENT PRODUCING ORGANISM. SO WE JUST HEARD AN EXAMPLE OF KIND OF NEURON CELLS WHICH WE UNDERSTAND AS PRODUCING CURRENT, WELL THIS ORGANISM ACTUALLY PRODUCES CURRENT AS WELL. AND THE PROCESS IN WHICH IT IS ABLE TO ESSENTIALLY PERFORM EXTRA CELLULAR ELECTRON TRANSFER OUT ON TO TO REDUCE MINERAL OXIDE SURFACES IS MEDIATED THROUGH SPECIALIZED TYPE FOUR PILL I THAT ARE DECORATE RATED WITH DIFFERENT MEMBRANE CYTOCHROME PROTEINS AND THE TYPE FOUR PILL I ARE IN FACT REGULATED BY THESE RIBOSWITCHES THAT WE DISCOVERED. OKAY, SO, THIS DISCOVERY AGAIN WAS SOMEWHAT UNEXPECTED BECAUSE ONE OF THE THINGS THAT WE PROVED WAS THAT THIS ORGANISM WAS A CYCLIC AMP-GMP, BUT IT LEFT A MYSTERY WHICH IS MEN AND WOMEN THAT--WHICH IS THAT IN THE GENOME, THERE WAS ONLY CYCLIC DMCB, SO CHASING AFTER THIS QUESTION WHICH WAS WHAT OF WAS THE IDENTITY OF THE ORGANISM THAT MAKES PSYCHE LIKE GMP, IT GIVE US INSIGHT INTO THE FACT THAT CYCLIC AG IS MORE BROADLY FOUND AND MEDIATING DIFFERENT PROCESSES. OKAY TO SCREEN FOR PSYCHE LIKE AG SYNTHESIS ABILITY IN DIFFERENT ORGANISMS WE NEEDED A WAY TO SCREEN FOR THIS ACTIVITY IN VIVO. AND THE WAY IN WHICH WE DO SO, GETS TO THE HEART OF THE TECHNOLOGY THAT WE DEVELOPED IN MY LAB. AND SO THESE ARE FLUORESCENT BORS THAT ALLOW FOR US TO DETECT THESE SIGNALING MOLECULES IN LIVING CELLS, AND THE WAY IN WHICH WE DEVELOP THESE SENSOR SYSTEM WE TAKE A RIBOSWITCH RNA SUCH AS THE ONE WE JUST DISCOVERED WHICH BYS SPECIFIC SIGNALING MOLECULE AND WE FUSE IT TO ANOTHER SEQUENCE SHOWN IN BLACK WHICH HAS BEEN NICKNAMED SPINACH. TIS RNA SEQUENCE SPINACH IS A SO CALLED RNA MIMIC OF GFP AND IT BINDS TO SOME SMALL MOLECULE AND IT'S STRUCTURALLY SIMILAR TO THE HEART OF GREEN FLUORESCENT PROTEIN. AND UPON BINDING TO THE RNA, THE DYE GOES FROM BEING LOW TO HIGHLY FLUORESCENT. SO IN ORDER TO DEVELOP A CYCLIC A. G. SENSING BIOSENSOR, WHAT WE HAVE DONE IS MAKE THE FORMATION OF THE DISCIPLINARY-BYPASSING POCKET DEPENDENT ON CYCLIC AAG, WHICH WE DO BY ESSENTIALLY BINDING--COMBINING THE LIGAND BINDING POCKET IN PROXIMITY TO THE DYE BINDING POCKET SO THE END RESULT IS A GENETICALLY ENCODED SEQUENCE OF A HUNDRED NUCLEOTIDES IN LINK THAT'S DETECTED TO IN THESE AG-CELLS SO IN ORDER TO SCREEN FOR THE CYCLIC AG SYNTHASE CAPABILITY, WE COULD HAVE DONE A WHOLE GENOME SCREEN USING OUR FLUORESCENCE BIOSENSOR BUT WE ALSO TURNED OUT HOW TO VERY COMPELLING HYPOTHESIS THAT WE PURSUED FIRST WHICH TURNED OUT TO PAN OUT. IN AND THE HYPOTHESIS WAS THAT BASED ON OUR FINDINGS FOR HOW CYCLIC AG SIGNALING WAS EFFECTED BY THESE RNAs WE HYPOTHESIZED THAT THE SYNTHASE MIGHTING--ARISE FROM A CLASS OF ENZYMES THAT CLASSICALLY HAS BEEN ASSOCIATED WITH ARE IF THE PAST 30 YEARS WITH CYCLIC DI-GMP PROTEINS WHICH ARE THESE PROEN TOOS AND IN MANY OTHER BACTERIA THIS ENZYME CLASS IS HIGHLY ABUNDANT AND VERY POORLY CHARACTERIZED. SO OUR HYPOTHESIS WAS, PERHAPS ONE OF THESE OR SOME OF THESE PROTEINS IN FACT HAVE SPECIALIZED IN THIS ORTHOGONAL SIGNALING PATHWAY. SO IN ORDER TO PERFORM THE SCREEN RATHER THAN DOING MICROSCOPY, WE WERE ABLE TO ADEPARTMENT OUR BIOSENSOR FOR USE IN FLOW CYTOMETRY EXPERIMENT SO WHAT YOU'RE LOOKING AT HERE IS THE BIOSENSOR LIGHTING UP OR IN RESPONSE TO DNCB WHEREAS A RELATED ENZYME, CALLED WHISPER AS YOU CAN SEE DOES NOT GIVE A RESPONSE. AND SO THE BIOSENSOR AND ANOTHER SENSOR THAT'S SELECTED FOR CYCLIC DI-GENE IS WE CAN PERFORM THIS SCREEN SO WHAT YOU'RE LOOKING AT HERE IS THE MEAN FLUORESCENCE INTENSITY FOR THE HISTOGRAMS AND I HOPE WHAT YOU CAN APPRECIATE IS THAT ONE OF THOSE TWEIVE ENZYMES LIT UP USING OUR BIOSENSOR. AND IN FACT, E.COLI NATURALLY DOES NOT HAVE CYCLIC AIG BUT E.COLI EXPRESSING THE ONE GENE MAKES CYCLIC AG AND WE WERE ABLE TO ANALYZE AND DETERMINE THAT THERE WAS A SIPPINGLE AMINO ACID SUBSTITUTION IN THIS THE GG-DEF TO HAVE THIS CATALICK UNDERSTANDING AND. IT WAS THIS UNDERSTANDING THAT THIS SYNTHESIS ABILITY HAD EVOLVED IN THIS SUBCLASS OF GG DOMAINS WHICH WE CALL HYPER GGDEF, AND THIS ALLOWED US TO THEN SURVEY OTHER ORGANISMS AND WE FOUND THAT MANY PROTEIN COMPLEXIO BACTERIA INCLUDING SOME PROTEIN COMPLEXIO BACTERIA THAT ARE FOUND IN ORAL BIOFILM MAKE CYCLIC A. G. THIS IS AN EXAMPLE OF ANOTHER FASCINATINGICALITIA BACTERIA CALLED THE DELO VIBIO, WHICH IS A SMALLEST BACTERIA OUT THERE BECAUSE IT IS A PARASITE WITH OTHER BACTERIA, SO THIS ORGANISM IS DESCRIBED AS A LIVING ABET BIOTIC AND IT IN FACT IS AN INTRACELLULAR PATHOGEN THAT BURR BURROS WHERE IT UNDERGOES RAPID DIVISION CONSUMING ALL THE MATERIALS IN THE CELL BEFORE LIESING AND BURSTING FORTH ALIEN STYLE IF YOU WILL FROM THE BACTERIA. AND WHAT THE SOCKET GROUP AT KNOTTING HAM HAD OBSERVED BUT NOT MADE THE CONNECTION TO THE SIGNALING PATHED WAY WAS THAT IN FACT THEY OBSERVED A MUTE ANT THAT HAD DEFECTS IN THE ABILITY OF THE BACTERIA TO ESSENTIALLY LEAVE THE [INDISCERNIBLE] BACK AND HAD SUCKED THE LIFE OUT OF. SO THE IMAGES THAT YOU SEE, THE DELAVIBRIO, THEY'RE PROPAGATING IN THE CELL, LIESING BY THE LOSS OF GFP AND OVER THE COURSE OF FOUR HOURS THESE MUTE ANTS ARE ABLE TO ADETACH FROM ONE ANOTHER TO FIND PREY. SO KIND OF SUMMARIZING OUR WORK IN THIS AREA BEFORE WE STARTED REALLY, THERE WAS PSYCHE LIKE H HAD NOT BEEN DISCOVERED BUT NOW WHAT WE KNOW IS THAT IT APPEARS TO BE AN IMPORTANT PLAYER IN DIFFERENT SURFACE SENSING INTERACTIONS. SO FOR EXAMPLE, I'VE GIVEN YOU ONE EXAMPLE WHERE CYCLIC AG IS MEDIATING CONTACT TO A HARD SURFACE, AND TWO EXAMPLES WHERE THE INTERACTIONS IS TO A HOST CELL OR TO ANOTHER BACTERIA. NOW WE HAVE THE MESSENGER CELL, CAN WE USE THE PROCESS TO IDENTIFY IN VIVO, THE PRIMARY SIGNAL, THE PROPERTY OF THE SURFACE OF THE HOST FOR EXAMPLE, THAT ALLOWS FOR ADHERENCE SO ON AND SO FORTH AND IS OUR ABILITY TO AGAIN WHEN SIGNAL SUGGEST ACTIVATED WILL ALLOW US TO INTERROGATE DIFFERENT PRIMARY END POINTS SO PREPRIMATESSOTIC INPUTS OR PERHAPS THE BACTERIA ARE ABLE TO ADAPT AND REGULAE THESE PROCESSES INCLUDING ADHERENCE AND DIFFERENT TYPES OF MOTELLITY AND CELL-CELL COMMUNICATION. THIS IS A SENSOR TO DEVELOP THE TRANSFERASE ACTIVITY BUT IN THIS EXAMPLE IS I'M SHOWING YOU REALTIME CHEMICAL INHIBITOR INHIBITING AND ENZYME THAT WE CAN MONITOR USING BY O SENSORS SELECTIVELY AND A NATIVE ENZYME IN THE CELL NOT AN OVEREXPRESSED ENZYME SO WE'RE DETECTING IN REALTIME ENZYME IN CYTOCELL. AND FIBALLY I WANT TO SAY WE HAVE A GENERAL PROGRAM ON DETECTING MESSENGERS INCLUDING DISCIPLINARY 60IC D-GMP, AND IT'S INVOLVED IN CELL HOMEOSTASIS AND PATHOGENS AND ALSO CYCLIC AG EVIL TWIN, ALSO KNOWN AS C-GAM AND SECOND MESSENGER INVOLVED IN INNATE IMMUNITY SO WITH THAT I WANT TO REALLY THANK MY STUDENTS, PAST AND PRESENT AS WELL AS FACULTY COLLABORATORS AT BERKELEY AND BEYOND AND OF COURSE THE NIH AND THE NEW INNOVATOR BECAUSE IT FUNDED ESSENT LALLY THE MAJORITY OF THE WORK THAT I TALKED ABOUT TODAY. [ APPLAUSE ] >> TIME FOR A COUPLE QUESTIONS. >> AS I UNDERSTAND IT YOU ARE TALKING ABOUT A MESSENGER THAT HAS A WIDE RANGE OF GENETIC INFRASTRUCTURE THAT'S RESPONSIBLE FOR THIS, IT SEEMS THERE'S A TOP DOWN SYSTEMS LEVEL NEED THAT'S DEFINING THE PROCESS RATHER THAN SOME RANDOM MUTATION PROCESS THAT IS THEN CAPITALIZED ON. >> YEAH, SO I THINK MY SPECULATION ON THE KIND OF EVOLUTIONARY ORIGIN OF THE SIGNAL SUGGEST ACTUALLY--A LOT INFLUENCED BOO I WHAT WE HAVE DISCOVERED SOA. WHAT WE HAVE FOUND IS THAT IT APPEARS THAT THE GMP SIGNALING IS AGAIN A VERY OLD PROCESS AND BACTERIA WHICH IS KNOWN TO REGULATE MOTILITY, WIDE VARIETY OF BACTERIA. IT APPEARS THAT SOME OF THE SIGNALING COMPONENTS FOR CYCLIC GMP, AND IT AROSE BY SPECIALIZATION OF CYCLIC GMP SIGNALING TO RECOGNIZE NEW RELATIVELY KNEW SIGNALING MLE PSYCHE LIKE AG, ANOTHER CONSEQUENCE OF THAT WHICH I THINK IS POWERFUL AND SEEN FOR EXAMPLE WITH THESE HYPER GG DEATH ENZYME, IS THAT IT'S POSSIBLE THAT THOSE THREE CYCLIC DINUCLEOTIDES THAT ARE KNOWN SO FAR, NOT THE ONLY ONES THAT ARE OUT THERE, AND SO, ONE OF THE THINGS THAT WE'RE EXPLORING IS THAT NOW WE KNOW THAT THERE'S ESSENTIALLY POTENTIAL FOR ADDITIONAL SPECIALIZATION AND SO IT WOULD BE VERY INTERESTING, IN WHAT WE'RE PURSUING NOW IS TO UNDERSTAND WHETHER NATURE HAS ALREADY ADAPTED AND MADE ADDITIONAL CYCLIC DINUCLEOTIDE SIGNALING OR IN FACT WE'RE ALSO INTERESTED IN ENGINEING NEW SIGNALS FOR BACTERIAL SENSING OURSELVES AS WELL. >> NOW FOR THINGS HAPPENING INSIDE THE CELL, DO YOU HAVE A TIME SCALE FOR HOW THEY'RE REACTING TO THE STIMULI, THE TIME LAG MIGHT BE INTRESTING TO KNOW. >> YEAH, SO I WISH I HAD A HIGH SPEED CAMERA. [LAUGHTER] NO, ACTUALLY IN ALL SERIOUSNESS, WE HAVE TO SAY THAT OUR SENSORS ARE NOT THAT FAST. SO THE LIMITATION IS LIKELY GIB THAT THE SENSORS WORK IS THROUGH ESSENTIALLY BINDING CONFIRMATION CHANGE BY FLUORESCENCE SO WE AND OTHERS HAVE SERVED CONFIRMATIONAL CHANGES TEND TO BE SLOWER BUT APPROXIMATER HAPPENS EVEN--OR ONE ORDER OF MAGNITUDE THAN PROTEIN CONFORMATIONAL CHANGES. SO WE'RE A BIT CERTAINED ACTUALLY THAT OUR SENSORS ARE LIMITING IN OUR TERMS OF OUR ABILITY TO SEE TIME SCALES. THAT BEING SAID, SOME OF THE PROCESS WE STUDY TAKE ON THE ORDER OF HOURS, SO BIOIS HOURS OR DAYS IN TERMS OF PILI FORMATION, IT'S A LARGE APPENDAGE OR ORGANIZATIONSLE OF THE ORGANISM THAT HAS TO BE ASSEMBLED SO THAT AGAIN IS A LONG-TERM PROCESS. BUT WHAT WE DON'T KNOW IS TRANSIENT, HOW LONG DOES THE SIGNAL HAVE TO BE PRES END TO TURN THINGS ON, THAT'S AN INTERESTING QUESTION WE HAVE NOT BEEN ABLE TO ADDRESS. >> SO IN SIGNIFY CYCLIC GMP IT REGULATES THESE CYTOCELL MAY BE IMPORTANT, HOW ABOUT IN THIS CASE? WE STARTED IN WOI TO ADDRESS THE QUESTION OF SPACIAL LOCALIZATION OF CYCLIC DISCIPLINARY NUCLEOTIDES CLE O TIDES, SO THIS IS DESCRIBED AS HAVING DIVERSE PHENOTYPES AND ONE OF THE ONLY WAYS THEY COULD THINK OF TO EXPLAIN WHY THERE WERE DIVERSE PHENOTYPE SYSTEM THE FACT THAT THERE MUST BE SUBCELLULAR LOCALIZATION. WHAT WE'VE SHOWN IN THIS CASE IS IS 10 WE STUMBLED UPON AND SOME CASES IT'S NOT SUBCELLULAR LOLLIZATION FOR PHENOTYPES BUT THESE SIGNALING PATHWAYS ARE MAKING ORTHOGONAL MOLECULE AND THAT BEING SAID I THINK THERE IS THE POTENTIAL ESPECIALTILY IN ASYMMETRIC ORGANISMS LIKE COLABACTER, THERE IS A GRADIENT OF THE SIGNALING MOLECULE AND WE NOW HAVE NEW GENERATION VERSIONS OF OUR SENSORS THAT WILL ALLOW US TO ASK THOSE KINDS OF QUESTIONS. THANK YOU. >> OUR THIRD SPEAKER THIS MORNING IS LIGUO WANG, AND HE WILL TALK ABOUT CRYO-EM STRUCTURE OF THE BK ION CHANNEL IN LIPID MEMBRANE. >> FIRST I WOULD LIKE TO THANK THE ORGANIZER FOR GIVING ME THE OPPORTUNITY TO SHARE MY RESEARCH WITH YOU TODAY. SO AS WE ALL KNOW THE PROTEIN MEMBRANE ARE IMPORTANT ABOUT THE WIDE SPREAD OF THE HUMAN GENOME, ENCODES MEMBRANE PROTEINS AND ABOUT 70% ALCOHOL ARE MEMBRANE PROTEINS, HOWEVER IF WE LOOK AT PROTEIN DATA BANK ALLOWING SIX CELLS IN THE POINTING STRUCTURES ONLY ABOUT ONE % OF THE STRUCTURES OFFER MEMBRANE PROTEIN SO THIS IS MAINLY BECAUSE OF THE DIFFICULTY IN INFORMING FOR CRYSTALLOGRAPHY. SO IN RECENT YEARS THERE'S A REALLY GOOD [INDISCERNIBLE] WHICH HAS BEEN USED TO STUDY MEMBRANE PROTEIN STRUCTURES FOR EXAMPLE, WE HAVE THE CHANNEL WITH THE SECTOR AND THE POTASSIUM ION CHANNELS. THE ALL OF THOSE STRUCTURES HAVE BEEN AN ATOMIC RESOLUTION. SO FOR CRYO-EM. IS IT STARTING? SO THIS SHORT MOVIE IS JUST TO SHOW YOU THE MICROSCOPY. SO IT'S AN ACTIVATOR OF THE INTRODUCTION AND SO FOR THE ELECTRON MICROSCOPY, WE HAVE A SOLUTION, BIOLOGICAL MOLECULES AND IT IS A YOUNG GRADE, AND THEN QUICKLY DROP IT INTO [INDISCERNIBLE] AROUND THE 80-DEGREES CELSUOUS BECAUSE OF THE GRADIENT SPEED, ALL THE ICE FORMED IS MORE [INDIS SO THERE'S NO CRESTALLING SO THE OTHER STRUCTURES WHO WILL BE IN TACT. SO WE PUT THAT IN THE MICROSCOPE AROUND 75-DEGREES CELSUOUS AND THEN WE COLLECT THE CRYO-EM IMAGES BASED ON THE INDIVIDUAL PROTEIN PARTICLES ASTERISKS DIFFERENT ANGLES THAN WE USE COMPUTATION MEASURED TO GET THE MUTATION, SO THE MUTATION IS CLASSIFIED [INDISCERNIBLE] SO IN ORDERED TO GET THE MUTATION OF ONE PROTEIN PARTICLE, WE HAVE TO COMPARE IT WITH AN INITIAL MODEL AT DIFFERENT ORIENTATION. FOR EXAMPLE, WE NEED TO COMPARE ONE PARTICLE IMAGE TO ALL OF THE POSSIBLE ORIENT AGES WITH A SEARCH STAFF OF 6-DEGREE SO WOO HAVE TO COMPARE IT TO ABOUT 100 SELLS IN THE DIFFERENT [INDISCERNIBLE]. SO IT'S COMPUTATIONALLY, SO WE PROPOSE [INDISCERNIBLE]. FOR THE MEMBRANE PROTEIN WITH A VESICLE, AND FOR EXAMPLE, WE HAVE ONE PROTEIN INSERTED IN THIS WAY, SO THE IMAGE DEPENDING ON THE [INDISCERNIBLE] OF THE PARTICLE, WITH RESPECT TO THE CNTER OF THE [INDISCERNIBLE] THEN WE CAN DESIGN THE ANGLES AND AND AFTER YOU YOU HAVE TWO FOLD AMBIGUITY AND WE DON'T KNOW--IN THE UPPER HALF, ON THE BOTTOM HALF, JUST THE VASCULAR SENSE, TO THE CENTER. SO WE DECREASE THE SEARCH FROM OE RESULT AND COMPARISONS TO ABOUT 100 COMPARISON. THAT'S A BIG IMPROVEMENT AND ALSO WE DECREASE THE SEARCH FROM [INDISCERNIBLE] SPACE TO ONE DIMENSIONAL, SO THAT MEANS THAT THE ACCURACY WILL BE MUCH HIGHER. SO THIS BENEFIT OF THE USES OF THE RECONSTITUTION IS ALSO THE [INDISCERNIBLE] RESTORE MEMBRANE FOR THE ENVIRONMENT FOR MEMBRANE PROTEIN BOTH STRUCTURAL FUNCTIONS AND STUDIES SHOWS IT'S THE RIGHT HELP FOR MEMBRANE PROTEINS, SO THE RECONSTITUTION, SO WE INCREASED THE ACTIVITY OF THE MEMBRANE PROTEIN AND ALSO HERE, CAN YOU SEE, BIGGER MIMIC OF THE CELLULAR ENVIRONMENT AND WE CAN CHANGE THE COMPOSITION AND INSIDE AND OUTSIDE, AND ALSO THE TOXINS, SO IT MEANS WE CAN [INDISCERNIBLE] THE STATE OF THE CONSITUTE TED MEMBRANE PROTEIN. SO FOR THIS [INDISCERNIBLE] WE CAN USE DIFFERENT COMPUTATIONAL ESTATE FOR THE MEMBER OF PROTEIN. SO AFTER RECONSTITUTION WE COMPILE PROTEIN COMPLEXIO LYSOSOMES TO THE CRYSTAL, THE [INDISCERNIBLE] TO THE CRYSTAL AND THEN WE DROP THAT INTO THE [INDISCERNIBLE] IMAGES SO THIS BACKGROUND IS THE CRYSTAL, WE HAVE OUR OWN PROGRAM TO REMOVE THE CRYSTAL BACKGROUND AND THE CONTRIBUTION FROM THE VESICLE. SO WE CAN USE OUR PROGRAM TO PICK THE PROTEIN PARTICLES TO OTHER COMPARISON, AND THIS IS MUCH SHORTER COMPARISON ABOUT 100 COMPARISONS, AND THEN THEY'LL WEAKEN THE RECONSTRUCT THE STRUCTURE OF THE MEMBRANE PROTEIN, SO HERE WE GET INSIDE AND MEASURE THE STRUCTURE OF THE POTASSIUM CHANNEL AND THAT'S LARGER CONDUCT ANTS SO IT CAN BE FOUNDED, A FUNCTION AS A FIFTH BACK REGULATORS OF THE MEMBRANE POTENTIAL AND THE INFRACTS SO THESE HAVE BEEN SENSITIVE STUDY OF THE ION CHANNEL, AS AN ORGANELLE AND THIS CHANNEL WILL OPEN WITH THE POTENTIAL BECOMES POSITIVE AND ALSO THE OPEN PROBABILITY INCREASED DRAMATICALLY WHEN WE HAVE THE CONSISTRATION FROM ZERO TO 100 MICROMOLAR. SO THIS CHANNEL IS A NUMBER OF THE 6:00 P.M. TRANSMEMBRANE CHANNEL SO THE ARCHITECTURE OF THE TRANSMEMBRANE REGION IS EXPECTED TO BE SIMILAR TO LIGHT UP KB-12, AND ANOTHER CHANNEL FOR THE CRYSTAL O GENICRAPHY, SO THIS IS FIVE, SIX, AND FORM THE CONDUCTING [INDISCERNIBLE] AUND IT ARE THE SENSORS MADE UP OF X-ONE TO FOUR. SO COMPOTTIBLE, WE HAVE ONE EXTRA HELIX AT ZERO, WHICH BRINGS THE NEURONS TO THE EXTRA CELLULAR SIDE. SO FOR THIS SENSOR AND CONDUCTING [INDISCERNIBLE] NOT FULLY UNDERSTOOD BUT THERE ARE MANY HYPOTHESIS, THE MOST PROMISING ONE IS THE MOVEMENT OF THE POSITIVE [INDISCERNIBLE] AND LIGATE OPEN. SO, THE INTRACELLULAR SIDE OF THE ION CHANNEL, THERE'S SENSING DOMAIN CALL IN THE RING. IN 2010, AND 2012, SO ACTUALLY [INDISCERNIBLE] CRYSTALLOGRAPHY HAS BEEN USED TO STUDY THE STRUCTURE WITH THE PRESENCE OF CALCIUM AND WITHOUT THE PRESENCE OF CALCIUM. SO COMPARED TO THE EXTRA STRUCTURES WE CAN SEE THE RESPONSE TO THE [INDISCERNIBLE] SO THE RCKONE DOMAIN OPENING THE PATHOS OF THE [INDISCERNIBLE]. TO THAT WAY. SO THE CHANCE OF THIS STRUCTURES ARE JUST FOR THE INTRACELLULAR DOMAIN, SO THE COMPUTATIONAL CHANGE OF SENSING IS [INDISCERNIBLE] TO THE CHANNEL, IS CANNOT BE DERIVED HERE. SO FOR THE LAST ONE THOUGH, THERE'S ONE STRUCTURE PUBLISHED IN THIS LAB, SO THE [INDISCERNIBLE] CHANNELS, THE LESS SODIUM ACTIVATED CELL STRUCTURE USING [INDISCERNIBLE] TO ABOUT 4-ANGSTROM, SO THIS IS A CLOSED ONE. SO THIS IS PROPOSED FOR THE COUPLING BETWEEN THE LIGAND SENSING AND THE CHANNEL OPENING. SO WE SEE THE LOBE OF THE RC-ONE DOMAIN, TO THE SODIUM, THAT WILL MOVE UP, AND OUTWARD, PUSH THESE AND X-FOUR AND FIVE UP AND PULL THE GATE OPEN, SO THAT CAN BE PROPOSED BUT HOW TO UNDERSTAND HOW IT WORKS, STUDY THE CHANNEL AND IN DIFFERENT MINERAL SPACE TOWARDS THE DIRECT COMPARISON, SO AS I MENTIONED EARLIER, SO WE CAN CHANNEL THE COMP CISION OF THE TIN ERNAL AND EXTERNAL SOLUTION, SO WE CAN [INDISCERNIBLE] SO WE PROPOSE TO USE OUR MEASURES TO STUDY THE STRUCTURAL OF THE PK-ECHANNEL AND THE PRESENCE OF THE MEMBRANE. SO WE--THE BK CHANNEL WAS [INDISCERNIBLE] AND THE ACTIVITY OF THE CHROMATOGRAPHY TO PURIFY, AND THEN WE MIX IT WITH THE LIPID AND DETERRENT AFTER REMOVAL OF THE DETERGENT, ALL THE PROTEINS WILL BE CONSITUTED INTO DIFFERENT VESICLES, AND THEN WE FUNCTIONALLIZE IT FOR THE PROTEOSOME, SO THE PROTEIN COMPLEXIO LAB STUDIES OF MULTIPLE ENDOCRINE, WITH THE PROTON DETECTION, ICMA, AND WE CHANNEL THE SOLUTION FROM CONCENTRATION TO LOW POTASSIUM CONCENTRATION THEN THE PK CHANNEL AND SO WE MOVE FROM THE INSIDE TO THE OUTSIDE POTENTIAL WILL BE ESTABLISHED, SO WILL OPEN SO PROTON WILL GO IN AND THE INTACT WAYS OF THE PROTON DETECTION OF THE ACMA, SO THE FLUORESCENCE SIGNAL WILL CHANGE. SO IN THESE TWO FIGURES WE SHOW THE RESPONSE OF THE PK PROTEOSOME, AND WE ALSO SHOW THE LYSOSOME FOR COMPARISON. SO WE CAN SEE AFTER THE ADDITION OF THE PROTEIN [INDISCERNIBLE], THE FLUORESCENT SIGNAL DROPS SIGNIFICANTLY AND ALSO HERE, OF THE [INDISCERNIBLE] DUE TO FUNCTION OF THE CHANNEL, YOU CAN SEE RESPONSE TO DIFFERENT POTASSIUM ACROSS THE MEMBRANE, THIS CLEARLY SHOWS THE BIOFUNCTIONALLITY OF THE BK CHANNEL. [INDISCERNIBLE] RAISE IT AND ELIMINATE IT USING F20 MICROSCOPE AND USING A TYPE OF CAMERA, CALLED THE KTWO SUMMIT DIRECT DETECTION CAMERA, SO NOW WE'RE RECORDING MOVIES INSTEAD OF ONE PICTURE. SO THIS ALSO INCREASED SIGNAL TO NOISE RATIO AND THEN AFTER THE COLLECTION OF THE IMAGES WE JUST REMOVE THE CRYSTAL INFORMATION AND THE VESICLE, THEN WE DO ON THE [INDISCERNIBLE], AND RECONSTRUCTED THE STRUCTURE, SO HERE'S THE EXAMPLE OF THE IMAGE, SO WE CAN SEE THE CRYSTAL BACKGROUND SO THIS BACKGROUND CAN BE REMOVED BY THE MANIPULATION, YOU CAN SEE THE BRIGHT SPOTS, THOSE ARE DUE TO THE CRYSTAL, AND WE GET THE INFRANCIS COLLINS SPACE AND THE CRYSTAL BACKGROUND. AND THEN ALL THE LABS OF THE SIZE, SO WE COULD DO THE RECONSTRUCTION, STUDY STRUCTURE OF THE ENTIRE PROTEIN COMPLEXIO LYSOSOME, BUT UNFORTUNATELY, THEY'RE NOT INSIDE, SO WE BUILD THE MODELS, SO WE [INDISCERNIBLE] WITH ALL THE OXIDATION, WE CALL THIS THE [INDISCERNIBLE]. WHEN SUBTRACTED THIS IMAGE FROM EACH INDIVIDUAL SIZE OF THE VESICLES AND YOU CAN SEE ALL THE [INDISCERNIBLE] INFORMATION AS HAN SUBTRACTED BUT HERE YOU CAN SEE [INDISCERNIBLE] BECAUSE THOSE ARE TOO SMALL, AND WE DON'T USE IT. WE ARE WONDERING THIS PROTEIN. FROM THIS, WE CAN THEN PICK THE PROTEIN PARTICLES, OR THE SEMIAUTOMATICALLY, SO, WE CAN USE OUR [INDISCERNIBLE] DO FIND ALL THE ORIENTATION OF ALL THE DIFFERENT INDIVIDUAL PROTEIN PARTICLES, THIS SPEAKER SHOWS THE ONITATION OF THE INSERTION, THIS IS THE [INDISCERNIBLE]. SO THE PK CHANNELS WHO ARE INSERTED AND RAPPED ORDER OF MICRONSLY, SO WE DON'T HAVE [INDISCERNIBLE], IT COVERS ALL THE POSSIBLE MUTATION. THEN AFTER RECONSTRUCTION, WE GOT A MAP FOR THE PK STRUCTURE, AND THE RESOLUTION, AND IT'S EXPECTED TO BE 8-ANGSTROM BASED ON THE PREVIOUS CORRELATION, SO LET'S LOOK AT FEATURES OF THE BK [INDISCERNIBLE]. SO THIS IS A CENTRAL SECTION AND THIS TRANSMEMBRANE REGION WE CAN JUSTIFY RECONSTRUCTION [INDISCERNIBLE] SO YOU CAN SEE THE CENTRAL INFECTION, CAN YOU SEE THE MEMBRANE [INDISCERNIBLE], WE CAN STILL SEE THAT IT IS, AND THE EMPLOYMENT PROGRAMS BRAIN IS SHOWING A MESH [INDISCERNIBLE] DOMAIN. THIS IS SOMEHOW [INDISCERNIBLE] SO WE KNOW THE NATURE OF THE CHANNEL IN THE ELECTRON TRANSAND STRUCTURALLY UPPER IN THE MAP, SO THE CHANCE [INDISCERNIBLE] TETRAMER, FOR DIFFERENT COLOR, IN THE SUBUNIT, THE SUBUNIT AND TWO MORE ON THE BACK. SO, THIS IS--THIS IS [INDISCERNIBLE] JUST ONE, THERE'S TWO, YOU CAN SEE THE MUTATION OF A POSITION, THE DSITY MAP IS QUITE CONSISTENT WITH THE [INDISCERNIBLE] AND THE CRYSTALLOGRAPHY SO YOU CAN SEE WE HAVE ACTUAL STRUCTURE WITH THE TRANSMEMBRANE REGION, AND THIS ACTUALLY NICE ACCOUNTS FOR ABOUT [INDISCERNIBLE] BETWEEN THE X-ONE, AND ZERO AS ONE, AND ALSO THIS [INDISCERNIBLE] END TERMENERS OF THE BK-ONE CHANNEL SO THIS IS THE EXTRA CELLULAR ALLELE TO SHOW THE INTERPRETATION OF ALL THE [INDISCERNIBLE] AND THIS CROSS THINKING, EXPERIMENT, AND THE MATERIAL IS CLOSE TO [INDISCERNIBLE] THE EXPORT [INDISCERNIBLE]--WE'RE WORKING ON IMPROVING THE RESOLUTION RICH TO 6-ANGSTROMS WE CAN ADENTIFY ALL THE [INDISCERNIBLE], AND GETTA A BETTER UNDERSTANDING OF THE STRUCTURE AND ALSO THE RESOLUTION IN WHICH 4-ANGSTROM, THEN WE CAN SEE IF THAT CHANGES WE CAN BUILD THE ATOMIC MODEL SO WE ARE WORKING ON GRADIENT. I WOULD LIKE TO THANK MY LAB WORKERS. THANK YOU FOR YOUR ATTENTION. [ APPLAUSE ] >> HI, BEAUTIFUL WORK. I HAVE A TECHNICAL QUESTION. DO YOU THINK YOU'LL BE ABLE TO VISUALIZE LIPITS INTERACTING WITH THE METHODOLOGY, DO YOU HAVE TO SUBTRACT THE LYSOSOME OR DO YOU--CAN YOU THINK YOU WILL BE ABLE TO SEE LIPIDS. >> YES, WE AWE AT FIRST THE SUBTRACTION, WE SUBTRACT THE IMPACT [INDIS], THAT TRANSMEMBRANE REGION, AND THE MOST SUCH AS MODEST DIFFERENCE BETWEEN THE [INDISCERNIBLE], SMOTHER RELATED QUESTION, HAVE YOU BEEN ABLE TO PUT ANY CHOLESTEROL IN THE HYPOSTUDIES OF MULTIPLE ENDOCRINES OR LOOKED AT HETEROGENEOUS LIPOSOMES WITH THE LIPIDS. >> ON THIS ONE I DIDN'T TRY LIPID SO WE USE PLPC, BUT IN THEORY THERE'S NO LIMIT TO BE ABLE TO [INDISCERNIBLE] LYSOSOME, [INDISCERNIBLE] SO WE ADJUST TO TAKE THE IMAGE THEN WE CAN [INDISCERNIBLE] SO THERE'S NO LIMIT ON THAT, CAN YOU USE IGG LIP IMPEDIMENTS COMPILATION. >> HI, I'M WONDERING, YOU KNOW YOU ARE GOING TO HAVE DIFFERENT SIGNAL TO NOISE RATIO FOR EACH PARTICLE AND SO GIVEN THAT YOU HAVE THE DIFFERENT SIGNAL TO NOISE RATIOS, HOW ROBUST IS THE COMPUTATIONAL SUBTRACTION OF THE LIP O STUDIES OF MULTIPLE ENDOCRINE? DO YOU GET ANY GHOST IMAGE AND ALSO WHAT IS THE DIFFERENCE IN STRUCTURAL INFORMATION BETWEEN THE PROTEIN WITHIN THE CELL? >> SO I THINK WE ONLY TRY TO SEE THE EFFECT AFTER LIPID BI-LAYER CURVEATURE ON THE STRUCTURE, SO FOR THIS NEUROSTRUCTURE WE DIDN'T [INDISCERNIBLE] YET BUT FOR WHAT WE HAVE DONE IN THE PAST, THE PATH AND [INDISCERNIBLE] THE CONFIRMATION, NO. >> THANKS. >> QUICK QUESTION, THIS IS RELATED TO MANY OF THE OTHER QUESTIONS THAT HAVE BEEN ASKED. SO A PART FROM THE ANNULAR LIPIDS THAT ARE RIGHT AROUND THE STRUCTURE, CAN YOU SEE THE ACTUAL STRUCTURE OF THE MEMBRANE, SAY UP TO 10-ANGSTROM UP TO THE PROTEIN AND DOES THE PROTEIN ITSELF CHANGE THE STRUCTURE OF THE MEMBRANE, OF THE LIPOSOME. OR CAN YOU NOT SEE IT? CAN YOU SEE THE STRUCTURE OF THE LIPID? NOT LIKE ANNULAR LIPIDS, JUST A FEW LIPIDS STUCK TO THE SIDE BUT THE ACTUAL LIPID ITSELF JUST PASSED THE ANNULAR LIPID, DO YOU SEE THE STRUCTURE. >> WE CAN SEE THE LIPID BI-LAYERS BUT WE CAN'T SEE THE LIPIDS IN THE [INDISCERNIBLE] >> SO WHEN YOU LOOK AT THE BI-LAYER DOES THE PROTEIN ITSELF EFFECT THE BI-LAYER AND IF SO HOW FAR AWAY FROM THE PROTEIN STRUCTURE? >> THAT ONE I DON'T HAVE THE ANSWER YET BUT THE DIFFERENCE IS THAT WE HAVE A LARGER LYSOSOMES AND A SMALLER LYSOSOMES AND I THINK WE CAN DO A COMPARSON IN THE FUTURE WHERE THE CHANGE MAY BE HIGH AND [INDISCERNIBLE] WE CAN SEE THE POSITIONS OF SOME [INDISCERNIBLE] BUT TO SEE THE DIFFERENCE WE NEED A HIGHER RESOLUTION. >> I'M NOT TALKING ABOUT THE PROTEIN I'M TALKING ABOUT THE STRUCTURE OF THE LIPID, THE LIPID MEMORY, DOES THE PROTEIN EFFECT THE LIPID MEMBRANE, NOT THE LIPID EFFECTS THE PROTEIN? >> SO I'M I DON'T THINK I HAVE THE ANSWER YET, SORRY. >> PLEASE JOIN ME IN THANKING HIM. >> AND OUR FOURTH SPEAKER IS PART OF THE HIGH RISK, HIGH RESEARCH REWARD PROGRAM IS ADAM DE LA ZERDA AND HE WILL TALK ABOUT HIGH RESOLUTION OPTICAL MOLECULAR IMAGING SYSTEM. >> THANKS SO MUCH FOR THE INVITATION TO COME HERE, VERY EXCITING TO BE HERE, PART OF THE SCIENCE THAT INTERESTS ME IS TO UNDERSTAND HOW CELLS TALK TO EACH OTHER AND WE'RE MADE OF TRILLIONS OF CELLS, MANY, MANY TRILLIONS OF CELLS AND THESE CELLS ARE IN CONSTANT COMMUNICATION WITH ONE ANOTHER, I'M NOT TALKING ABOUT NEURONS THAT ARE SENDING ELECTRICAL PULSES THAT BUT THE OUTER CELLS THAT ARE SENDING BIOCHEMICALS FROM ONE TO THE OTHER. AND ALL WE KNOW BEG YOUR PARDON IS COMMUNICATION THAT EXIST, IT'S REALLY IMPORTANT FOR ALMOST ANYTHING THAT HAPPENS IN THE BODY AND WE KNOW THE BASIC LANGUAGE. RIGHT? WE KNOW THE PROTEINS ARE IMPORTANT, SOME OF THE LIP I EDUCATIONALS AND SO ON, WHICH HAS NEVER SEEN A CONVERSATION TAKING PLACE IN REALTIME AND IT WILL BE QUITE INSIGHTFUL TO DO THAT, SO IF YOU GO BACK TO MAYBE 10, 15 YEARS AGO, THE FIELD OF MOLECULAR IMAGING IS STARTED TO GAIN A LOT OF MOMENTUM, THIS IS WHERE PEOPLE WATCH, LIVING SUBJECTS FOR EXAMPLE AND TRY TO WATCH AND SEE WHICH PROTEINS ARE EXPRESSED IN WHICH AREAS OF THE BODY. AND FOR THIS GROUP, THE WHOLE HOST OF APPROACHES FOR EXAMPLE, TAKING AN ANTIBODY, TIED IN WITH FLUORA FORCE AND GETTING IN TO BIND AND DEVELOPING EITHER TECHNIQUES SO ON TO TRY TO SEE HOW AND WHY PROTEINS ARE EXPRESSING DIFFERENT REGIONS IN THE BODY. SO THIS IS THE GENERAL SCOPE OF WHAT THE INTEREST MEAN AND KIND OF--IF YOU'RE GOING TO GO THROUGH THE FINAL AND OF COURSE YOU CAN TAKE FLUORESCENT ANTIBODIES, SETTING IT INTO THE BODY AND SEE WHERE IT BINDS AND IN THIS CASE, THERA A TUMOR IN THIS INTEREST, AND SEE IF THIS IS EXPRESSING SOME IMPORTANT PROTEIN, ALSO A BETA THREE INTEGRIN AND SO CAN YOU SEE THIS JUST LIGHTS UP AND WHAT ACTUALLY LIGHTS UP HERE IS ESSENTIALLY SHOWING HERE IS ONLY A SINGLE PICKS OF TUMORS IF YOU WILL, BUT IT'S ACTUALLY THIS ONE PICK REPRESENTS CANCER CELLS AND IN THIS CELLS AREA, THERE IS ARE THAT ARE EXPRESSING THIS ALPHA THREE BETA INTEGRIN. SO THE OUTER TECHNIQUES FOR EXAMPLE, THE ONE THAT HAS BEEN MADE FOR MANY YEARS IS THE FLUORESCENCE IMAGING AND IT ALLOWS YOU TO GO DEEPER IN AND CREATE A CROSS SECTION AND CREATE A THREE DIMENSIONAL IMAGE OF THIS WHERE YOU LOOK INTO THE TISSUE AND SEE DEEPER AND DEEPER IN THE TISSUE AND IN A WAY THAT IS NOW SHOWING YOU, ACTUALLY THE PARTICULAR AREAS WITHIN THE TUMOR SO HERE THE SURFACE OF THE TUMOR, THE BOTTOM OF THE TUMOR AND NOW EVERY PIXEL HERE THAT'S LIGHT UP IN GREEN REPRESENTS THE EXPRESSION OF THE PROTEIN WE TALK ABOUT REPRESENT ABOUT 10,000 CELLS OR SO. SO THAT'S QUITE A BIT NICER AND AND NOW CAN YOU START TO SEE THERE'S SOME REGIONS IN THE TUMOR THAT BEHAVE DIFFERENTLY THAN OTHERS. SO THIS WAS MISSING FOR SEVERAL YEARS NOW, IS ABLE TO GO EVEN FURTHER, AS YOU MENTION SO IF YOU WANT TO GOOGLE EARTH AND ZOOM IN MORE. THERE ARE A LOT OF TECHNIQUES THAT ALLOW YOU TO MAKE MICROSCOPY AND WITH THE CELLS, AND LOOK RIGHT UNDER THE SURFACE AND IF YOU, AND JUST THAT ALL THESE CELLS LOOKING INTO THE SKIN, AND BY THE TIME YOU GET TO THE SKEAN, THE SECOND LAYER OF CELLS, THE SECOND LAYER ARE SHADOWING FIRST LAYER AND TELL BE HARDER TO SEE THEM AND BY THE TIME YOU GET TO THIRD, FOURTH, SIXTH LAYER YOU ARE SEEING ANYTHING AND IN THE CONTEXT OF SEEING SOMETHING THROUGH SKIN, THIS IS OLD DEAD TISSUE ANYWAY, SO BY THE TIME YOU GET TO THE INTERESTING AREA, IT'S HARD TO SEE THINGS AND IT'S MORE OR LESS IS THE CASE FOR OUTER TECHNIQUES AND YOU CAN JUST MAKE MICROSCOPY AND THE SO ON, BUT AT THE END OF THE DAY THEY'RE LIMITED IN HOW DEEPLY THEY CAN SEE AND THE DEEPER YOU WANT TO SEE, AND YOU WILL START LOSING RESOLUTION EMPLOY AND SO WHAT WE'VE BEEN TRYING TO ASK IS CAN YOU TRY TO FIND SOME INTERESTING REGION HERE, SOME TECHNIQUE THAT MIGHT ALLOW US TO SEE, DEEP ENOUGH INTO TISSUE SO WE CAN SEE NOT FOR THE FIRST LAYER OF CELLS OR TWO LAYERS OF CELLS BUT TRULY SEE AN AREA THAT COVERS OF HUNDREDS OF MILLIONS OF CELLS, BILLIONS OF CELLS EVEN AND STILL MAINTAIN A SINGLE CELL RESOLUTION, CAN YOU SEE EACH OF THESE CELLS THEORETICALLY. SO THIS IS WHAT WE REFERRING TO AS MOS ARD AND I'LL BE TELLING BUT THIS TODAY, THE FIRST IS THE IMAGE ITSELF AND WHAT WE HAVE IN MIND AND THIS IS RELATIVELY NEW RESEARCH. ALL THE RESULT VS EITHER JUST BEEN ACCEPTED FOR PUBLICION OR IN THE PROCESS MUCH AND WILL BE SUBMITTED SOON. THEN I'LL TELL YOU, I WILL TELL YOU ABOUT THE IMAGING AGENTS THAT WE'RE TRYING TO PRODUCE ON THE NANO PARTICLES WE'RE MAKING TO ALLOW US TO SEE AND THOSE PROTEINS. AND AGAIN WE'LL TRY TO EPITHELIAL GREAT THEM AND GET INTERESTING IMAGES FROM THE CHIP. SO WE'RE GOING TO START A STORY WITH OPTICAL TOMOGRAPHY WHICH HAS BEEN AROUND FOR A WHILE NOW, 25 YEARS OR SO, SO THE BEAUTIFUL TECHNIQUE, WHAT IT ALLOWS YOU TO DO IS TO GET VERY, VERY HIGH DETAILED STRUCTURAL IMAGES FROM THE TISSUE. IN THIS CASE, WE ARE LOOKING AT A MOUSE HERE AND WE WILL TAKE SCANS, IT'S A CROSS SECTIONAL IMAGES THROUGH THE TISSUE. SO HERE, YOU CAN MAKE THIS THREE DIMENSIONAL IMAGE OF THE MOUSE HERE, YOU CONSIDER IT A VERY FIRST LAYER HERE, THAT'S THE EPIDEMIC AND THE DERMIS LAYER, THE CARTILAGE AND THE DERMIS AGAIN AND EPIDERMIS, YOU CAN SEE THE ENTIRE MOUSE HERE, CAN YOU MAKE MANY SUCH IMAGES LIKE THIS FOR A SECOND AND IF YOU MOVE EVERY TIME YOU CAN GET A FULL 3D IMAGE THAT WILL BE PRESENTED HERE IN THIS IMAGE HERE AND ONCE YOU HAVE THE 3D IMAGE YOU CAN CROSS IT WHICHEVER WAY YOU WANT, THE TOP, AND JUST TO GIVE YOU A CONTEXT OF HOW BIG THOSE IMAGES ARE, THIS IMAGE RIGHT HERE IS WELL OVER A HUNDRED PICO BITES IN SIZE, SO THESE ARE REALLY, REALLY LARGE DATA SETS. AND THE REASON THEY'RE SO LARGE IS THEY CAN COVER VERY LARGE AREA, SO THE FIELD OF VIEW IS QUITE HIGH, BUT FILL THE SPACIAL RESOLUTION IS QUITE REMARKABLE, IT'S TWO MICROMETERS, SO WHILE THIS TECHNIQUE MAY NOT ALLOW YOU TO SEE THE MITOCHONDRIA VERSES THE NUCLEUS, VERSES THE LIPID MEMBRANE AND SO ON, IT HAS JUST THE RIGHT RESOLUTION TO SEE THE I THINK SINGLE CELLS SO CAN YOU SEE A REGION WHY A SINGLE CELL IS, YOU WON'T BE ABLE TO WITH THE MEMBRANE WHICH IS THE PHOTON MICROSCOPY. YOU CAN SEE THIS THROUGH THICK CONDENSED TUMORS. SO THIS A TECHNIQUE WE'RE GOING TO EMPLOY, AND IT'S REALLY BEAUTIFUL IN THE CONTEXT OF GETTING STRUCTURAL IMAGE IT IS, BUT UNFORTUNATELY IF THERE'S A CERTAIN PROTEIN THAT YOU'RE INTERESTED IN, LIKE WITH THE INTEGRIN, THAT WAS EXPRESSED RIGHT HERE, YOU WOULD NOT BE ABLE SEE THAT AND FOR THIS YOU NEEDED AN EXTERNAL AGENT. SO WE'RE GOING TO TALK ABOUT WHAT ONE COULD USE AND THE BASIC MECHANISM TODAY WHICH OCT WORKS IS BASING LIGHT THAT GETS REFLECTED OR SCATTERED OFF OF A TISSUE, SO OUR OWN TISSUES ARE HIGHLY REFLECTIVE AND SCATTERING AND'S WHY YOU CAN SEE THOSE IMAGES AND WE ASK OURSELVES WHAT ELSE IS HIGHLY REFLECTIVE AND SCATTERING AND GOLD PARTICLES ARE HIGHLY SCATTERING ASSENTITYS AND FOR EXAMPLE, THESE GOLDEN RODS WHICH WE'LL REFER TO NOW ARE ABOUT 50-NANOMETERS BY 45-NANOMETERS OR SO, BY CHANGING ASPIRATIONS YOU CAN SEE WHICHEVER COLOR YOU WANT TO BE AT SO CAN YOU TUNE THEM TO THE FREQUENCY OF THE OCT SYSTEM AND THE WAY THAT THEY WORK IS IF YOU SHINE THE LIGHT ON THESE NANO STRUCTURES FOR THIS EFFECT, THE LIGHT THAT YOU'RE SHINING, SOME WILL BE ABSORBED AND SOME OF IT WILL GET SCATTERED, MOSTS OF THE LIVES WILL GET SCATTERED, SOME WILL GET CARRIED TO THE EDGE AND VERY LITTLE WILL GET SCATTERED AS TRACK SCATTERING, UNFORTUNATELY AND THE IMAGES FROM THE LIGHT AT TOP AND MEASURING IN REVERSE ORDER AND WRE MEASURING THE BACK SCATTERED LIGHT WHICH IS NOT THAT MUCH AND SO WE INITIALLY START WIDE THIS AND I WILL SPARE YOU THE SLIDES WITH THE NEGATIVE RESULTS IT'S HARD TO SEE THE PARTICLES BECAUSE THE AMOUNT OF THE BACK CAT SCATTERING IS SO, SO, MANY, BUT THEY HAVE ART PROPERTIES THAT YOU CAN TUNE TO THEM TO DERIVE FREQUENCY AND SO OSO WHAT WE ASK OURSELVES IS, WELL, WHAT IF WE MAKE THEM BIGGER, WOULD THAT HELP AND AS IT TURNS OUT THAT BY MAKING THEM BIGGER AND TWICE AS BIG IN EACH DIRECTION, AND YOU ARE GOING TO GET THE HUGE EFFECT HERE, NOT ONLY THAT THE LIGHT WILL GET SCATTERED IN AND YOU WILL GET MUCH MORE LIGHT SCATTERING BACK BUT ALSO PROPORTIONALLY, THE LIGHT WILL GET BACK SCATTERED IS MORE OR LESS EQUAL TO THE ONE THAT'S FORWARD SCATTERED AND SO ON. SO YOU'RE GETTING A HUGE AMPLIFICATION HERE AND THE AMOUNT OF LIFE TO GET BACK SCATTERED AND THIS TO US IS GREAT, NICE IF WE CAN ONLY MAKE THIS PARTICLE, SO IT'S BIGGER, NOW WE HAVE A SHOT, LET'S TRY THEM. SO WE SCAN THROUGH THE LITERATURE AND IT TURNS OUT THE LITERATURE IS FILLED WITH TECHNIQUES HOW TO PRODUCE THESE AND ONLY A SINGLE PAPER HOW TO MAKE THIS AND EVEN THAT IS ONLY THE MATERIAL SCIENCE LEVEL IF YOU WANT TO HAVE THEM IN PURE WATER. AND ANYTHING ELSE OTHER THAN THAT WILL NEVER WORK. BUT IF WE CAN GO IN THERE, WE WILL TRY IT, SO MOST OF THEM ARE 15 BY 45-NANOMETERS BUT SOME OF THEM ARE MORE SPHEREICAL, SOME ARE FATTER AND SKINNIER AND SO ON, AND IN THE LARGE MANNER HERE REFERRED TO AS LGNRs, INTERESTING ENOUGH, WE CAN MAKE A REALLY, REALLY MONITOR THESE, AND SO MUCH THAT WHEN YOU ANALYZE THESE, AND THE GRIDS LOOKING AT THEM, YOU WILL SEE THAT THE ASPECT RATIO IS IN THE MIDDLE OF WHERE YOU WANT IT TO BE. IN FACT, THE FACT WE CAN GET THEM DISPURSED WE ARE LOOKING AT THE SPECTRUM OF THEM TO BE PRECISE AND NARROW. YOU WILL NOTICE THESE RODS HERE, ARE ABOUT 150-NANOMETERS FULL WITH HALF MAX WHICH IS REALLY STANDARD FOR ANY TYPE OF GOLD PARTICLE AT THAT RANGE AND THESE ARE QUITE UNIQUE. YOU WILL SEE THAT THE FULL WIDTH AND ONLY A HUNDRED NANOMETERS WHICH IS MUCH, MUCH, MORE NARROW. I'LL NEVER SEEN A PARTICLE RANGE THAT NARROW BECAUSE REALLY BECAUSE THERE'S SO, SO BUSINESS PROGRAMS PURSE EVERY SINGLE ONE OF THESE PARTICLES IS DISPERSED. SO AGAIN I WILL SPARE YOU ABOUT 15 OR 20 SLIDES WHERE THE CHEMISTRY WAS DONE, WE WANT THE DOSE TO BE STABLE IN BIOLOGICAL FLUID AND THAT WAS NOT SIMPLE AND IN FACT WHAT WE'VE SEEN IS THAT THE BASIC CHEMISTRY, THE POLYMER CHEMISTRIES WE USE TO STABILIZE THE SMALL PARTICLES DO NOT WORK FOR THE LARGE PARTICLES AND YOU NEED A LARGER SURFACE TO STABILIZE THEM. SO WE HAVE THAT NAILED DOWN QUITE WELL, IN FACT WE'VE SHOWN THAT THEY'RE STABLE, AND WE'VE DONE BIOLOGICAL TOXICITY TESTING, EVERYTHING SEEMS TO BE GOOD. SO AT THIS STAGE WE WANT TO TEST THIS AND SEE EVERYTHING WE GET FROM THAT, SO WE WILL AGAIN TRY TO COMBEAR THE TWO HERE AND THE SMALL PARTICLES VERSES LARGE, WHEN WE TAKE--WE TAKE THEM ON EQUAL PARTICLE PER MILL LETTER BASIS, YOU WILL SEE WE'RE TAKING THIS CAPILLARY TUBES THIN, SMALL AND THOSE WE'RE FILLING WITH THE PARTICLES, WHEN YOU PUT THE SMALL PARTICLES HERE YOU'LL BARELY SEE ANYTHING, IN FACT ALL YOU'LL SEE HERE ARE THE TWO SPECCEDS OF COLOR, THESE ARE REFLECTIONS, OFF THE GLAND. SO THERE'S SOME PARTICLES HERE, VERY, VERY HARD TO SEE THEM. AND THEN WE USE THE SAME PARTICLE CONCENTRATION TO LARGE PARTICLE AND YOU WILL SEE MUCH, MUCH, MORE COLOR, MUCH MORE SIGNAL, IN FACT ABOUT 30 TIMES MORE SIGNAL, SO CAN YOU LOOK AT IT AND SAY, WELL, YOU HAVE THESE PARTICLES ARE MUCH LARGER. WE HAVE FOUR TIMES THE SURFACE AREA OF GOLD. AND YEAH, THAT'S RIGHT BUT A-YOU HAVE MORE GOLD SURFACE OFF HERE BUT YOU'RE GETTING 32 TIMES THE SIGNAL AND SO THAT THE AMPPLIFICATION WE'RE REFERRING TO MORE, AS WE GET BIGGER, YOU'LL CATCH MORE LIGHT AND MORE OF THAT LIGHT THAT GETS SCATTERED GETS BACK SCATTERED TO WHERE YOU WANT IT TO. SO NOW WE HAVE A CHANCE OF SEEING THESE PARTICLES. AND SOW NOW WE COVERED THEM WITH THE RIGHT PROPERTIES AND SO ON, AND WE CAN SEE IF WE SEE THEM IN VIVO. SO WE'RE LOOKING HERE AT A MOUSE HERE, THE MOUSE, YOU'LL SEE THE DIFFERENT STRUCTURES HERE, AND EPIDERMIS, LIMP VESSELS, BLOOD VESSELS, AND WE'RE NOT GOING TO INJECT THESE AT A FAIRLY HIGH CONCENTRATION, AND YOU'LL SEE--ALSO LOOKING THE SAME, AND THIS WAS REALLY DEPRESSING WHEN WE DID THAT IN ORDER TO GET INTO THE LARGE PARTICLES, WE COVER THE STAPPLING AND THE YET WE'RE NOT SEEING ANYTHING, AND IF YOU LOOK CLOSELY, TO THE AREAS WE'RE APPOINTED HERE, YOU MIGHT BE ABLE TO CONVINCE THE SLIGHTLY MORE SIGNALING DOSE AREAS AND IN FACT THERE IS MORE SIGNALING IN THOSE AREAS IF YOU'RE REALLY CAREFUL, YOU TAKE TWO IMAGES AND SUBTRACT ONE FROM THE OTHER AND YOU WILL SEE NO RESIDUAL SIGNAL THERE, SO THAT'S REALLY TOUGH TO SEE. SO AT THIS STAGE HA IT SAYS, ALL RIGHT, REMEMBER WE HAD TO REALLY, REALLY NARROW SPECTRUM TO THOSE PARTICLES, LET'S SEE IF WE CAN DO POST PROCESSING ALGORITHMS TO HELP US SEE WHERE THOSE PARTICLES ARE. SO WE'VE DONE THAT AND YOU'RE GOING TO LOOK NOW, IMAGES FOR RED, IS REPRESENTING AND YELLOWISH, GREENISH WILL BE SIGNALS FROM THE GOLD PARTICLES, PREINJECTION THE IMAGE LOOKS LIKE THIS, THE SAME IMAGE POST INJECTION, NOW IT LOOKS NICER. SO NOW YOU CAN ACTUALLY START TO SEE WHERE THE PARTICLES ARE, AND YOU SEE ALL THES THAT LIGHT UP WITH YELLOWISH, GREENISH COLOR, THOSE ARE THE BLOOD VESSELS WE INJECT, WE EXPECT ALL THE BLOOD VESSELS TO LIGHT UP. SO NOW WE CAN SEE THOSE PARTICLES IN THE SECTIONS IN FACT YOU'LL SEE THAT SOME OF THE BLOOD VESSELS YOU CAN SEE ARE DEEP, ABOUT A MILLIMETER DEEP AND THYSELFER TINY CAPILLARIES AND YOU CAN CAN SEE THAT'S THE CAPILLARIES DEEP, DEEP INSIDE THE MILLIMETER, SO NOW WE WANT TO ASK YOURSELF WHY DOES THE SMALLEST AMOUNT OF GOLD PARTICLES DO THIS USING THE IMPROVED WAY OF SEEING THEM. BEFORE THE INJECTION, AFTER THE INJECTION, WE'RE INJECTING MORE AND MORE, AND THEY'RE CONNECTED TO THE MOUSE TAIL SO WE'RE INJECTING SMALL AMOUNTS EVERY TIME AND WE'RE ANALYZING ALL OF THESE AND CREATING THIS GRAPH THAT SHOWS US AS WE INJECT MORE GOLD PARTICLES IN THE BLOOD SYSTEM, WE'RE SEEING MORE SIGNAL AND YOU'LL NOTICE DOWN AT 250 PICO MOLARS SO WE HAVE A SMALL AMOUNT OF PARTICLES AND IN VIVO WE CAN SEE THINGS. NOW BEAR IN MIND, THE SENSITIVITY IS 250 PICK O MOLARS, YOU HAVE TO REMEMBER WHAT IS THE SIZE OF THE VOXEL, HERE IT'S A VERY SMALL VOXEL TWO-5-MICRONS DEPENDING ON WHICH DIRECTION, SO IF YOU ACTUALLY CAN COUNT HOW MANY PARTICLES YOU HAVE PER VOXEL, THESE ARE 40 NANO PARTICLES SO ONE, TWO, THREE, FOUR, FIVE, TIMES 40, IT'S ENOUGH TO GIVE 40 NANO PARTICLES IN A TINY AREA AND YOU CAN SEE THAT DEEP INSIDE THE TISSUE IN VIVO. SO THAT'S QUITE NICE. ALL RIGHT AT THIS STAGE WE'RE LOOKING AT DIFFERENT MICE, INJECTING MORE AND MORE PARTICLES, CAN YOU GET FULL THREE DIMENSIONAL IMAGES, YOU CAN SEE CROSS SECTION, YOU SPEAK TO MANY OF THEM, CAN YOU MAKE THEM INTO FULL 3D IMAGE, SO CAN YOU SEE HOW PREINJECTION LOOKS LIKE THIS. ONE HOUR AFTER THE INJECTION OR PARTICLES ARE STILL THERE, EIGHT HOURS LATER, YOU CAN SEE LESS BUT BY AND LARGE, THIS WILL FILL WITH THOSE PARTICLES, 16 HOURS THEY ARE BEGINNING TO CLEAR OUT AND 24 HOURS, MOST OF THEM ARE ALREADY DONE. BUT, THOSE OF YOU WITH SHARP EYES WILL NOTICE SOMETHING INTERESTING, AS WE'RE LOOKING AT DAT AT WE NOTICE THE SMALL AREA HERE, LET'S BRING IT UP HERE, YOU WILL SEE THAT NOT ONLY THE BLOOD VESSELS SLIDE UP HERE BUT YOU SLEEP APNEA AND OBESITYY A DIFFERENT TYPE OF STRUCTURE IT'S NOT ONLY HERE, THERE ARE OTHER AREAS, IT HAPPENS ONLY THE 16 HOURS, NOT AT EIGHT OR 24, THIS WAS LIKELY O'CLOCK IN THE MORNING FOR MY STUDENTS, BUT INTERESTING ENOUGH, CAN YOU SEE THE STRUCTURES AND THESE ARE LIMP VESSELS SO AS IT TURNS OUTS WE CAN SEE THOSE LIMP VESSELS IN THOSE TISSUES AND YOU CAN DO A WHOLE BUNCH OF THINGS WITH IT, YOU CAN INJECT GOLD PARTICLES IN SOME AREA AND WE CAN INJECT THEM HERE, CAN YOU SEE THEM SIDE OCCUPY, YOU CAN DO ANOTHER TYPE OF COLORFUL PARTICLES AND YOU CAN SEE THEM LIGHT UP IN A DIFFERENT WAY. IF YOU LOOK AT A DIFFERENT MOUSE HERE FOR EXAMPLE, YOU SEE THE ALL THE LIMP VESSELS LIGHT UP NICELY AND YOU SEE EVERYTHING STOPPED HERE AND INJECT MORE PARTICLES OF A DIFFERENT COLOR NOW, AND SEE THAT THEY ALL TRAVEL DOWN IN THIS DIRECTION, AND THIS INDIVIDUAL ONE IS MAKING A DECISION TO CUT UP THIS VALVE HERE THAT IS GOLD PARTICLE TRAVEL FROM THE OUTSIDE, YOU CAN START WATCHING ALL THESE DYNAMICS IN REALTIME AND THIS IS A LIVE ANIMAL. I WILL SUMMARIZE THIS, NOW, THE NEXT STEP IS THE MOLECULAR IMING STUDY SAYS SO HERE WE TRY TO STAIN FOR DIFFERENT REGIONS, WITH THE VESSEL AND TRY TO SEE WHETHER WE CAN ACTUALLY SEE THE EXPRESSION OF THIS CASE OF LIVE ONE WHICH IS AN IMPORTANT ANTIBODY AND I'LL START BY SAYING THESE THREE DIMENSIONAL IMAGES ARE NICE BUT THEY'RE REALLY MEANING LESS WHEN YOU LOOK AT THEM IN TWO D, SO IF YOU WANT TO COME OVER TODAY DURING THE POSTER SESSION AT 3:00 I'LL SHOW YOU A SYSTEM THAT WOULD ALLOW YOU TO SEE IMAGES, LIKE THIS, WHERE THE IMAGE WILL TRULY COME TO LIFE AND I'VE NEVER SEEN ANYTHING THAT EXCITING, YOU CAN ACTUALLY LOOK, CAN YOU PULL THE IMAGE LIKE, YOU'RE WEARING THOSE 3D GOGGLES AND YOU CAN TRAVEL ALONG THE IMAGE HERE AND SEE IN REALTIME HOW YOU'RE TRAVELING THROUGH THE LIMP VESSELS AND SEE THE EDGE OF A LIMP VESSELS LIGHT UP. IT'S QUITE INCREDIBLE. ALL RIGHT, THANKS SO MUCH. [ APPLAUSE ] >> SO TIME FOR ONE OR TWO QUESTIONS? >> COULD YOU PRELABEL ONE OF THESE CELL CYCLES AND WATCH IT MIGRATE TO A SITE. >> WE'RE HOPING TO. THAT'S A GREAT QUESTION. AND WE PLANNING TO TRY TO DO THAT. SO WOO WE'RE TRYING TO TAKE THE CELLS AND WATCH THEM TRAFFIC INTO TUMOR AND SEE HOW THEY WILL GO IN CERTAIN AREAS BUT OTHERS ASK WATCH IT IN REALTIME SO THE SENSITIVITY OF ONLY 40 PARTICLES FOR A REGION WOULD HOPEFULLY ALLOW US TO DO THAT. >> TED DECODING YOU USE FOR FLUIDS EFFECT THE PROPERTIES-- >> SORRY I DIDN'T HEAR THE QUESTION. >> COULD THE CODING YOU USE TO DEFER BIOLODGEICAL STABILITY AND BIOLOGICAL FLU EDUCATIONALS EFFECT THE SPECTRAL PROPERTIES? >> GOOD QUESTION, NO IT DID NOT. THE POLYMER CODING DID NOT--WELL NO EFFECT ON RESPECT TO THE PROPERTY PARTICLES. >> PLEASE JOIN ME IN THANKING ALL THE SPEAKERS FROM THE FIRST SESSION. >> SO WE'RE RUNNING A BIT BEHIND SCHEDULE BUT WE'LL TAKE A 15 MINUTE BREAK AND COME BACK AT 10:45. LET'S GET STARTED WITH OUR SECOND SCIENTIFIC SESSION AND AS YOU MIGHT HAVE NOTICED THE THEME IS STRESS AND STRESS RESPONSE. OUR FIRST SPEAKER IS STEVEN KOSAK, WHO WILL TALK ABOUT REGULATORY PROTEIN TRANSLATION IN THE HUMAN NUCLEUS. >> THANK YOU ROBBIE AND THANK YOU FOR THE INNOVATOR AWARD, OF WHICH I WOULD NOT HAVE BEEN ABLE TO DO THIS PROJECT. BUT GETTING IT PUBLISHED IS AN ENTIRELY DIFFERENT STORY. SO THE HUMAN NUCLEUS IS FUNCTIONALLY AGER ANISEED AND THE ELECTRON DENSE, ELECTRON UCOMMA TIN THAT REPRESENTS THIS DOMY OF THE CHROMATIN. OTHER FUNCTIONS ARE ALSO CALLED NUCLEAR BODIES. WHAT'S UNIQUE ABOUT THE NUCLEAR BODIES, UNLIKE THE COUNTERPARTS THEY'RE NONMEMBEREROUS AND THEY FORM BY VIRTUE OF THEIR FUNCTION. FOR EXAMPLE THE NUCLEOLUOUS IS THE SITE OF RDNA AND RIBOGENESIS SPORTSMANSHIP IF YOU OBLATE RNA TRANSCRIPTION, YOU LOSE THE NUCLEOLIS, ANOTHER FEATURE OF NUCLEAR BODIES IS THAT THEY'RE HIGHLY PERMISS CUOUS, IN TERMS OF FUNCTION, SO HERE YOU SEE A WHOLE POST OF DIFFERENT ACTIVITIES AND MANY DIFFERENT BODIES AND DIFFERENT NUCLEAR PROTEINS ASSOCIATE WITH THESE GIVEN FUNCTIONS SO THE PMO BODY IS AN EXCELLENT EXAMPLE OF THIS. SO THERE ARE SEVEN ISOFORMS, THE LEUKEMIA BODIES OR PMO BODIES AND THEY HAVE A WHOLE HOST OVER 150 PROTEINS ASSOCIATE WITH THEM AND THEY'RE HETEROGENEOUS ROG NOWS AND THERE ARE SEVEN ISOFORMS AND PNH IS EACH BODY AS YOU MIGHT SUSPECT, GIVEN ALL THESE DIFFERENT CRITICAL PROTEINS THAT MANY OF THESE HAVE BEEN ATTRIBUTED TO THE PML BODY, REPAIR, RESPONSE, APOPTOSIS, PROTEOLYSIS, ET CETERA. SO I HAVE A LOT OF SAYINGS IN THE LAB THAT ANNOYS MY PEOPLE IN THE LAB AND ONE IS THAT IF A STRUCTURE HAD BEEN SHOWN TO DO ANYTHING, PERHAPS WE DON'T KNOW WHAT IT ACTUALLY DOES. SO ONE INTERESTING FEATURE OF THE PML BODY SYSTEM THAT THEY HAVE A BUNCH OF FACTORS THAT ARE CRITICAL FOR TRANSLATION, PROTEIN TRANSLATION. MRNAs COAT THE OUTSIDE OF THE BODY. SO I'VE LONG REASONED THAT PERHAPS THE PML BODY IS THE SITE OF TRANSLATION WITHIN THE NUCLEUS SO OF COURSE WE AREN'T THE ONES TO CONSIDER NUCLEUS TRANSLATION THAT GO BACK AS FAR AS 1954 BUT EVERY TIME IT RAISES ITS HEAD, IT GETS CHOPPED OFF BECAUSE PEOPLE FIND EXPERIMENT STRATEGY THAT'S BEEN USED, FORTUNATELY A RELATIVELY NEW TECHNIQUE, RIBOPURE MICEOLATION METHOD, IS USED IN THE TRANSLATION FIELD, BOTH CYTOPLASMIC AND NUCLEAR AND IT'S GOOD EVIDENCE THAT INDEED NUCLEAR TRANSLATION OCCURS, BUT WHERE AND WHY IS A REMAINING QUESTION. SO WE ABOUT THEN TO ASK WHETHER RIBOSOMAL SOFT PROTEINS CAN LOCALIZE A PML BODY. I DIDN'T MENTION BEFORE BUT THE MANY PROTEINS THAT ASSOCIATE WITH P ML RIBOSOMAL PROTEINS ARE AMONG THE CATEGORIES EMPLOYED SO WE ADOPTED THIS PROTEIN WHERE WE USE THIS ARRAY SYSTEM, THE LONG AWAY OF BINDING SITES ARE EPITHELIAL GREATED INTO THE HUMAN GENOME AND THEN YOU CONFUSE A FLUORESCENT PROTEIN TO YOUR PROTEIN OF INTEREST, AND THE LAST IPROTEIN WHICH BINDS THE OPERATOR ARRAY AND BASICALLY CAN YOU LOCALIZE PROTEINS WITHIN THE GENOME THAT WAY. SOPHISTICATEDY WE USED RPLP ONE AND TWO. HERE WE HAVE IT FUSED TO THE ARRAY, AND HERE WE HAVE THE PML STAIN WIDE AN ANTIBODY AND SURE ENOUGH WE CAN EITHER RECRUIT OR GENERATE DE NOVO PML BODIES AT THIS SITE, WITHIN THE GENOME. AND OF COURSE THAT'S SIGNIFICANTLY OVER JUST HAVING THE LACI. BUT THE QUESTION WAS ARE THESE TRANSLATIONALLY COMPETENT SITE. SO HERE'S THE RIBOPURE MICEALATION METHOD AND ESSENTIALLY WHAT WE USE IS PURE MICEIN WHICH ACTS AS A TRNA MEDIC AND IT ENTERS THE SITE AND YOU DO IMAGING HAD OF-YEAH, YOU USE THIS TO INHIBIT THE TRANSFER AND THAT DOES NOT CAUSE AN EFFECT ON TRANSTREPIDATION, YOU INTEGRATE YOUR PURE MICEIN INTO A GROWING POLYPEIDE AND THEN YOU USE AN ANTIBODY TO DETECT NAISSANTENT TRANSLATION. AND SO THIS HA BEEN AS I SAID DEVELOPED A COUPLE YEARS AGO AND WHAT THEY HAD FOUND WAS HERE YOU CAN SEE, SUSTAINING BUT ESSENTIALLY WHAT THEY FOUND WAS THAT THEY DETECTED THIS, THE PMI OR THE NASCENT TRANSLATION IN THE NUCLEOLUS, WHICH I SAID SITE OF OF THE RIBOSOMAL GENESIS, BEFORE THEY'RE EXPORTED TO THE CYTOPLASM SO PERHAPS THEY THOUGHT THIS WAS AIAN WAY OF TESTING RIBOSOMES BEFORE THEY'RE EXPORTED. SO WE USE THE RPM STRATEGY WITH OUR LACL APPROACH, SO WE'RE DID FIND THE DETECT AND THE OVERLAYS, AND WE DID FIND A SMALL PERCENTAGE OF OUR PML BODIES CO LOCALIZING WITH NASCENT PIPE TYPE SIPGHT SIS BUT IT WASN'T ROBUST. BUT THEN OF COURSE WE REMEMBER THAT THE PML BODY IS CENTRAL TO A WHOLE HOST OF CELLULAR STRESS RESPONSES THAT THEY--THEY ARE--YEAH, PRODUCED OR INTERACTING WITH THE PML, AND THEN THE OBVIOUS OUTCOMES OF THOSE STRESSES. SO THEN USING THE SAME STRATEGY AS BEFORE, WE DECIDED OKAY, WE WILL USE THE STRESS TO SEE IF WE CAN INCREASE THE AMOUNT OF PML BODIES THAT SHOW NASCENT POLYPEPTIDE SIPGHT SIS AND SURE ENOUGH WE GET A SIGNIFICANT INCREASE UPON UV EXPOSURE SO THERE APPEARS TO BE MORE POLYPEPTIDE SYNTHESIS AT PML BODIES UNDER UV STRESS. SO WE NEXT TURN TO ENDOGENOUS PML, AND WE USED A HOST OF DIFFERENT STRESSES, AND SO, THE C-23 IS THE MARKER FOR THE NUCLEOLUS, PMI DETECTION, SO UNDER CONTROL CONDITIONS YOU CAN SEE, THE PMI, PRIMARILY IS LOCALIZED TO THE NUCLEOLI, HOWEVER UNDER UV STRESS, THE ENDOGENOUS SYSTEM, I'M SORRY THESE ARE PRIMARY HUMAN FIBROBLASTS, YOU CAN SEE THAT UNDER UV STRESS, YOU GET A LOSS OF THE RIBOSOMAL STAINING AND YOU GET COLOCALIZATION OF NASCENT POLYPEPTIDE SYNTHESIS AT PML BODIES. SO WE NEXT PERFORMED THE SAME EXPERIMENT WITH A NUMBER OF STRESS EVENTS AND YOU CAN SEE THIS IS THE AMOUNT OF PMY SIGNAL AT THE NUCLEOLUS, IT DECREASED UPON EACH OF THE STRESSES WHEREAS THE PERCENT OF THE PMY SIGNAL IS SIGNIFICANTLY INCREASED UNDER EACH OF THE STRESS EVENTS. WE WE BELIEVE WITH THE STRESS, IT'S TEST TRANSLATING ABERRANT TRANSCRIPTS TO PREVENT THE ULTIMATE STRESS RESPONSE FOR THE NEGATIVE EFFECTS OF IT ON THE CELL. INTERESTINGLY, ANY NEUROLOGICAL DISEASES EXHIBIT NUCLEAR AGGREGATES AND IN FACT THERE ARE 15 NEUROLOGICAL DISEASES WHERE WHERE THE PRIMARY CELL ASK AGGREGATE, AND SO YOU DECIDED TO EXPLORE PROTEIN COMPLEXIO TOXIC STRESS THEN, AND THE CONTEXT OF NEURNEUROLO DISORDERS IN THIS THE FORM OF SPINE O CEREBELLAR ATAXIA. IN TREEINGLY, POLYACUTE DISORDERS OF WHICH THERE ARE MANY DISEASES, NEUROLOGICAL DISEASES, ASSOCIATED POLYQs--MUTANT FORM CO LOCALIZES BUT EVERYONE ASSUMES THAT THESE GET AGGREGATED INTO THE CYTOPLASM AND COME BACK INTO THE NUCLEUS, SO WE WERE THEREFORE WITH THE SYSTEM TO TEST OUR HYPOTHESIS. SO THE POLYQ DISEASE, YOU HAVE A CAG REPEAT AND AFTER IT GETS OVER A CERTAIN SIZE, THE PATHOLOGICAL AND WHAT HAPPENS IS YOU HAVE AN ABERRANT RNA OR HAIR PIN WHICH CAUSES RNA TOXICITY AND MISFOLDED PROTEINS WHICH CAUSES PROTEIN COMPLEXIO TOXICITY SO IT'S A GOOD MODEL TO INCLUDE BOTH RNA TOXICITY AND PROTEIN TOXICITY. SO SURE ENOUGH, WE DID THE EXPERIMENT WHERE WE LOCALIZED 84 Q, THE MUTANT FORM OF ATTACKSIN ONE WITH THE NUCLEUS AND FOUND IT SIGNIFICANTLY ASSOCIATES WITH PML BODIES. SO THEN WE PERFORM SUPER RESOLUTION BI COSCOPY, CONTAINING THE MUTANT FORM OF ATTACKSIN AND PML FORMS A DO NUT TYPE STRUCTURE AND THE 84 Q, WE SEE IS WITHIN THIS DO NUT WHERE THE PMY, THE POLYSYNTHESIS OCCURRING AROUND THE DONUT, AND HERE'S THE IMAGE. IF TRANSLATION AND CONSIDERING AT THESE SITES, YOU WOULD EXPECT TO SEE THE RNA SO YOU PERFORM RNA FISH, AND RNA IMMUNO FISH AND WE LOOK AT THE PML, AND THE ATTACKSIN RNA AND HAPPENED IN THE FIBROBLAST AND WE FOUND THAT THAT DO CO LOCALIZE WITH THE PML BODIES. ALL RIGHT, SO PML HAS A LIGASE ACTIVITY AND HAS BEEN SHOWN RECENTLY TO BE IMPORTANT IN NUCLEAR PROTEOSOME BEHAVIOR. SO AGAIN, HERE--YEAH. SO WE NEXT ASKED WHAT HAPPENS WHEN THE OVEREXPRESS IN THE CONTEXT OF THE PATHOLOGICAL 84 Q ATTACKSIN DISEASE, WE OVEREXPRESS THE ISOFORMS FOUR AND FIVE AND THE 84 Q, AND AS YOU CAN SEE, IN THE IN YOU CLEUS, WHEN WE OVEREXPRESS FOUR OR FIVE, YOU GET A DECREASE IN THE AMOUNT OF 84 Q AND HERE CAN YOU SEE A VISUALIZED IMMUNO FLUORESCENCE AND THE 84 Q IS ESSENTIALLY LOST AND THE PML BODIES ARE INCREASED UPON OVEREXPRESS OF THESE ISOFORMS. SO YOU GET A BIGGER BODY, MORE CAPACITY, IT'S CLEAR FOR THE PATHOLOGICAL AGGREGATE. INTERESTINGLY, THIS WILL BE IMPORTANT AT THE END OF MY TALK, IRPT FERON BETA IS AN NATURAL ACTIVATOR OF PML AND WE CAN DO THE EXPERIMENT WHERE WE SIMPLY TREAT THE CELLS WITH INTERFERON BETA AND SURE ENOUGH UNDER THOSE CONDITIONS, YOU BASICALLY WIPE OUT OR COMPLETELY CLEAR THE 84 Q PATHOLOGICAL AGGREGATE BY TREATMENT OF INTERFEERON BETA. OKAY, SO, PML, OUR SITES OF POLYPEPTIDE SYNTHESIS, THE RNA, THE MRMA IS THERE AND IF YOU OVEREXPRESSED OR ATOPICALLY OVEREXPRESSED PML, YOU CAN ACTUALLY CLEAR THESE AGGREGATES. SO THEN THE QUESTION IS, WELL, WHAT IS THE NATURE OF THE RNAs THAT ARE THERE, WHAT IS THE HALLMARK THAT THE PML BODY WOULD WANT TO TEST TRANSLATE FOR, A RELATIVELY NEW MRNA SURVEILLANCE STRATEGY HAS BEEN OBSERVED THAT'S CALLED NO-GO TO K, IT'S PRIMARILY WHERE TWO CRITICAL FACTORS ARE HPSIL AND [INDISCERNIBLE] AND POLAUTA THAT LEAD TO THE TRANSCRIPT LEADING TO DEGRADATION OF THE MISFOLDED PROTEIN. SO WE THOUGHT, WOULDN'T PCULAR AWESOME IF THIS IS OCCURRING AT PML BODIES AND I KNOW THIS IS REALLY MESSY FROM THE PAPER BUT THE BOTTOM LINE IS IF YOU LOOK AT 84 Q, THE PMI AND EITHER HPSONE OR POLUTA, YOU FIND THAT THEY ACTUALLY CO LOCALIZE WITHIN THE NUCLEUS UNDER THE MUTANT CONDITION AND NOT OBVIOUSLY WHAT THE GFP MLS, AND IF YOU KNOCK DOWN HPSONE L OR PELOTA, YOU GET AN IPT GREATER CREASE IN THE INCLUSION BODY SIZE IMPLYING YOU ABNORMALITIES BRO GREATED THE RNAs BY GETTING MORE PROTEIN. ALL RIGHT, SO FOCUSED ON FORMALLY TEST THIS IDEA THAT AERRANT RNAs BECAUSE THEY'RE STRUCTURALLY CURIOUS ARE TEST TRANSLATED IN THE NUCLEUS, WE ADOPTED A STRATEGY ENTIRELY NOT TO HAVE TO RELYOT MICEALATION SO WE CO OPTED HIV BIOGENESIS ESSENTIALLY AND HIGH HAS BEEN CRITICAL IN LEARNING A LOT ABOUT BASIC BIOLOGY AND THIS IS ONE CASE WHERE IT REALLY HELPS US SO LONG STORY SHORT IN THE EARLY DAYS OF THE VIRUS SUSPECT TRANSPORTED OR EXPORTED INTO THE NUCLEUS BUTALATE TRANSCRIPTS OF HIV ARE RREs, AND WITHOUT REV, ANY TRANSCRIPT THAT HAS THE RRE CANNOT LEAVE THE NUCLEUS. SO WE DECIDED WE WOULD ADOPT THIS STRATEGY TO FORMALLY TEST NUCLEACE TRANSLATION SUCH THAT CREATE AID CONSTRUCT AS WELL AS GFP AS WELL AS REVERENCE, THERE'S NO HAIR PINS, THERE'S NOTHING FUNNY ABOUT IT, ONLY IN THE CASE OF REV WOULD YOU ACTUALLY GET A GREEN CELL ESSENTIALLY, WITH NO REV, YOU WOULD GED NO TRANSLATION BECAUSE THE TRANSSCRIPTS AREN'T LEAVING THE NUCLEUS BECAUSE THERE'S NO REV, OR IF YOU HAVE MUTANT REV, HAVE YOU NO GREEN CELL BECAUSE AGAIN THE TRANSCRIPTS ARE NOT LEAVING THE NUCLEUS, SO SURE ENOUGH, HAPPILY UNDER NORMAL WILD-TYPE REV, YOU GOT A FULLY GRAIN FLUORESCENT CELL, YOUR PML BODY AND YOU CAN SEE THAT THE REV PROTEIN IS WITHIN THE NUCLEOTIDES US AND THAT'S YOUR MERGE, IMPORTANTLY WITH MUTANT REF WHICH ISN'T SHOWN HERE THE TRANSSCRIPTS CANNOT LEAVE AND YOU GET NO GREEN CELL. HERE CAN YOU SEE WHERE WE GET THE FISH, AND THE NUCLEI, BUT ONLY IN THE CONDITION WITH THE REV, YOU GET THE GREEN CELL BUT NOT WITH THE MUTANT REV. NOW WITH OUR 84 MUTANT ATTACKSIN ONE, WHICH WE'VE BEEN GOING INTO AS WELL IT WOULDN'T MATTER WHETHER REV IS THERE OR NOT BECAUSE IT'S A HAIR PIN, SO WITH OR WITHOUT REV, YOU SEE NUCLEAR AGGREGATES OF THE CUE. SO THAT IS WHAT WE SEE, WILD-TYPE OR THE MUTANT, YOU SEE THAT WANT TRANSCRIPTS ARE THERE AND CO LOCALIZING WITH PML SO WE'RE--SO WE'RE SORT OF CONFIDENT THAT INDEED WE ARE SHOWING THE TRANSLATION. SO IN SUMMARY WE BELIEVE THAT THE PML BODIES ARE--HAVE A NOVEL FUNCTION AND THAT THEY SCAN--THEY SCAN TEST TRANSLATE AND DEGRADE THE AND DEGREED THE PEPTIDES ALL AT THE BOTTOM, AND AS I SAID ABOUT THE INTERFERON BATTA, WE'RE VERY KEEN TO START IMPORTANT RELATIONSHIPS TO TRY AND TEST THIS HYPOTHESIS IN THE CASE OF NEURODEGENERATIVE DISEASES TO USE INTERFEREON BETA AS A TREATMENT. AND AREXY AND DANIEL ARE THE AWESOME DUO WHO HAVE DONE MOST OF THE WORK AND OF COURSE THE COMMON FUND AND THE INNOVATIVE AWARD. IT PROBABLY DOES MAKE ANY SENSE BUT--30 SECONDS TO SPARE. [ APPLAUSE ] TIME FOR A COUPLE OF QUESTIONS. >> I HAVE A QUESTION, I HAD TO WALK ALL THE WAY FROM THE BACK, I'M TIRED, WHOO! SO CAN YOU COMMENT ABOUT THE IMPORT OF THE MACHINERIES INTO THE NUCLEUS, AND I'M WONDERING ABOUT THE PROCESS VERSES A PASSIVE PROCESS DURING THE BREAK DOWN OF THE NUCLEAR ENVELOPE AND CELL DIVISION WHICH WOULD DISTINGUISH THE DIVIDING CELLS THERE ARE SOME ISOFORMS THAT ARE IN THE CYTOPLASM BUT THEY ALL HAVE NLSs BUT THEY ARRIVE IN THE NUCLEUS BECAUSE THAT'S WHERE THE HOME SHOULD BE. >> IN THE TRANSLATION APPARATUS,. >> IT'S CREATE INDEED THE NUCLEUS SO THEN IT'S EXPORTED SO YEAH, IT'S CREATED IN THE NUCLEOLUS, AND YOU HAVE DNA TRANSCRIPTION, WHICH TRANSCRIBE THE RNAs AND THE RIBOSOMAL PROTEINS THAT COME IN AND THE NUCLEOLUS AND IT'S EXPORTED. >> SO YOU'RE PROPOSING A MODEL WHERE THE RIBOSOMES ARE-- >> WE DON'T KNOW ABOUT THE CONONICAL, THERE ARE NONCONONICAL BUT WE DO KNOW THAT USING THE RAY, WITH THE RPLP PROTEINS CAN YOU NUCLEATE MULTIPLE RIBOSOMAL PROTEINS AS WELL AS PML BODY. I DON'T FINISH THAT SOUNDS RIGHT. >> ALL RIGHT, WE CAN TALK ABOUT IT LATER, THINGS. >> DO YOU KNOW IF THERE ARE PROTEINS THAT DISRUPT PML, DO THEY EFFECT NUCLEAR TRANSLATION? IS THE STRUCTURE OF THE PML NECESSARY FOR THIS NUCLEAR PROCESS TO GO ON? >> SO THAT'S A GREAT QUESTION, EARLIER SLIDE WHERE THERE WAS SHOWED VIRAL APOPE ESTIMATE THADIS, ALL OF THAT INTERSECTED THE CELL BODY, YES, I THINK THAT ONE REASON WHY VIRUSES HAVE TO OFTEN TIMES ATACT THE PML BODY IS BECAUSE IT'S DOING THIS PROCESS TO TEST TRANSLATE THE VIRAL RNAs. >> IT WAS MORE OF A CONCEPTUAL QUESTION. >> DO YOU KNOW WHETHER DISRUPTION OF PML CHANGES THE NUCLEAR TRANSLATION-- >> NO, THAT'S A GREAT THING TO TRY. >> VERY QUICK QUESTION, IS THERE A PRACTICAL WAY TO DISTINGUISH THOSE NUCLEAR TRANSLATED PEPTIDES FROM CONVENTIONAL RIBOSOMAL PRODUCTS BY ANY KIND OF MASS SPEC BASED WAY OR ASSAY. >> WE ARE DOING PML PRODUCTS TRANSLATING BECAUSE THEY'RE HETEROGENEOUS ROG NOWS, NOT ALL BODIES HAVE IT TO SO WE'RE GOING TO TRANSLATE THE PML BODIES AND IDENTIFY PROTEINS ASSOCIATE WIDE THEM AND SEE THE TRANSCRIPTS AS WELL UNDER DIFFERENT CONDITIONS. >> THANK YOU STEVEN. >> WE WILL TALK ABOUT THE ROLE OF THE FOSTER PHOSPHOLIPIDS AND STRESS RESPONSE. >> I WOULD LIKE TO START BY THANKING ORGANIZERS TO TALK ABOUT WHAT WE'VE BEEN DOING, MY INTEREST FOCUS ON BASIC QUESTIONS ABOUT MEMBRANE BIOLOGY AND WE'RE USING THE NEMATODE TO TRY TO GET SOME ANSWERS. HERE WE GO, SORRY. PHOSPHOLIPID BILAYERS ARE MORE THAN SIMPLE FENCES, SO IT WASN'T LONG AGO THAT IT FIGURED OUT THAT IT KEPT SOME OUT. BUT IT'S INCREASINGLY CLEAR AND IMPORTANT THAT NOT ONLY THE SENSE IS THERE BUT THE DIFFERENT BUILDING BLOCKS OF THAT SENSE AS THEY INFLUENCE THINGS LIKE FLUIDITY AND PERMIABILITY, PROTEIN FUNCTION, SIGNALING, SUSCEPTIBILITY TO DAMAGE AND SO ON. AND THE THING I LIKE TO THINK ABOUT IS THE COMP CISION IS IMPORTANT BUT IT ALSO CAN VARY AND IT VARIES BASED ON WHAT WE'RE--WHICH ORGANELLE YOU'RE LOOKING AT, WHAT TISSUE YOU'RE LOOKING AT, AND MOST INTERESTING FOR ME, THE AGE OF THE ANIMAL OR THE DISEASE STATE. SO WE CONSIDER THESE PHOSPHOLIPIDS IN JUST SLIGHTLY MORE DETAIL. THEY COME IN A TREMENDOUS VARIETY, SO THE MODULAR NATURE OF A PHOSPHOLIPID GENERATES, THOUSANDS OF SPECIES IN ANY GIVEN MEMBRANE CONTAINS HUNDREDS OF PHOSPHOLIPIDS AND THAT DIVERSITY COMES AT THE TOP FROM THE POLAR HEAD GROUP THAT CAN BE ASSOCIATED THROUGH THE PHOSPHATE LINKAGE AND THESE ARE A FEW EXAMPLES, TODAY I WILL BE FOCUSING ON THE TWO MOST ABUNDANT EXAMPLES, FOSTER NURSED FOCUSED ON CO LINES AND EGHTALLA MINE. FELT AND ADDITIONALLY THERE'S A NUMBER OF DIFFERENT FATTY ACIDS THAT CAN BE ATTACHED AND THIS IS WHERE MOST OF THE DIVERSITY COMES INTO PLAY SO THERE ARE A COUPLE DIFFERENT FATTY ACIDS CAN YOU FIND IN A PHOSPHOLIPID AND THERE'S TWO OF THISEM SO THAT INCREASES THE COMPLEXITY QUITE A BIT AND YOU CAN HAVE SATURATED FAT ALL THE WAY TO POLYUNSATURATED FAT AND THERE'S ALSO A NUMBER OF UNIQUE FATTY ACIDS THAT CAN BE FOUND IN THIS SYSTEM AS WELL. SO SOMEWHAT LESSER APPRECIATED THAN THE OTHER TWO CONTRIBUTORS TO DIVERSITY IS THE FACT THAT YOU CAN HAVE LINKAGES CONNECTING THE TWO. SO TYPICALLY YOU FIND A ESTHER MUTATION BUT EQUALLY WE SY POSITIVE BOPPED. SO I'LL GET TO THAT IN A BIT. ON KEEP THINGS SIMPLE TODAY I WILL REFER TO THESE MOLECULES BY THEIR CARBON, SO IN THE PHOSPHOLIPIDS WE WILL HAVE THE NUMBER OF CARBONS AND THE UBIQUITINNATION NUMBER OF DOUBLE BONDS WHEN WE TALK ABOUT FATTY ACIDS WE WILL LOOK AT CARBONS AND DOUBLE BONDS AND THAT'S JUST A QUICK WAY TO LOOK AT WHAT YOU'RE LOOKING AT WITHOUT HAVING TO KNOW THE NAMING SYSTEMS, THIS IS ILLUSTRATED SIMPLY HERE WHERE IF YOU START AT A CERTAIN TIME POINT, HAVE YOU A CERTAIN COMPOSITION. IF YOU LOOK LATER THIS CAN BE ON I SCALE OF SECONDINGS OR MONTHS OR YEARS DEPENDING ON THE ORGANISM AND THERE'S NO CHAIRMAN WITHIN THE COMPOSITION, BUT WE KNOW FROM LOOKING AT MICROSCOPY BASED STUDIES, JUST EMs FOR EXAMPLE, YOU CAN SEE THERE'S A TREMENDOUS AMOUNT OF THE PATHWAYS BETWEEN MEMBRANES, TRAFFICKING PATHWAYS AND DIRECT CONTACT SITES FOR EXAMPLE, AND YOU CAN ALSO QUANTIFY THE DYNAMICS IN THESE POPULATIONS. THIS IS AN INTERESTING PROBLEM FOR ME SO THE WHYED HERE IS THAT THIS HAS TO HAPPEN, THE MAINTENANCE OF THE COMPOSITION OVER TIME REQUIRES A HUGE AMOUNT OF REGULATION THAT ARE ENCODED SO YOU NEED TO MODULATE THESE AND COME IN AND OUT OF THE MEMBRANE AT THE RIGHT RATE WITH THE RED PHOSPHOLIPID AND THEN THIS HAPPENED IN IN ALL OF THE DIFFERENT SPECIES IN THE MEMBRANE AND IN THERE WE'RE TALKING ABOUT 40 DIFFERENT SPECIES, SO THAT'S QUITE A BIT TO KEEP TRACK OF. AND NOT ONLY DO YOU HAVE TO DO THIS ONCE OR ESTABLISH THE PARAMETERS ONCE FOR DOING THIS BUT YOU HAVE TO MODULATE YOUR COMPOSITION AS WELL. SO AS YOU ENCOUNTER DIFFERENT TEMPERATURES, DIFFERENT DIETINGS YOU NEED TO KNOW BE ABLE TO ADAPT THESE PARAMETERS IN ORDER TO IN ORDER TO SURVIVE IN A LOWER TEMPERATURE FOR EXAMPLE. AND WE SEE EXAMPLES OF THIS GOING WRONG SO IN MANY DISEASES IF YOU LOOK AT THE MEMBRANE COMPOSITION IT NO LONGER LOOKS LIKE THE MEMBRANE OF THE HEALTHY INDIVIDUAL AND SO IN THESE KISSS THERE'S DISREGULATION OF THESE PATHWAYS THAT POTENTIALLY CONTRIBUTE TO DISEASE. AND THIS MIGHT HELP WITH THE DISEASE STATE. SO TO DO THIS, WE WANT TO INCORPORATE A LABEL IN A GENERAL WAY THAT WILL ALLOW US TO MONITOR NOT JUST ONE PARAMETER OF LIPIDS GOING IN AND OUT OF THE MEMBRANE BUT AS MANY AS POSSIBLE. AND THIS IS WHAT WE DID. INSTEAD OF INNERCORPORATING ONE MOLECULE THAT HAD A STABILIZE O TAUPE TRACER WE GAVE THE ANIMAL A DITET WHERE ALL WERE STABILIZE O TAUPES SO BASICALLY WE START WITH C.ELEGANS AFTER THEY HATCH, THE EB COAL I SO WE ALLOW THOME TO EAT E.COLI UNTIL THEY'RE DAY ONE OF ADULTS SO WE LET THEM GO TO DAY THREE AT 25-DEGREES AND THIS IS A STAGE WHERE THEY'RE GONE GROWING, POST MITOTIC SO THEY'RE NOT MAKING ANYMORE CELLS AND THEY'RE DONE REPRODUCING. SO AT THIS POINT ANY NEW PHOSPHOLIPIDS ARE PRODUCED ARE NOT GOING INTO NEW MEMBRANES BUT THEY'RE GOING TO MAINTAINING THE MEMBRANES THAT ARE ALREADY THERE AND THAT'S WHAT WE'RE ULTIMATELY INTERESTED IN UNDERSTANDING SO WE FEED THEM THE DIET WHERE 99% OF THE CARBONS ARE [INDISCERNIBLE] WE FOLLOW INTO THE TAILS OF THE MEMBRANES. AND I DON'T HAVE TIME TO GO INTO ALL OF IT BUT WHAT WE SEE IS ONE FATTY ACID POPULATION OVER CONTROL, YOU CAN SEE THE APPEARANCE OF ALL THESE DIFFERENT ISOTOPE MERS OR FATTY ACIDS THAT INCLUDE ONE-18 ISOTOPES AND THAT ALLOWS US TO TAKE THAT INFORMATION AND APPLY IT TO THE TOTAL ABUNDANCE OF THAT FATTY ACID WITHIN THE MEMBRANE AND SAY THIS AMOUNT OF THAT FATTY ACID IS NEW OVER THE LABELING PARLIAMENT AND THAT ALLOWS US TO SAY, FOUR% OF THIS PARTICULAR FATTY ACID IS NEW EACH OUR IN THESE DAY THREE ADULTS, AND WE CAN DO THIS NOT ONLY WITH THE CAT BUT WE CAN DO IT WITH THE VAST MAJORITY OF MEMBRANE FATTY ACIDS. WHAT WE CAN'T DO IS THIS SYSTEM, SO WE CREE THEM FROM THE FOSTER NURSED FOCUSED ON GROUP WE LOOK AT THE DYNAMICS SO PHOSPHOLIPIDS ARE COMPLICATED THAN A FATTY ACID, SO WE HAVE TO DETECT THEM BY HPLC COMBINED WITH HANDY MASS SPECTROMETRY AND INCORPORATING ISOTOPES WE MAKE IT IMPOSSIBLE TO SEE WHAT WAS ACTUALLY GOING ON, SO IN THIS CASE INSTEAD OF USING A CARBON ISOTOPE, WE USE A NITROGEN BASED ISOTOPE WHERE WE HAVE ONE THAT'S FOUND IN THE HEAD GROUP OF THESE PHOSPHOLIPIDS IN THIS POPULATION AND WE DO SOMETHING SIMILAR SO KEY CAN LOOK AT FOSTER NURSED FOCUSED ON LIM EDUCATIONAL SPECIES SIMULTANEOUSLY IN THE SAME ANIMAL AND WE CAN SEE THERE ARE DIFFERENCES THAT NOT EVERYTHING IS TURNED OVER AT THE SAMEERATE WHICH IS CONCERN WIDE OUR PREDICTION AND YOU CAN SEE THINGS THAT ARE HIGHLY TURNED OVER VERSES SPECIES THAT ARE LOWLY TURNED OVER. SO TAKEN TOGETHER WE HAVE A SYSTEM NOW TO LOOK AT MEMBRANE STASIS AND MANY OF THE PHOSPHOLIPIDS AND THIS IS LIMITED BY WORKING ON A MORE AUTOMATED WAY OF DOING THE ANALYSIS SO THIS HAS TONS OF ROOM FOR IMPROVEMENT WE'VE SEEN A FEW THINGS THAT ARE QUITE INTERESTING. WE'VE SEEN UNIQUE SPECIES THAT ARE DYNAMIC THAT CAN CHANGE OVER PRETTY LARGE AMOUNT OF LIPID EACH HOUR AND THE COMBINATION OF MEASUREMENT SHOWS THERE'S SIGNIFICANT MEMBRANE REJUVE NATION EACH DAY SO WE'RE LOOKING AT MORE THAN 60% OF THE MEMBRANE BEING NEW AFTER A DAY SO THIS IS QUITE AN ACCOUNTIVE PHENOMENON. SO NOW WHAT WE WANT TO DO IS PUSH ON THIS SYSTEM, SO IF WE CAN NOW INTRODUCE SOMETHING IN THIS CASE, WHAT I'LL BE TELLING YOU ABOUT TODAY ISOXIDATIVE STRESS IF WE CAN PUSH THE SYSTEM, WHAT HAPPENS TO THE PARAMETERS AND HOW CAN THEY COMPENSATE FOR THE FACT THAT THEY HAVE TO DO DIRECT CHANGES IN MEMBRANE COMPOSITION AND THIS LED US TO AN INTERESTING GROUP OF LIPIDS CALLED MLASMALOJENS, THEY HAVE A PLACENTAS MEANAL BOND AND IT IS ACID LABELED SO MANY TYPICAL LIPID PURIFICATION PROTOCOLS WILL RESULT IN THEIR DESTRUCTION BY ACCIDE. SO EVEN THOUGH WE'VE KNOWN ABOUT THEM FOR NEARLY A CENTURY, THERE'S RELATIVELY KNOWN ABOUT THEIR BIOLOGICITY IN VIVO. BUT THEY'RE NOT A SMALL AMOUNT OF THE MEMBRANE. SO THIS IS DATA FROM C.ELEGANS BUT IT'S VERY SIMILAR TO HUMANS. IF YOU CONSIDER THE PH OSPH ATIDYL, OUR MASS IS BUILT FROM GENERATE FRIDAY PLASMANYLGENS AND YOU CAN SEE SEIVET% OF THE LIPIDS IN MILEIN COME FROM THESE MLASMANYL GEN LIPIDS. WE DO KNOW WHERE THEY'RE MADE. SO THEY'RE MADE BY THESE THREE ENZYMES PRIMARILY WITH THE MAMMALIAN COUNTERPART ON TOP OF THE C-ELEGANS GENE AND WE KNOW THE SYNTHESIS PATHWAY BECAUSE MUTATIONS IN IF THESE ENZYMES CAUSE DISEASE. STATE OF EMERGENCY THERE'S A SET OF DISORDERS THAT ARE CAUSED BOO I THESE BIOSYNTHESIS GENES. AND THERE'S A SUITE OF DISEASES, AND THESE CHILDREN ALMOST NEVER MAKE IT PAST THE AGE OF ONE AND VERY, VERY RARELY MAKE IT THROUGH ADOLESCENCE. SO IN--IN ADDITION TO THIS DIRECT SORT OF CAUSAL ROLE, THERE'S ALSO A CORRELATIVE ROLE BETWEEN PLASMALGEN ABUNDANCE IS CORRELATE WIDE ALZHEIMER'S PROGRESSION AND IT'S BEEN SUGGEST THAD THESE CAN BE USED AS A BIOMARKER AND ADDITIONALLY AS A THERAPEUTIC SO IF YOU CAN ADD THEM BACK IN THROUGH THE SUPPLEMENTATION, YOU MAY HAVE A RESTORAATIVE IMPACT AND THEY'RE IN CLINICAL TRIALS IN THIS CAPACITY. REGARDLESS WE STILL DON'T REALLY KNOW WHAT THEY'RE DOING IN THE ANIMAL OR IN HUMANS. LET SO WE WANTED TO SEE IF WE CAN USE C.ELEGANS AS A WAY TO MODEL PLAYS MALGEN DEFICIENCY AND THE FIRST THING CAN YOU DO IS, YOU CAN TARGET SPECIFICALLY GOING TO TALK ABOUT JUST ONE OF THE GENES FOR SIMPLICITY BUT WE'VE DONE IT WITH ALL OF THEM. IF YOU TARGET FARD-ONE, AND THE ANIMALS AT DAY THREE, YOU SEE THERE'S TOTAL REDUCTION AND NEAR COMPLETE ABSENCE OF PLASMALGENERATEDS SO THEY REALLY HAVE NO WITHIN THEIR MEMBRANE AT ALL. SO TO LIMIT SOMETHING'S IN THE ALZHEIMER'S MODEL WE CAN INITIATE THAT LATER IN ADULTHOOD AND SEE IF WE CAN PERTURB THIS POPULATION, AND IN THIS DAY ONE INITIATED RNAi, WE SEE A TREND TOWARDS REDUCTION IN THEIR LEVELS BUT IT'S NOT SIGNIFICANT SO WHAT WE DID WAS WE SAW USING THE ISOTOPES AND ALTHOUGH THE LEVELS ARE LARGELY INTACT, THEY'RE NOT ABLE TO PRODUCE ANY NEW PLAYS MALGEN WITH THIS RNAi TREATMENT. THAL ALLOWED US TO TEST WELL IN MALOGENERATED, AND IF YOU TAKE CELLS FROM PATIENTS THAT HAVE RDCP, YOU SEE THEY'RE SUSCEPTIBLE TO STRESS PARTICULARLYOXIDATIVE STRESS AND THE IDEA BEHIND THE STRESS SENSITIVITY IF YOU HAVE A CLASSIC PHOSPHOLIPID AND THEY ENCOUNT OR ANOXIDATIVE SPECIES, OR A FREE RADICAL, THAT--THEY'RE VERY EASILY DAMAGED, PARTICULARLY IN THE POLYUNSATURATED FATTY ACID CHAIN. AND WHAT ENDS UP HAPPENING UPON THE DAMAGE IS IT CONTRIBUTED TO THE OVERALLOXIDATIVE LOAD IN THAT PEROXIDIZED LIPIDS CAN GO ON AND DAMAGE DNA PROTEIN AND OTHER LIPIDS. THIS IS THE CASE IN PLAYS MALLOW GEN SO IN PLA SMALMALOGEN, SO THIS IS CONSUMED DURING AND BY T REACTIVE OXYGEN SPECIES BUT IT DOES NOT PROPAGATE THE OXIDATIVE LOAD WITHIN THE CELL. AND SIMILAR TO THE CELL CULTURE SAYS SYSTEMS, WE CAN SEE IN THE NEMATODE THAT THERE IS A SIGNIFICANT REDUCTION IN SURVIVAL WITH PARAQUAT STRESS AS A GENERALOXIDATIVE STRESS, BUT IT'S NOT NEARLY AS BIG SUGGESTS IT'S NOT--THE PRODUCTION OF PLASMALOGENS BUT IT'S REDUCTION IN THE MEMBRANE. AND WE SEE THIS SIMILARLY WITH LONGEVITY AND WE LOOK AT LIFE SPAN THERE'S DECREASE WITH L-ONE BUT NOT WITH THE DAY ONE. SO NOW WE CAN ACTUALLY USE THE POWER OF THE WORM. AND LOOK BIOCHEMICALLY AT WHAT'S HAPPENING IN THESE ANIMALS. SO OW MODEL WOULD BE THAT UPON THIS STRESS WE WOULD SEE A CONSUMPTION OF PLAYS MALLOW GEN POPULATION, THIS IS NOT WHAT WE SAW. WE IN FACT SAW AN INYEAS IN THE AMOUNT OF PLASMAROGEN, WHICH IS COUNTER TO OUR EXPECTATION. SO THIS SUGGESTS IT WAS SOME MECHANISM WHERE OXIDATIVE STRESS DIRECT LEER OR INDIRECTLY DRIVE THE SIPGHT SIS, PERHAPS TO GET THE MEMBRANE READY FOR THIS STRESSFUL EVENT. AND IN FACT THIS UPREGULATION REQUIRES ACTIVITY OF THE FARD ONE GENE SOPHISTICATED IT REQUIRES BIOSYNTHESIS. SO TAKEN TOGETHER WE HAVE A MODEL WHERE WE HAVE INCREASEDOXIDATIVE STRESS WHICH IS DRIVING AN INCREASE OF PLASMALOGEN, CONTRARY TO OUR EXPECTATIONS AND THIS INCREASE IS IMPORTANT FOR THE SURVIVAL OF THE ANIMALS. NOW MOVING FORWARD, WE WANT TO UNDERSTAND, OH, WE WANT TO UNDERSTAND WHAT MECHANISMS ARE IN PLACE FOR THIS OXIDATIVE DAMAGE AND RESPONSE AND LIKEWISE FURTHER INTERROGATE THIS IN THE SURVIVAL AS THEY'RE SACRIFICIAL ANTIOXIDANT HYPOTHESIS ISN'T SEEM TO BE TELLING THE FULL STORY. THESE PEOPLE CONTRIBUTED TO THE WORK AND THE NIH AS WELL AS SUPPLEMENTAL FUNDING FROM THE NATHAN SHOCK CENTER AND IF FROM'S QUESTIONS I WILL BE HAPPY TO TAKE THEM. >> SO INTERACTING WITH PROTEINS, SOME OF THEM ARE TRANSCRIPTION FACTORS SO I WAS WONDERING IF YOU LOCK THE AT CROSS BETWEEN THE TYPES OF LIPIDS WHEN THOSE ARE MODIFIED, THEY CAN TURN ON GENES? >> YEAH, I THINK THAT'S DEFINITELY A POSSIBILITY. I THINK THERE'S A NUMBER OF PUBLICITYS BETWEEN HOW THE SIGNAL IF YOU WILL OF IF THERE'S DAMAGE TO CAN BE TRANSLATED INTO AN ACTUAL RESPONSE SO MAINLY WHAT WE'RE DOING IS SCREENING APPROACHES THAT MAY GET AT, YOU KNOW WORK THROUGH ANTIAND STRESS RESPONSE ELEMENTS THAT CAN SENSE AND DRIVE TRANSCRIPTIONAL AND ON A MORE BASIC LEVEL WHAT THE MECHANISM MAY BE, SO THIS IS SORT OF A BLUNT INSTRUMENT WHEN IT COMES TO OXIDATIVE STRESS AND IF WE CAN APPLY THE STRESS IN MORE OF A SPECIFIC WAY, WE MIGHT BE ABLE TO PREDICT IF IT MIGHT BE THROUGH INTERACTION LIKE YOU ARE SUGGESTING. >> IS THE REELINGATIVE POPULATION OF SUBSPECIES OF THE PHOSPHOLIPID SENSITIVE TO THE NATURE OF LIPID INTAKE IN THE DIET? >> YES AND NO. SO IT DEPENDS ON THE ORGANISM, SO WE'RE REALLY GOOD AT NOT CARING ABOUT WHAT OUR DIET IS AND OUR MEMBRANE CAN BE LARGELY INTACT YEAST ARE STRONGLY AND THEY'RE SOMEWHERE IN MIDDLE AND IN HUMANS THERE'S SYRUPLY, YOU KNOW YOU CAN'T--GET ANY KIND OF LIPID AND WE'RE PRETTY GOOD AT IT AND IT'S SORT OF AN INTERMEDIATE JOB, AND IN THE THESE ARE INTRODUCING PRECURSORS AND YOU CAN INFLUENCE THE MEMBRANES IN THAT WAY. THIS SEEMS A LOT GROSSER THAN THE POSSIBILITIES OF REGULATION THAT YOUR RESEARCH SUGGESTS BY POSSIBLE MORE SUBTLE VARIATIONS IN THE SPECIES MIX. >> SURE. >> SURE. >> OKAY. >> OUR FINAL SPEAKER THIS MORNING IS DR. BRUCE YANKENR WHO WILL TALK ABOUT--YANKNER WHO WILL TALK ABOUT FUNDAMENT PATHWAY OF STRESS RESISTANCE. >> SO THANK YOU ROBBIE AND I WOULD LIKE TO THANK YOU FOR GIVING ME THE OPPORTUNITY TO TALK ABOUT MY WORK AND EXPRESS MY GRATITUTE TO THE COMMON FUND WHICH WILL SUPPORT THE WORK YOU'RE GOING TO HEAR ABOUT. SO TWO YEARS AGO, THE RANDOMIZED TRIAL CORPORATION COMPLETED THE MOST EXPENSIVE DISEASE STUDY AND THE SURPRISING RESULT, THAT DEMENTIA AND ALZHEIMER'S DISEASE IS THE MOST EXPENSIVE DISORDER EXKEYEDING THAT OF CANCER AND HEART DISEASE CURRENTLY COSTING WELL OVER A HUNDRED BILLION DOLLARS A YEAR IN THE U.S. AND PROJECTED IF NO EFFECTIVE DRUG IS FOUND IN THE NEAR FUTURE TO EXCEED A TRILLION BY 2015. IT WAS ESTIMATE THAD THIS DEGREE OF EXPENDITURE IS LIKELY TO CONTRIBUTE TO THE BANKRUPTCY OF MEDICARE AND WAS ASHES TRIBUTED TO TWO PRIMARY FACTORS. THE FIRST IS THE ENORMOUS EXPENSE AND TIME NEEDED TO GET A DEMENTED PATIENT THROUGH EVERY DAY ACTIVITIES AND SECOND THE LACK OF ANY THERAPEUTIC PROGRESS IN THE TREATMENT OF THIS DISORDER. NOW AT FIRST BLUSH THIS IS SURPRISING GIB THE AMOUNT OF EFFORT THAT HAS BEEN BEVOTED TO THIS ENORMOUS PROBLEM AND I CONTINUING WARRANTS ON OUR PART SOME REEXAMINATION OF THE SCIENCE OF THE PROBLEM, WHAT YOU SEE HERE ARE THE POST MOTOR UMKC BRAINS OF TWO INDIVIDUALS WHO DIED OF MYOCARDIAL INFARCTION BUT COGNITIVELY INFACT, THEY WERE PART OF THE COGNITUDAL STUDY OF AGING 60 AND 98, AND WHAT'S OBVIOUSLY APPARENT IS THE STREAM DEGREE OF PAIN ATROPHY DUE TO LOSS OF PROCESSES AND NEURONS IN THE ALZHEIMER'S BRAIN RELATIVE TO THE OLD BRAIN. AND THE FOCUS OF RESEARCH IN THE PAST 20-30 YEARS ON THIS DISORDER HAS BEEN ON THE HALLMARKS OF PATHOLOGY, THE PLAQUES, THE TANGLES HOW THEY ARISE AND HOW THEY CAUSE A BRAIN DEGENERATION. BUT AN EQUALLY INTERESTING ASPECT OF THE DISEASE CAME FROM A PARADIGM SHIFT IN THE 90 WHEN IS STEREO LOGICAL QUANTIFICATION SHOWED THAT A NORMAL AGING IN THE BRAIN HAS BARELY ANY NEURONAL LOSS. AND THAT GIVEN THE SMALL DEGREE OF NEUROGENESIS IN THE BRAIN, THIS MEANS MOST NEURONS ARE BORN WITH, YOU DIE WITH SO A POST MITOTIC NEURONS MUST MAINTAIN IT'S FUNCTION WITH VIABLE STAY FOR 80 TO A HUNDRED YEARS OR MORE. AND GET THROUGH ALL KINDS OF STRESSES, THE STRESSES OF YOUR TEENAGE YEARS, THE STRESSES OF TEENAGE KIDS, SO, THERE MUST BE A REMARKABLE STRESS MECHANISM, IN ADDITION TO WHAT THE PATHOLOGY IS DOING HOW THIS REMARKABLE STRESS OR PROTECTIVE MECHANISM IS LOST IN THIS DISORDER AND THAT'S WHAT I WILL TALK ABOUT TODAY. SO SOME YEARS AGO WE PREPORTED AGING AND GENE EXPRESSION INLET HUMAN BRAIN, AND WE FOUND THAL CORE PROFILE IS SOMEWHAT PRESERVED IN MANY CORTICAL AREAS WITH SOME REGION SPECIFIC HETEROGENEITY BUT BY EXPANDING THIS GROUP TO OVER A HUNDRED CASES AND BY LOOKING AT AGING MOUSE BRAIN AND RECESS MACAQUES WE WERE ABLE TO LOOK AT UPSTREAM REGULATORS OF THIS PROFILE AND FROM THAT ANALYSIS WE DERIVED FOUR MAJOR TRANSCRIPTIONAL REGULATORS THAT COULD POTENTIALLY ACCOUNT FOR THIS CORE PROFILE, FIRST THE TUMOR SUPPRESSOR P53, THE ETWO ESTROGEN RECEPTOR AND MOST SURPRISING THREE MEMBERS OF A PATHWAY THAT IS SUPPOSED TO BE OFF IN THE ADULT BRAIN, A DEVELOPMENTAL PATHWAY MEDIATED BY THE TRANSCRIPTIONAL REPRESSOR AND THAT THIS WAS THE FACT ONOR THAT WAS RAIDED AT NUMBER ONE IN OUR INGENUITY IPA PATHWAY LIST AND THE SECOND WAS THE KOREPRESSOR, SAME COMPLEX, AND A TARGET OF MICRORNA 174, SPECIFIC MICRORAN. JUST BY WAY OF PACK GROUND, ARREST BINDS TO THE LARGEST TRANSCRIPTION FACTOR SPECIFIC MOTIF IN THE GENOME, IT'S A 21, SOMETIMES UP TO 23 BASE PAIR RE ONE MOTIF THAT TARGETS IT TO SPECIFIC GENES, ANYWHERE IN THE GENE MOST COMMONLY IN THE UPSTREAM REGION, REST SERVES AS A PLATFORM TO ASSEMBLE A NUMBER OF DIFFERENT KOREPRESSOR COMPLEXES OF CHROMATIN REMODELING ENZYMES, THE NET EFFECT OF WHICH IS THE TURN OFF THE TARGETED GENE. WE FOUND THAT CONTRARY TO EXPECTATION AS THE BRAIN AGES, TOTAL REZONING PROTEIN GOES UP IN THE PREFRONTAL CORTEX BUT NOT IN CASES OF ALZHEIMER'S DISEASE AQUANTIFIED HERE AND BY SUBCELLULAR FRACTIONATION, WE ARE ABLE TO SHOW THIS WAS DUE TO AN ENRICHMENT OF REST IN IT'S NORMAL PREDOMINANTLY NUCLEAR LOCALIZATION WHICH SURPRISINGLY WAS NOT DETECTABLE IN ALL CASES OF DISEASE, WE COULD SEE THE DRAMATIC UPREGULATION OF LABELING SPECIFICALLY AND DOUBLE CORTEX DOUBLE LABELED BY THE MARKER MAP TWO, BUT IN ALZHEIMER CASES ESPECIALLY EARLY IN ALZHEIMERS THERE WAS A LOSS OF REST IN THE CYTOPLASM AND IN DATA I WON'T TALK ABOUT TODAY, WE SHOW THAT MUCH OF THAT IS IN AUTOPHAGOSOMES. SO TO UNDERSTAND THE CONSEQUENCES OF THIS AGE DEPENDENT UPRECKULATION, WE PERFORM SHIP SEEK ANALYSIS IN NEUROCELLS WHICH HAD NEVER BEEN DONE BEFORE BECAUSE IT HAD BEEN ASSUME THAD REST IS OFF IN NEUROCELLS BUT IN FACT IT IS NOT. WE WERE ABLE TO RESOLVE THE CANONICAL ESTABLISHED RISK TARGETS. THESE ARE GEOGROUPS THAT CAPE OUT OF CHIP SEEK ANALYSIS OF A NEURAL CELL LINE. BUT WE IDENTIFIED A NEW CATEGORY OF REST TARGETS, GENES THAT ARE INVOLVED IN APOPEITOSEIS, INFLAMMATION, BE AND VARIOUS PATHOLOGICAL FEATURES OF NEURODEGENERATIVE DISORDERS, PARTICULARLY ALZHEIMER PATIENT HYMER'S DISEASE. THIS SCHEMATIC SHOWS A NUMBER OF DIFFERENT TARGETS THAT WE SHOWED BY TAKING THE CHIP SEQ DATA AND DOING PC R EXPRESSION PROFILES OR OVER EXPRESSING REST, OR KNOCKING DOWN REST AND SEE FIGURE THIS WAS INDEED THE TARGET. THESE ARE ALL TARGETS OF REST. TARGETS THAT ARE ESSENTIAL FOR THE INTRINSIC PATHWAY, THE ERINESSIC PATHWAY MEDIATED BY INFLAMMATORY MEDIATORS SUCH AS PATH, AND ALL THE CHROMATIN PATHWAY, AS I MENTIONED A NUMBER OF DIFFERENT TARGETS THAT ARE RELATED TO THE PATHOLOGY OF ALZHEIMER'S DISEASE, AND IN OTHER EXPERIMENTS WE'RE ABLE TO SHOW THAT IN FACT, INVITRO IF YOU KNOCK DOWN RREZONING, IT WILL BE NOR VULNERABLE TOOXIDATIVE STRESS AND THE KNOCKOUT MICE IN WHICH REST UNDER THE CONTROL, THE PHLOX ALLELE WAS RECOMBINED WITH MKCREE SO ONLY IN NEURONS OF THE POSTNATAL ADULT MOUSE BRAIN, AND THOSE ANIMALS ARE VIABLE INENTIALLY, THEY LOOK GOOD AS YOUNG ANIMALS BUT WITH AGE THERE'S PROGRESSIVE LOSS OF NEURONS IN THE HIPPOCAMPUS. AND CORTEX TAKEN TOGETHER SUGGESTS THAT REST PLAYS AN ESSENTIAL ROLE IN MAINTAINING IN NEURONS DURING THE AGING PROCESS. TODAY I'D LIKE TO TALK ABOUT OTHER STUDIES IN WHICH WE ASK THE QUESTION OF WHETHER THIS IS RELEVANT TOW COGNITIVE DECLINE IN THE HUMAN AGING POPULATION. AND TO DO THIS WE COLLABORATE WIDE DAVE BENNETT WHO RUNS THE RELIGIOUS ORDER STUDY OF THE MIDWEST WHICH IS A STUDY WHERE HE TAKES COMMUNAL POPULATIONS OF MONKS, NUNS AND PRIESTS BECAUSE THEY HAVE FEWER PRESUMABLY ENVIRONMENTAL EVALUATION AND LONG NUDEINALLY THEY GET PSYCHOMETRIC TESTING AND AT DEATH THEY LEAVE THEIR BRAINS TO SCIENCE AND WE ISOLATE THE HYPOCAMPUS AND PREFRONT AUTOCORTEX AND QUANTIFY REST LEVELS AND WE LOOK AT THE RELATIONSHIP BETWEEN THE TRAJECTORY OF THEIR COGNITIVE STATE AND REST LEVELS AT DEATH. THESE ARE WHAT YOU CALL SPAGHETTI PLOTS, THEY'RE CURVES OF INDIVIDUAL SUBJECTS WHO ARE COGNITIVELY TESTED. THIS IS A MEASURE OF 19 DIFFERENT COGNITIVE SUBDOMAIN TESTS IN A GLOBAL COGNITIVE SCORE AND THE FIRST THING YOU'LL NOTICE IS THAT THE HETEROGENEITY THAT AS PEOPLE AGE IS A GROUP OF 125 CASES, WE LOOKEDAD, THEY HAVE QUITE A BIT OF VALID AND RELIABLE I CAN'T B ELIEVE--VARIABLES WITH THE COGNITIVE EFFECTS TO THIS PERSON WHO GOT BETTER WITH AGE, PROBABLY A P. I. SINCE AS WE KNOW P. I.s GET BETTER WITH AGE. [LAUGHTER] AND YOU CAN GET A MEAN SLOPE FOR EACH OF THESE AND YOU CAN STRATIFY IT, IN HAD OUR CASE WE STRATIFIED IT FOR REST LEVELS, MEASURED IN THE NUCLEI, FOR PREFRONTAL CORTEX PLACES AFTER DEATH AND AND THESE REGRESSION CURVES WHICH IS A MULTIVARIANT ANALYSIS WHERE YOU INTRODUCE EXTRA TERMS FOR EACH CASE, THE POINTS ARE NOT TECHNICALLY INDEPENDENT, THEY'RE LINKED, WE STRATIFIED FOR INDIVIDUALS WHO WERE IN THE HIGHEST WENTILE ARREST EXPRESSION, MIDDLE AND THE LOWEST AND THIS POINT IS AN ART OF PUTTING A LIB THROUGH A CURVE THAT BEEPEDS OVER THE FURTHER OUT YOU GOET FROM THE TIME OF DEATH WHICH IS AT ZERO POINTS. THE IMPORTANT POINT HERE IS THAT THE INDIVIDUALS IN THE UPPER WENTILE, 20th PERCENTILE OF REST EXPRESSION DO NOT SHOW IGF 95 CANT--AS A POPULATION DO NOT SHOW COGNITIVE DECLINE WITH AGE. THE IRPT MEDIATE EXPRESSION GROUP SHOWS SOME DEGREE OF COGNITIVE DECLINE AND THE LOWEST WENTILE ARREST EXPRESSION SHOWS STRIKING COGNITIVE DECLINE. ALL ALL THESE CASES HAVE ALZHEIMER'S DISEASE, WE THEN WENT ON TO DO GENE EXPRESSION PROFILING TO GAIN INSIGHT INTO THE RELEVANT REST TARGETS THAT MAY BE ASSOCIATED WITH MEMORY. SPECIFICALLY FOR THIS ANALYSIS, WE--I JUST SHOWED YOU GLOBAL COGNITIVE FUNCTION WE LOOK AT EPISOTTIC MEMORY BECAUSE THAT'S THE MOST SENSITIVE DOMAIN FOR EARLY ALZHEIMER'S DISEASE THAT'S BIOGRAPHICAL MEMORY OF TIME, PLACES AND EFFECTS THAT LOST THE EARLIEST IN A PERSON WHO IS DESTINED TO GET THIS DISEASE. SO WHAT WE DID IS WE TOOK THIS LONGITUDINAL COGNITIVE DATA AND WE PUT IT TOGETHER WITH THE GENE EXPRESSION DATA AND DID NETWORK ANALYSIS TO PULL OUT GENE MODULES THAT WERE ASSOCIATED STATISTICALLY WITH MEMORY LOSS AND THAT'S SHOWN HERE: FOUR MAJOR MODELS CAME OUT OF THIS, WHICH IS SCHEMATIC LOAMACYY ASSOCIATED WITH THEM, AND THE MOST SURPRISING RESULT, WAS THAT CELL DEATH GENE WHERE THE MOST SIGNIFICANTLY AS A GENE MODULE ASSOCIATED WITH MEMORY LOSS SUCH THAT REPRESSION OF THESE GENES AS A POPULATION WAS ASSOCIATE WIDE BETTER MEMORY. THE AND FOUR OTHER--THREE OTHER MODULES IN ORDER OF SIGNIFICE WERE MITOCHONDRIAL CHANGES, NUCLEAR ENCODE TED MITOCHONDRIAL GENES AND MEANING BY O LARGE OF THE GENES IN THIS GROUP WHEN THEY'RE UPREGULATED THEY SEEM TO BE ASSOCIATE WIDE BETTER MEMORY, AND THE REST TARGETS FOR EACH OF THESE MODULES ARE SHOWN AT THE NETWORK INTERSECTIONS, A VERY LARGE NUMBER OF REST TARGET MEDIATING CELL DEATH, MEANING A THIRD OF THE CELL DEATH GENES APPEARED IN THIS GROUP BUT THERE ARE SOME REST TARGETS IN THE OTHER GROUPS AS WELL. THESE RESULTS SUGGEST THAT IN THE AGING BRAIN, THE REPRESSION OF CELL DEATH GENES MAY BE ASSOCIATE WIDE COGNITIVE PRESERVATION AND MAY CONTRIBUTE TO THE PHENOMENON I ALLUDED TO, THE SECOND SLIDE WHICH WAS THAT A NEURON HAS TO SURVIVE FOR A HUNDRED YEARS OR OR MORE DESPITE LOTS OF DIFFERENT STRESSORS, POTENTIALLY LOTS OF DAMAGE. BECAUSE IT'S LARGELY IRREPLACEABLE. SO TO UNDERSTAND THE SYSTEM IN GREATER DEPTH, WE TURN TO THE WORM C-ELEGANS WE HEARD ABOUT IN THE LAST TALK FOR TWO REASONS, FIRST C-ELEGANS IS ONE OF THE BEST ESTABLISHED MODELS OF STRESS RESISTANCE AND AGING. AND SOCKED, THERE ARE ORTHOLOGS, FUNCTIONAL AND SPHRUCTURAL ORGANIZATIONS O LOGS OF THE ARREST CODE OR REPRESSOR FOR C-ELEGANS FOR ALMOST EVERY MEMBER OF THE COMPLEX, SPECIFICALLY FOR ARREST, THERE ARE TWO GENES, SPR THREE, STANDING FOR SUPPRESSOR, AND THREE AND FOUR WHICH ARE ORTHOLUS, SO HERE WE LOOK AT A INTEGRATED SPF-4G FP CONSTRUCT IN C-ELEGANS AND FOUND THAT IT CO LOCALIZED WITH THE NEURON SPECIFIC MARKER RAB, THREE. SO INTERESTINGLY THE REST ORTHO-LOG AND THE C.ELEGANS AND PREDOMINANTLY EXPRESSED IN NEURONS SIMILAR WITH THE EXPRESSION PATTERN AND THE RANGING BRAIN. FURTHER MORE, AS IS THE CASE FROM THE MAMMALIAN IT'S ALSO A STRESS RESPONSE GENE, THIS EXPERIMENT WE TOOK WORMS, EITHER CONTROL OR TREATED WITH PARAQUAT WHICH IS A MITOCHONDRIAL ELECTRON COUPLER AND REDUCES AND INDUCES OXIDATIVE STRESS FOR THE GENERATION TO PEROXIDE AND YOU CAN SEE THAT THE SPR 4G SP, GENE IS MARKEDLY TURNED ON BY THISOXIDATIVE STRESS. THE SO WE LOOKED AT A NUMBER OF LOSS OF FUNCTION MUTANTS IN THIS THESE LOGS AND WHEN WE FOUND WHEN HE TREATED WITH STRESS AND LOSS OF FUNCTION IN THESE MUTANTS,EE FOUND THAT LOSS OF MUTANTS SIGNIFICANTLY REDUCED THE SURVIVAL OF THE WORM. INCLUDING A LOSS OF FUNCTION MUTANT OF SPF-ONE OF MUTANT IN THE LOG, AND IT'S MALLALLIAN CO REST. BY THE WAY, THE THAT EVIDENCE THESE ARE LOSS OF FUNCTION MUTATIONS IS THAT THESE PHENOTYPES ARE REPLICATED BY RNAi, FOR SPR FOUR AND CAN BE RESCUED BY TRANSGENIC EXPRESSION OF WILD-TYPE SPR-FOUR AND THREE. , THE LOSS OF MUTANTS IS THE SPR FOUR, ARE ALSO RESISTANT TO THIS CASE, HEAT SHOCK THAT ARE MUTANTS IN THE WORM SYSTEM, AFTER INCREASING PERIODS OF TIME, THERE'S INCREASING MORTALITY IN WILD-TYPE WORMS BUT THAT MORTALITY IS AUGMENTED IN THE DIFFERENT LOSS OF FUNCTION MUTANTS, ACHIEVES ITS HIGHESTEST LEVELS IS THE SPR 34 MUTANT AND INTERESTINGLY IN ONE MUTANT OF CO-RES WHICH HAS HIGH LEVELS OF SENSITIVITY TO HEAT SHOCK. LOSS OF FUNCTION MUTANTS OF THE REST ARE ALSO HIGHLY SENSITIVE TO PROTEIN COMPLEXIO TOXIC STRESS THAT ARE INDUCED BY YOU HAD HAN AMYLOID BETA PROTEIN, THE MAJOR COMPONENT OF AMYLOID DEPOSITS IN THE ALZHEIMER BRAIN. IN THESE WORMS THERE IS AN INTEGRATED GFP CONSTRUCT IN FLUTA MITTURGIC NEURONS WHICH SERVES AS A MARKER OF THOSE NEURONS WHEN WE INTRODUCE A HUMAN A-BETA 42 TRANSGENE THERE IS A PROGRESSIVE LOSS OF NEURONS WITH AGE IN THE WILD-TYPE WORMS THAT'S ILLUSTRATED HERE, QUANTITATIVELY. AT DAY THREE OF ADULT LIFE, 40% OF THE NEURONS ARE GONE IN THE WILD-TYPE, IT'S GREATER AFTER DAY EIGHT. BUT THE SPR FOUR LOSS OF FUNCTION MUTANT HAS A SIGNIFICANT LOSS, GREATER LOSS THAN THE WILD-TYPE AND THAT CAN BE RESCUED BY TRANSGENICLY INTRODUCING THE WILD-TYPE. SO WORMS THAT SHOW LOSS OF FUNCTION FOR THESE REST ORTHOLOGS ARE SENSITIVE TO A VARIETY OF DIFFERENT STRESSORS, SO WE HAD TO MORE GENERALLY REGULATE THE AGING PROCESS BECAUSE AS YOU KNOW STRESS RESISTANCE IS INTRICATELY LINKED TO AGING AND FURTHER MORE WE OBSERVE THAT THE HUMAN BRAIN, HIGHEST LEVELS OF REST OCCURRED IN INDIVIDUAL WHO IS LIVE INDEED THEIR NINTH AND 10th DECADE. SO TO ADDRESS THIS WE COLLAB WAIT WITH GEORGE CHURCH'S LAB AND WE USE THE VARIANT OF THE CRISPR, CAS-NINE IN THIS WAY NOT TO CHAIN THE SNA SEQUENCE BUT TO CHANGE ACTIVATION, THE WAY WE DID THIS WAS TAKE CAS NINE, AN ENZYME THAT CLEAVES DNA, FUSED IT TO THE TRANSCRIPTIONAL ACTIVATOR, BP-64 AND THEN USING GUIDE RNAs WE DIRECTED THE COMPLEX TO THE SPR FOUR PROMOTER USING GUIDE, RNAs TO THE MULTIPLE POINTS PROMOTER, AND THIS RESULT INDEED INCREASE EXPRESSION IN THE SPR GSP AND AN ADVANTAGE OF THIS SYSTEM, IS YOU INCRISE THE ENDOGENOUS TRANSCRIPT AND IT REMAINS LOCALIZED TO THE NORMAL SITES OF EXPRESSION. WE DON'T SEE ECTOPIC EXPRESSION SO IT'S A RELATIVELY PHYSIOLOGIC TYPE OF EXPRESSION, AND WHEN WE DID THIS, THE LIFE SPAN OF THE WORMS, THE MEAN LIFE SPENT SIGNIFICANTLY INCREASED. SUGGESTING THAT A MODEST INCREASE OF THIS WILL SPAN THE LIFE OF THE WORM. IF YOU LOOK AT THE MUTANT IN THE IGF SIGNALING PATHWAY, THE DAF-TWO FIRST DESCRIBED BY 60IA KENYAN, YOU CROSS THE LOSS OF FUNCTION SBR-FOUR MUTANT INTO TAT, HAVE YOU A SIGNIFICANT ABNORMALITIES BROIGATION OF THAT LIFE SPAN EXTENSION WHICH IS MOST PRONOUNCED FOR THE DOUBLE SPRFOUR AND THREE MUTANT SUGGESTING THAT NOT ONLY DOES SPR-FOUR INCREASE LIFE SPAN BUT LOSS OF FUNCTION WILL ABNORMALITIES BROIGATE THE ABILITY OF THE INSULIN SIGNALING PATHWAY MUTANTS TO INCREASE LIFE SPAN, SO IN CONCLUSION, THE TRANSCRIPTIONAL REPRESSOR IS ACTIVATED AND SPECIFIC NEURONAL POPULATIONS, OF THE AGING MAMMALIAN BRAIN. IT ACTIVATES A STRESS RESISTANCE NETWORK THAT PROTECTS NEURONS AGAINST MULTIPLE TOXIC INCULTS, A FUNCTION THAT APPEARS TO BE CONSERVED AND ELEVATED REST LEVELS DURING AGING ARE ASSOCIATED WITH COGNITIVE PRESERVATION AND LONGEVITY. AND THE SCHEMATIC REGISTRATION SHOWS THE PLEOTROPIC REST OF THE PATHWAY, SOME OF WHICH I DESCRIBE TODAY BUT THERE ARE A NUMBER OF OTHERS, REST AND MAMMAL RAN ORTHOLOGS APPEAR TO PROTECT AGAINST A VARIETY OF DIFFERENT STRESSORS, MAMMALIAN REST ALSO PREVENTS THE APPEARANCE OF PATHOLOGICAL PROTEIN AGGREGATES SUCH AS AMYLOID DEPOSITS AND MOUSE MODELS THAT I HAVEN'T SHOWN YOU AND FINALLY THROUGH REGULATION OF THE INSULIN SIGNALING PATHWAY, WE BELIEVE THAT THIS STRESS RESPONSE NETWORK MAY BE A NOVEL WAY IN WHICH BOTH STRESS RESISTANCE AND AGING IN THE BRAIN IS COUPLED TO THE AGING OF THE ORGANISM. SO I'LL JUST CONCLUDE BY ACKNOWLEDGING THE PEOPLE WHO DID THE WORK, TAO LU, AND DEREK DRAKE, AND JOE ZULLO, TOGETHER WITH MONICA COLAIACOVO, AND WE COLLAB WAITED WITH CHURCH CHURCH AND THIS WAS A DIRECT OUTGROWTH OF MY PIE O NEAR AWARD. THANK YOU. --PIONEER AWARD. THANK YOU. [ APPLAUSE ] >> COUPLE OF QUESTIONS. >> I WAS WONDERING IF YOU HAVE ANY THOUGHTS AS TO THE VERYIABLE AND REST EXPRESSION LEVEL THAT YOU SEE IN AGING HUMANS AND WHETHER YOU THINK THAT'S GENETIC OR ENVIRONMENTAL OR BOTH. >> IT'S A VERY GOOD QUESTION: IS THERE GENETIC COMPONENT OR ENVIRONMENTAL COMPONENT--I THINK IT'S BOTH BUT THERE'S ALREADY EVIDENCE FOR GENETIC COMPONENT. THERE'S ONE REST GENETIC VARIANT TO SNP THAT WAS REPORT FRIDAY THE ADNE BRAIN CONSORTIUM WHICH IS PROTECTIVE AND WE'VE SHOWN IT INCREASES REST LEVELS AND WE'RE LOOKING AT A NUMBER OF RARE VARIANTS. SO I THINK THERE'S LIKELY TO BE A GENETIC COMPONENT. IS REST EXPRESSED IN THE IMMUNE LEVELS AND DOES IS IT CHANGE WITH ANALLING? >> IS REST EXPRESSED IN THE IMMUNE CELLS AND DOES IT CHANGE WITH AGING. >> THE ANSWER IS YES, THE AGING PATTERN WE DON'T HAVE A DEFINITIVE PATTERN ON YET. >> DO YOU HAVE ANY IDEA HOW ALZHEIMERS IS AT REST? >> GOOD QUESTION. WE FOUND THAT NOT OHM IN ALZHEIMER'S DISEASE BUT ALSO IN OTHER NEUROGENRATIVE DISORDERS, FRONTAL DEMENTIA DISORDER, LOUIS BODY, IT'S NOT AS PROFOUND AS IN ALZHEIMER'S WE THINK IT IS IN PART A LOSS OF REGULATION OF NUCLEAR TRANSLOCATION THAT A COMPANY'S PROTEIN COMPLEXIO STASIS BREAK DOWN AND THAT'S SORT OF A BROADWAY OF SAYING THAT WE DON'T KNOW. [LAUGHTER] >> LAST QUESTION. >> THERE'S A LINK BETWEEN APOE-FOUR ALLELE AND ALZHEIMER'S DISEASE, HOW DOES THAT FIT INTO ARREST? >> VERY GOOD QUESTION HOW DOES APOE-FOUR FIT INTO REST. WE HAVE PRELIMINARY INFORMATION THAT THERE MAY BE GENETIC INTERACTION BETWEEN APOE-FOUR AND REST, AND IT IF YOU'RE APOE-FOUR POSITIVE, THE HIGH QUINTILE REST IS STILL VERY PROTECTIVE AND YOU SEEK MININAL COGNITIVE DECLINE BUT THE LOWER TWO CURVES COLLAPSE WHICH MEANS THAT THE INTERMEDIATEIARY LEVELS ARREST IN THOSE INDIVIDUALS. >> THANK YOU. >> [ APPLAUSE ] >> SORRY TO CUT THE QUESTIONS SHORT BUT IN THE INTEREST OF TIME WE'RE ALREADY SUBSTANTIALLY BEHIND, WE NEED TO END THIS SESSION. SO I THINK YOU'LL AGREE THAT WE'RE OFF TO AN OUTSTANDING START. WELL THEA GIVE THE MORNING SPEAKERS ANOTHER ROUND OF APPLAUSE. >> LET'S GET STARTED WITH THE AFTERNOON SESSION, OUR FIRST SPEAKER IS ALEXEI, ARAVIN AND HE WILL TALK ABOUT PIRNA BIOGENESIS AND TRANSGENERATIONAL EPIGENETIC INHER STANCE. >> SO IN THE LAST TWO SYSTEMS, WORK IN BACTERIA IN ANIMALS TO FIGHT DIFFERENT GENOMIC INVADERS AND--HOW THEY USE DIFFERENT PROTEINS SO ON SOME LEVEL THEY USE SIMILAR APPROACH TO FIND AND FIGHT GENOMIC INVADERS SO IN BOTH SYSTEM, THE SMALLER PRODUCE AND THE SMALLER DIRECTLY RECOGNIZE WITH TRANSPOSEON TARGETS. AND SO, THEY JOIN THE PROTEINS SO IT'S UNRELATED SO THE SPEEDY PROTEIN OF THE SYSTEM OR INCREASE PROTEINS AND CASE OF THIS, AND THEN AFTERWARDS COMPLIMENTARY SMALLER TARGETS AND FOR SALADS, SO IN BOTH SYSTEMS AGAIN IN BACTERIA, AND ANIMALS ABLE TO FIND DIFFERENT GENOMIC INVADERS LIKE TRANSPORTER LIKE [INDISCERNIBLE] SO WE LIKE TO COMPARE THE SYSTEM TO IMMUNE SYSTEM, LIKE THE IMMUNE SYSTEM AND PATHOGENS BUT WE REALLY WANT TO PROVE THE SMALL RNA CAN WORK AS IMMUNE SYSTEM, THEY SHOULD BE ABLE NOT JUST TO REPRESS CURRENT TRANSPOSEON BUT ALSO ADAPT IN THE NEW INVASIONS, SO, ANOTHER QUESTION LIKE MORE SPECIFIC QUESTION, HOW THE SMALLER RNA PRODUCED IN CASE OF CRISPR, AND IT'S RELATIVELY WELL IN THE MOUSE, SO THEY POSITION A SPECIAL LOCI IN A REGULAR MANNER AND WITH THE SPACIAL SEQUENCES BETWEEN TED CREASE OR SMALL RNA WORK IN THE PROCESS OF SIGNAL FOR RECOGNITION, FOR THE PROCESS, SO OCCASIONALLY YOU THINK LIKE THIS, AND SO WHAT THE PROCESSING SIGNAL FOR A LOCI THAT'S IN THE GENOME. AND SO WITH DISCRETION, ONCE OR TWICE A FEW YEARS AGO, AND SUPPOSED TO SHOW YOU WHERE THEY PRODUCE, THEY PRODUCE FROM SPECIAL REGIONS WHICH THEY CALL THE CLUSTERS THAT ARE RELATIVELY LONGER CAN BE FULL OF DIFFERENT TRANSPOSEON SEQUENCE AND EVERY LOCUST LIKE THIS PRODUCE STYLES AND SO THE EXPERIMENT WHICH THEY DID A FEW YEARS AGO WHICH IS A COMPLETELY RANDOM SEQUENCE INSIDE THE CLUSTER BECAUSE WHAT YOU WANT THE MODEL HOW THE SYSTEM ACQUIRE ASSISTANCE AND IMMUNITY AGAINST NEW TRANSPOSEON AND WHAT'S TRANSPOSE ON WITH THE GENOME SOONER OR LATER WILL LEAD TO GENERATION, AGAINST THIS NEW TRANSPOSE ON. SO YOU JUST WANT TO MODEL THIS SYSTEM AND INSTEAD OF JUST WAGING FOR A NEW TRANSPOSEON TO REACH LIKE THIS, WE SOURCE COMPLETELY GFP OR LIKE Z IN THE FLIGHT GENOME IN THE CLUSTER OR MOUSE, GENOME AND INDEED WHAT THEY FOUND WAS THAT THIS ARTIFICIAL SEQUENCE LEAD TO GENERATION OF NEW PILOT PROJECT RUE EIGHT THAT'S ABLE TO SILENCE THE MACHINE. SO IT DOESN'T MATTER WHAT YOU PUT IN THE REGION LIKE THIS, IN THIS PART OF THE CLUSTERS, THIS GENERATE NEW PYRUVATE, SO THE NEW SEQUENCE CAN BE CLEAR LOOKS SO IT'S CLEAR THERE'S NO VERY SPECIFIC PROCESS AND SIGNAL INSIDE THE SEQUENCE, LIKELY INCRISPR, AND NOW WHO THE NEW TRANSPOSEON SAYS THAT DIFFERENTIATE FROM TRANSCRIPT FROM OTHER TRANSCRIPT IN THE CELL, AND HOW THORS PROCESS AND NECKANISM, AND AGAIN, SO IN THE FIELD OF [INDISCERNIBLE] THAT'S REGION LIKE THIS, TRANSCRIBE LOW, AND IT'S PRECURSOR TO BE PROCESSED TO SMALL RNA, SO AS WE SHOW THE LOCAL SEQUENCE IS NOT IMPORTANT, SO THEY PROPOSE THE MIGHT BE IMPORTANT IS SO WHAT WE DID, WE PROFILE THE CHROMATIN MARK IN THE GERM CELLS EXPRESSED PYRUVATE TO DEFINE THE EPIGET GETTIC SIGNATUREOT CLUSTERS SO IN THIS CASE, IT'S ANALYZE USING THIS FOR THIS CHROMAIN, UNFORTUNATELY I DON'T HAVE TIME TO EXPLAIN IN DETAILS BUT WHAT DID SHOWS IS THAT NORMAL GENE ON THIS PLACATED HERE HAVE A MARK THAT YOU EXPECT IT'S ESSENTIALLY ACTIVE MARK LIKE FOR METHYLATION AND THE CK FIRST SIX METHYLATIONS AND THEY HAVE VERY DIFFERENT CHROMATIN SEQUENCE, ESSENTIALLY THE CHROMATIN CLUSTERS AND THE REPRESSIVE MARKER AND THE HISTONE THREE, METHYLATION THAT PH-ONE, MARK, VERY DIFFERENT FROM ACTIVE GENE TRANSCRIBED SO THIS IS JUST RELATIONED THAT LEAD TO THE CHROMOSOME STRUCTURE, MIGHT PLAY A ROLE IN LAY FUNCTION CAN CAN REALLY PROOF IT. SO TO PROVE IT WHAT THEY WANT TO FIND SOME OVERIDENTICAL REGION THAT'S NEVERTHELESS DIFFERENT THAN THEIR ABILITY TO PRODUCE PILOT PROJECT RUE EIGHT. SO WE WERE ABLE TO FIND A SYSTEM LIKE THIS IN ONE OF THE SYSTEMS, SO WHAT WE FOUND, WE FOUND TWO DIFFERENT STRAINS WHAT WE CALL ACTIVE ELECTIVE AND SAME GENOMIC REGIONS EITHER PRODUCED PIRNA, OR IT DOESN'T REDUCE PiRNA, SO THIS IS ACTUALLY GENOMIC SEQUENCE EXPRESS INDEED BOTH CLUSTERS. IT'S ALSO NOT A MUTATION BECAUSE YOU CAN SEE HERE, THE CLUSTER NUMBER FOUR PRODUCED BOTH STRAINS AND IT'S AN A-STRAIN. OKAY AND WHEN WE PROFILE THE CHROMATIN MARKS AND AND LIKE WE WERE HAPPY TO SEAL IT, THIS METHYLATION, THIS SUPPOSEDLY REPRESSIVE MARKER COULD RELATE THIS PRODUCTION OF PiRNA IN THIS AREA, YOU CAN SEE THE HIGH METHYLATION IN THIS REGION AND THIS LOAD LEVEL SO THIS IS JUST IN THE SAME, SO NOW WHEN THEY FOUND THESE TWO STRAINS, AGAIN THIS CARTOON IS TO MODERNIZE WHAT I SHOW YOU BEFORE AND THERE'S NO METHYLATION AND NO PiRNA AND ANOTHER STRAIN, VARIOUS CHROMATIN, SO NOW WE CAN CROSS THESE TWO STRAINS AND SEE WHAT HAPPENED IN THE APPROACH HERE AND BECAUSE WE SEQUENCE THESE WITH BOTH STRAINS AND IDENTIFY A SINGLE POLYPEPTIDE MORPHY MORPHISM, WE CAN KNOW ON EACH SPECIFIC CHROMOSOME, BECAUSE WE CAN THE REED SPECIFICALLY TO ONE CHROMOSOME OR OTHER AND SURPRISINGLY WHAT WE FOUND DEPEND ON HOW YOU CAN DO THIS STRAIN, YOU GET THE DIFFERENT RESULT. SO THE PROTEIN DIRECTIVE AND THE CHROMOSOME REMAIN INACTIVE AND IF YOU CROSS A LOT OF THIS DIRECTION, YOU ACTUALLY GET AT LEAST SOME MODERATE LEVEL, FOR THE CHROMATIN MARK, BUT THIS AIR A MODEL DEPOSIT SOMETHING TO THE ACTIVE CHROMATIN, SO THE SIMILAR SYSTEM WAS ALSO FOUND IN THE CASE OF SOME TRANSGENIC PiRNA STRUCTURE SO IT'S ALSO DIFFERENT IN THE ABILITY TO PiRNA IN THE PROGENY, IT'S PRODUCTIVE PiRNAF THE PREIOUSLY CLUSTER. SO THIS IS IN THIS NATURE PAPER AND WHAT WE SHOW THAT THIS ACTIVATION OF PiRNA PRODUCTION COULD RELATE THIS CHANGE IN THE METHYLATION MARK. SO THE CHROMATIN PiRNA CHANGE TOGETHER. SO WHAT'S THE NATURE OF THIS TSMITTED CYTOPLAYS MIC FACTOR AND HOW WE CHANGE CHROMATIN MARK. SO I'M NOT GOING TO DISCUSS MUCH ABOUT IT BECAUSE IT'S LIKE PUBLISHED IN SEVERAL PAPERS BUT ESSENTIALLY IT'S VERY LIKELY THAT PiRNA THEMSELVES, SO THEY FLY OOCYTE AND NACAUSE ACTIVATION OF PiRNA ACTIVATION. SO THIS IS BASED ON TWO FINDINGS, SO ONE IS IT'S PiRNA PROTEINS, WE KNOW THEY DEPOSIT IS BY THE MOTHER SLIDE, SO THIS IS AT LEAST FOUND, AND THEN ALSO WORK FROM SEVERAL GROUPS SO THAT THE COMPLEXING CAN INDUCE A METHYLATIONOT TARGETS. SO WE USE THE SYSTEM THAT WE ACTUALLY PRODUCE ARTIFICIAL PiRNA AGAINST LYSINE BUT IN THIS CASE IN THE GROUND WE LOOK AT TRANSGENE TARGET AND SHOW IF YOU PRODUCE THIS ARTIFICIAL PiRNA AGAINST THIS, IT'S GOING TO [INDISCERNIBLE], DECREASE ON ACTIVE MARK ON THE TARGET TRANSGENE AND THE METHYLATION MARK. SO THIS LOOKS LIKE PiRNA, SO THE MOTHER SOMEHOW CHANGE CHROMATINS OF HOMOLOGOUS SEQUENCE, AND THE APPROACHING THAT, SOMEHOW LEAD TO PiR NA GENERATION AGAIN SO HOW THIS WORKS, THE ROAM CHROMATIN MARK LEAD TO PiRNA. SO A FEW YEARS AGO, FROM THE PUBLISHES PAPER, IN THE PROTEIN, OF THE PH-ONE HOMOLOGUE, THAT'S REQUIRED FOR TRANSPOSEON SILENCE, AND IT'S HP-ONE IS VERY WELL CHARACTERIZED PROTEIN THAT'S BINDS AND THE HEALTHALATION MARK IS NOT PLAY THE SIMILAR ROLE AND THE GENE AND SLOAN-KETTERING WAS ABLE TO SHOW THAT THE CHROMATIN DOMAIN AND METHYLATION MEP PIEDS SPECIFICALLY AND NOT--NOT METHALATE AND EVEN GENERATE A CRYSTAL STRUCTURE OF THE CHROMATIN WITH THIS METHYLATION, SO AS A RESULT OF THIS MODEL THAT'S LOCI, THAT'S LIKELY FOUND BY TRANSGENERATIONAL PROCESS AND THE PiRNA BY THE MOTHER INTO THE OOCYTE LEAD TO RECOGNITION OF [INDISCERNIBLE] BY THE COMPLEX, IT'S PRODUCING METHYLATION OF THIS REGION THAT'S AFTER RIGHT IN THE PROTEIN AND THE SODIUM RETRACTS IN THE PROTEIN BUT TRYING TO FORM THE COMPLEX COUPLE OF THE PROTEINS INCLUDING PROTEINS TYPES IN THE SECOND. SO, THE QUESTION IS WHAT DOES THIS COMPLEX DO, SO HOW DOES THIS IN THE NEXT GENERATION, SO WE SHOW THE COMPLEX FOR THE TRANSCRIPTION OF THE CURSOR, SO IF YOU ELIMINATE, IF YOU MUTATE AND YOU LOSING TRANSCRIPTION OF THE PRECURSORS, AND SO INTERESTINGLY ENOUGH WHEN THEY LOOK BY RNA SEQUENCE, YOU FIND THE PRECURSORS, IN THE LOPING PRECURSOR WITH THE HERE, THEY ARE NONPOLEYINATED, BUT IF YOU--[INDISCERNIBLE] BUT IF YOU ANYTHING DOWN PROTEINS SO IN THIS CASE, YOU GET SOME SHORTER ELATED PRODUCTS AND THEN THEY START TO THINK HOW THE PRECURSORS CAN BE TRANSSCRIBBED BECAUSE THIS REGIONS LIKE THIS PRODUCE THE TRANSPOSEON AND THEY HAVE A LOT OF POTENTIAL FOR THE POLYMERASE SIGNALS AND VERY OFTEN, IF IT WOULD BE NORMAL TRANSCRIPTION AND IT'S POLYMERASE CHPSEQ BY THE WAY BECAUSE WE DO KNOW IF IT'S A GOOD SIGNAL SO WE DECIDE TO LOOK AT INSERTION OF REALLY WELL CHARACTERIZED POLYSIGNALS INSPITE OF RNA CLUSTERS IN THE WILD-TYPE SITUATION AND WHEN WE KNOCK DOWN THE PROTEIN AND WHAT WE FOUND HERE, SO IF YOU ELIMINATE CUFF, YOU GERMINATE THE SIGNAL MUCH BETTER KNOW THAN IN WILD-TYPE SITUATION SAYS AND THE POLYMERASE TEND TO IGNORE THE POLYSIGNAL AND CONTINUE TRANSCRIBING CAN THE WHAT BUT WHEN YOU ELIMINATE THIS PROTEIN IT GERMINATES MUCH BETTER SO WE LOOK MORE CAREFULLY TO LOOK WHAT'S GOING ON AND THE CUP MUCH PROTEIN TO COMPLETELY ARTIFICIALLY [INDISCERNIBLE] THROUGH THIS SITES AND RNA IS RECOGNIZED BY THE PROTEIN SO THIS REPORTER HAS NOTHING TO AND THE CUT OF PROTEIN TO THIS REPORTER AND WHAT YOU FOUND WHAT HAPPENED IN THIS CASE, IT AGAIN WENT TO THE CONONICLE, THE SIGNAL STARTS TO BE IGF NORRED WHEN YOU RECRUIT CUP OF PROTEIN, SO IT'S INCREASED AMOUNT OF TRANSCRIPTION WITH THE SITES AND IT'S ACTUALLY EFFECTS TWO THINGS SO THERE'S A LOT MORE OF THIS TRANSCRIPTION, IT'S NOT THE SITE BUT ALSO THE CLEAVE DRAINS SCRIPT, SO NOW, I HAVE TO BRIEFLY TALK WHAT IS KNOWN ABOUT TRANSCRIPTION GERM NATION BECAUSE IT'S FROM THE MENTAL PROCES, QUITE COMPLEX AND NOT VERY WELL UNDERSTOOD, SO THE TERM NATION CONSISTS OF TWO STEPS AND THE FIRST IS CPS COMPLEX, RECOGNIZE SITES AND DESPITE THAT AND DOESN'T STOP WILL POLYMERASE AND THEN ACTUAL TERM NATION STEPS SHOULD HAPPEN THEN, THERE'S TWO DIFFERENT MODELS WHO AT LEAST WITH ONE MODEL IS THE MODEL OF TERM NATION, WITH THE EXONUCLEASE AND THE CONONICLE [INDISCERNIBLE] FORMED BY THE CLEAVAGE THAT STARTS TO DEGRADE DEGRADE IN FRAGMENT AND THAT LEADS TO TERM NATION OF POLYMERASE. SO NOW WHEN WE DECIDE TO LOOKA BACK--AT WHAT IS IMPORTANT HERE, THE CPSS, COMPLEX, NONCONONICAL END WITH THE SUBJECT OF SUBSTRATE FOR DEGRADATION. WE DECIDE WHAT'S GOING ON IN REAL CLUSTERING AND WE FOUND THIS REGION FULL OF THESE NONCANNONICLE AND SO WE CAN DO RNASEQ THAT'S SELECT, BUT THESE ARE MONOPHOSPHATE ENDS AND IT INCORPORATES THE NORMAL SO THIS IS--THIS MONOPHOSPHATE. SO IT'S POTENTIALLY SHOULD BE [INDISCERNIBLE] FOR DEGRADATION BY RAT ONE, AND SO WE LOOK AT HOW RAT ONE UPREGULATES EXPRESSION AND SO IT REPEAT THIS IS CLONING OF RNA SEGMENTS LOOKING SPECIFICALLY AT MONOPHOSPHATE AND THE PRESENCE OF WHEN WE KNOCK DOWN THE EXONUCLEASE AND KNOCK DOWN COUGH PROTEIN AND THEY HAVE OPPOSITE EFFECT. WHEN YOU KNOCK DOWN RAT ONE, YOU INCREASE MONOPHOSPHATES AND THE CLUSTERS AND YOU KNOCK DOWN [INDISCERNIBLE] YOU DECREASE AMOUNT. SO SUGGESTS THAT RAT ONE DEGRADES STRENGTHS AND THIS CUTS OPEN THE EFFECT AND THEN YOU COMBINE MUTATIONS AND CUT OFF MUTATION SO WHEN YOU JUST MUTATE COUPLE BECAUSE THE WHOLE RNA BY CHANCE, THIS COLLAPSE, HAVE YOU STRONG TRANSPOSEON COLLABORATION, SO THEY INCREASE LIKE TWO HELPED FOLD WHEN YOU KNOCK DOWN, BUT NOW IF YOU COMBINE WITH THIS RAT ONE KNOCK DOWN, FAYE FIT MUCH WEAKER, SO THIS CUT OFF PROTECTS WITH [INDISCERNIBLE] FROM DEGRADATION BY RAT ONE. SO COMBINING THESE THINGS ALTOGETHER IN THE MODEL WHAT WE BELIEVE GOING ON, THIS IS CLUSTERS, THE CLUSTER SYSTEM TRANSCRIBED SO IT'S AFTERWARDS HAVE TO BE PROCESS TO MATURE PYRUVATE, SO THE NEW TRANSPOSEON CARRY THE POLYSIGNALING. AND IN FACT, YOU EXPECT TO BE CONSIDER PROPER POLICING SO BY POLYMERASE AND ELIMINATE INARM OLDER PEOPLE RNA PRODUCTION, SO THE PRESENCE OF CUT OFFOT REGION, IT'S AGAIN THROUGH THE METHYLATION MARK AND THE PROTEIN, PREVENTS TWO THINGS, IT'S SO PREVEN CLEAVAGE AND PREVENT CLEAVEERAGE BUT IF CLEAVAGE HAPPENS, IT SOMEHOW PROTECT THIS TRANSCRIPT FROM DEGRADATION BY RAT ONE NUCLEACE. AND SO WHAT I DESCRIBE HERE WAS THE FUNCTION OF THIS COMPLEX BUT I DON'T QUITE EXPLAIN HOW THIS DIFFERENTIATED FROM OTHER TRANSCRIPTION IN THE CELL, AND MORE RECEIPTLY THEY ALSO FOUND THAT IT'S NOT ONLY METHOD FOR TRNSCRIPTION BUT ALSO ALLOW THE LOAD OF THIS CONSORT RNA BINDING, ON THE PRECURSOR TRANSCRIPT AND MAYBE THIS IS WHAT ACTUALLY FOR SPECIFICATION OF THE TRANSCRIPT, AND THIS IS VERY INTERESTING PATHWAY, CHROMA SIN MARK, BINDING OF CHROMATIN, BUT EITHER LIKE RIDING, BUT ALSO METHOD FOR LOADING UP RNA BINDING PROTEIN ON RNA. SO THIS IS RATHER SPECIFIC PATHWAY, BUT MORE IF WE PRESENT IT IN MORE ABSTRACT WAY THIS IS INTERESTING BECAUSE WE SUGGEST THAT THE STATEUS SUGGESTS THE CHROME - TIN MARK CAN RNA BINDING PROTEIN, NOT JUST TRANSCRIPTION SIZE AND THAT'S WHAT HELPED REALLY LONG QUESTION IMPLICATION ON THE LIFE OF RNA. SO HENS THE CONCLUSION HERE IS THAT IN THE CASE OF PiRNA SYSTEM, NOT CHROMATIN CAN PROVIDE REAL SIGNALING THROUGH GENERATION. SO IT'S INTERESTING THAT BOTH [INDISCERNIBLE] IN THIS PROCESS BUT WHAT ACTUALLY TRANSMIT SIDE ONE CODE OF RNA, BUT AFTERWARDS CHANGE THIS CHROMATIN MARK, SO THEN WE IDENTIFY THE TRANSCRIPTIONAL CONTROL, THAT'S UNLIKE MOST OF OTHER TRANSKRIMS AND MAKE IT WORK ON THE LEVEL OF GERM NATION AND MULTIGENERATION OF TRANSCRIPTION, SO THE PROPOSE OF THE STRUCTURE IS POST TRANSCRIPTIONAL STRUCTURE AND THE GIDDING THE BINDING PROTEINS, AND SO HERE I'M GOING TO STOP, SHOW THE PEOPLE INVOLVED IN WORK, I OUR COLLABORATORS ESPECIALLY NIH, INNOVA FOR SUPPORT. THANK YOU. [ APPLAUSE ] >> SO IN THE GENOME THERE ARE A LOT OF REGIONS THAT HAVE GENOME BINDING REGIONS BUT COOPERATED -DON'T HAVE CAD WHAT BYPASSING? >> YES VERY GOOD QUESTION, SO ESSENTIALLY IT'S PROBABLY METHYLATION WAS NOT ENOUGH FOR THE PROTEIN, SO IN THE MODEL THAT I SHOW YOU, SO YOU CAN GO BACK A LITTLE BIT, SO I CANNOT GO BACK, IS ABOUT THAT SO WHAT WE PROPOSE, IT'S A PROTEIN RECOGNIZING THE NASCENT--YEAH, SO IT RECOGNIZE TED NASCENT TRANSCRIPTING FOR THE [INDISCERNIBLE] METHYLATION MARK AND THAT WILL HAPPEN ONLY ON SPECIAL REGION SO MAYBE WHAT WE CAN PROPOSE WHILE WE DON'T HAVE EVIDENCE NOW BUT THAT IS HYPOTHESIS AND WE CAN EXPLORE IT, BUT AT THE SAME TIME THIS PROCESS [INDISCERNIBLE] RIGHT AWAY, BECAUSE [INDISCERNIBLE] ALREADY BROUGHT EARLY STEP BY STEP BY PIWI, SO SEVERAL HP-ONE PROTEINS SO THEY ALSO HAVE SAME BINDING TO [INDISCERNIBLE] METHLAGS LACE PEPTIDES INVITRO, THE IMAGES ARE DIFFERENT BUT WHY IN VIVO THAT DON'T BIND TO SAME SITES IS NOT QUITE UNDERSTOOD. >> ANOTHER QUESTION, IS, DO YOU TRY TO OVEREXPRESS THE RAG ONE FOCUSED ON SEE WHETHER IT HAS PHENOTYPES FOR THE TRANSPOSER OF ACTIVATION OF LY FOR [INDISCERNIBLE] WHATEVER. >> SO WHAT WE DID SO FAR IS WHAT I SHOW. WE TRY TO COMBINE RAG ONE DOWN [INDISCERNIBLE] EFFECT, BUT WE DON'T EXPECT MUCH FROM OVEREXPRESSION BECAUSE IT CAN'T PROTECT IT WELL ENOUGH THAT OVEREXPRESSION IS PROBABLY NOT GOING TO CHANGE MUCH. >> THANK YOU. >> I WAS WONDERING, I WAS WONDERING IF YOU KNEW, IF YOUR DROSOPHILA HAD [INDISCERNIBLE] IN THEM AND IF YOU THOUGHT ABOUT ANY DIFFERENCES BETWEEN LIKE WITH THE PATHWAY AND THE ABSENCE O [INDISCERNIBLE]. LET. >> SO WE DON'T KNOW. I MEAN OBVIOUSLY WE USE ALSO DFFERENT STRAINS LIKE WHEN WE KNOCK DOWN, WE USE DIFFERENT SHRNA STRAINS AND [INDISCERNIBLE] I KNOW FROM OTHER PEOPLE THAT EVEN SOURCE OF [INDISCERNIBLE] PATHWAY, MIGHT BE EFFECTED BY INFECTION, BY [INDISCERNIBLE], SO WE DO KNOW WHAT TYPE IF IT MIGHT PLAY A ROLE IN THE PATHWAY. >> OKAY. >> THANK YOU ALEXI. >> OUR NEXT SPEAKER IS HAIFAN LIN, HE WILL TALK ABOUT APOPTOSIS DURING FETAL DEVELOPMENT IN EELIMINATING CLONEALLY RELATED GERM CELLS. ? SO TODAY I WOULD WOULD LIKE TO USE A POST DOC'S WORK TO ILLUSTRATE TO YOU THE PROGRESS WE HAVE MADE WITH THIS AWARD. THIS IS THE MECHANISM THAT UNITES OF THE GENOME IS MADE OF 23,000 DIFFERENT GENES AND OVER A MILLION TRANSPOSE ON, SO MOST OF THESE TRANSPOSEONS, THEY CAN PRODUCE TRANSPOSEON RNAs AND TRANSPOSEONS ARE STILL GENERALLY VIEWED AS SELFISH PARASITES. THEY ARE ACTIVE FUNCTIONING OF T, AND THE INTERGENIC REGIONS THERE ARE ALSO MANY KINDS OF NONCODING ANTIGENS NOW ENCODES FOR THOUSANDS AND LINK RNAs AND FINALLY, WE SHALL REMEMBER THAT IN OUR GENOME, THERE ARE ALSO A SOLVENT OF THE GENES WHICH ARE OFTEN TRANSCRIBED BUT NOT TRANSLATED SO DESPITE THE CONSPICUOUS OF THESE MAJOR CONSTITUENTS OF THE GENOME, THE FUNCTION OF THE REGULATORY RELATIONSHIP AMONG THEM REMAINS VERY LITTLE IS KNOWN FOR EXAMPLE, [INDISCERNIBLE] ARE KNOWN TO BE DIFFERENT [INDISCERNIBLE] OF [INDISCERNIBLE], AND WE DON'T KNOW ACTIVE FUNCTION BEING PRESCRIBED. SO TODAY I LIKE TO TELL YOU THAT ACTUALLY TRANSPOSEONS PLAY AN ACTIVE ROLE TO REGULATE EXPRESSION OF MANY PROTEIN CODING GENES AND IN ADDITION LIKE TO SHOW YOU THAT PSEUDOJEANS ALSO PLAY A VERY IMPORTANT ROLE IN REG RULING THE EXPRESSION OF THE COGNATE GENES AT THE POST TRANSCRIPTIONAL LEVEL. FURTHER MORE I SHOW THAT TRANSPOSEONS PLAY AN ACTIVE ROLE IN REGULATING THE EXPRESSION OF THE [INDISCERNIBLE] AND FINALLY I LIKE TO ILLUSTRATE TO YOU THAT ALL OF THESE ARE THROUGH THE [INDISCERNIBLE] PATHWAY. SO THESE FINDINGS STARTED WITH THIS PROTEIN THAT ALEXY MENTIONED TO THE ARGON AUGHT PROTEIN FAMILY DISCOVERED SELF-YEARS AGO, AND THESE ARE BINDING TO SMALL RNAs AS WELLA IN THE C-TERMINAL, THE GENE FAMILY CAN BE DIVIDED INTO THE AND AS YOU ALL KNOW THE PROTEINS BIND TO SIRNAs AND MICRORNAs AND PLAY A CENTRAL ROLE IN THE SiRNA AND THE MICRORIN NA PATHWAYS WHERE AS PIWI, PROTEINS BIND TO YET ANOTHER CLASS OF CODING RNAs AND SO THAT ALEXY JUST TALKED TO AND THE OTHER LABS DISCOVERED SEVERAL YEARS AGO, SO JUST TO BRIEFLY INTERVIEW, MORE ABOUT THE PiRNAs AND MUCH MORE COMPLEX THAN MIRNs SO IN MY LAB ALONE, WE HAVE 1000 SPECIES AND AGAIN THIS IS SOME SHARP CONTRAST TO MICRO RNAs WHICH CORRESPOND TO THE UTR OF MATURE MESSENGER RNAS AND THIS IS PRETTY AMAZING TOO, AS ALEXY ALLUDED TO, THEY HAVE LONG STRAPPEDDED PRECURSORS IN CONTRAST TO MicroRNAs OR SiRNAs AND TO ILLUSTRATE THIS POINT, THIS SLIDE SHOWS YOU A MAJOR PiRNA GENERATING LOCI WITH THE MOUSE, INDICATING A PROPOSED NUMBER OF THE INDIVIDUAL PiRNA THAT ARE PRODUCE FRIDAY THE THAT LOCUST, AND IT MEANS POST [INDISCERNIBLE], OF DNAs CAN BE DESCRIBED INTO PiRNAs AND ON CHROMOSOME 17 INTO THIS CLUSTER, AND 17-ONE, CAN YOU SEE IT'S A GENIC REGION WHICH ARE SINGLE STRANDED RNAs ON INTO THOUSANDS OF RNAs AND WE OFTEN THALEGION THERE ARE REPETITIVE SEQUENCES SO CAN YOU IMAGINE THAT MANY OF THESE RESULTING PYRUVATES CAN ACT IN MANY OTHER SITES IN THE GENOME. SO THIS REALLY WAS EXCITING TO US BECAUSE AS YOU ALL KNOW THAT THE MOLECULE BIOLOGY AS A FIELD OF STUDY HAS BEEN FOCUSED IN THE PAST SIX DECADES THAT ARE GENETIC INFORMATION DNA IS TRANSCRIBED, WHICH HAVE BEEN CHOSEN INTO PROTEIN WHICH IS LEADS TO LIFE PROCESSES. THE DISCOVERY OF THE MICRORNA AND THE SIRNAs PATHWAYS FOR THE [INDISCERNIBLE] DOGMA FOR ILLUSTRATING THAT THERE ARE TWO MORE, SLOW AND IMMEDIATEIATE PATHWAY WHICH REGULATE THE STABILITY AND MESSENGER RNAs, SO HOWEVER, AS I SHOWED IN THE LAST SLIGHT, THE PiRNAs GO FROM THE REGIONS AND SO THIS IS REALLY EXCITING TO US, BUT THE KEY QUESTION IS WHAT'S A FUNCTION OF THIS PiRNAs, AND I WOULD LIKE TO SHOW YOU ANOTHER NOVEL FUNCTION OF THESE PiRNAs IN ADDITION TO WHAT ALEXI TOLD YOU SO A FELLOW IN MY LAB, HE DECIDED TO FUNCTION THE FUNCTIONS IN THE MAMMALIAN SYSTEM IN THE MUTAGEN SIS AND IN THE MOUSE GENOME THERE ARE THREE [INDISCERNIBLE], THE FIRST ONE ENCODES NEARLY TWO PROTEINS THAT EXPRESSED DURING THE STEM CELL STAGE AND THE SECOND ONE CALLED THE [INDISCERNIBLE], IS DISPURSED FROM THE COLONIAL STEM CELL ALL THE WAY TO [INDISCERNIBLE] SPERMAT O SIGHTS THEN ENTER MEIOSIS, AND THE THIRD ONE IS TO THE SPERM FOR THE EARLY PRODUCT OF MARKETS EIOSIS, THERE ARE TWO POPULATIONS OF PiRNAs AND THEY'RE MOSTLY EXPRESSED IN THE GERMINAL STEM CELLS AND THE IMMEDIATE DAUGHTER CELLS, THE SECOND POPULATION CALLED THE [INDISCERNIBLE] ARE MOSTLY EXPRESSED SO WHEN WE FIRST CLONED THE P A CHYTENE, PiRNAs ONLY FOUR% TARGET TRANSPOSEONS, WHICH IS THE BEST FUNCTION FOR THE PATHWAY. PATHWAY. SO WE THEN--BECAUSE THE KNOCK OUT EXPRESSION OF MOST PiRNAs DURING THIS STAGE OF THE MUTE O GONE SIS AND IT HAS A CLEAR DEFECT IN THE EXIT WHICH GIVE US A BIOLOGICAL STUDY, SO GIB THAT THE PROTEIN IS A CYTOPROTEIN, THAT START TO EXPRESS IN THIS STAGE, OF THE PERM SPERMAT O SIGHTS, IF WE HAVE ANY FUNCTION IT COULD N IT COULD BE TRANSQUESTIONABLE. FELT AND TOKER SURPRISE WE FOUND THAT OVER 400 MRNAs WERE UPREGULATED AND THESE ARE REQUIRED NORMALLY TO REQUIRE SUPPRESSION OF THESE MRNAs. THIS IS THE KEY TO THE PATHWAY WHICH ALLOW FURTHER TO FURTHER COME THROUGH THAT AND MAY BE INED IN THE OVEREXPRESSION, SO TO CONFIRM THAT WOULD WE KNOCK OUT ANOTHER COMPONENT OF THE SPERMATOCYTES IT SHOWS A SIMILAR DEFECT. SO, NOW, SINCE [INDISCERNIBLE] AND THE [INDISCERNIBLE] COMPONENT INVOLVE THAT, THEN WE SUSPECT FOR THIS MESSENGER RNA, UPREGULATED IN THE MUTANT, THEY MUST COMPARE THIS TARGET SITE AND THAT IS THE CASE AS CAN YOU SEE, IF YOU PROFILE THE TRANSCRIPT OHM IN THESE MIRROR IMAGIC CELLS ONLY THESE ARE UPREGULATED IN THE PIWI MUTANT IN THE MEMBER OF THE TARGET SITES. NOW IF YOU'RE LOOKING TO THE NATURE OF THESE TARGET SITES, IT'S GOOD TO KNOW THAT THESE TARGET SITES ARE DERIVED FROM TRANSPOSON SEQUENCES SO WE CAN CONCLUDE THAT THE MRNA IS REGULATED BY THAT ARE DERIVED FROM TRANSPOSONNINGS. SO NOW THE QUESTION BECOMES WHERE ARE THESE TRANSPOSE OWN SEQUENCES HIDING IN THESE MRNA, WHY HAVE PEOPLE BEEN STUDYING TRANSCRIPT OHM AND NOT FOUND THESE SEQUENCES BEFORE? SO WHEN WE CAREFULLY PROFILED OUR TRANSCRIPT OHM, WE FOUND OUT THAT IN MANY MRNAs, THE PREFRIEND UTR, TRANSPOSON BITS AND PIECES, AND WHEN THE MRNA IS MORE UPREGULATED THE MORE SUCH SEQUENCES ARE THERE IN THE [INDISCERNIBLE] UTR, WE THEN WONDER WHAT THE TRANSPOSE AN SEQUENCES AND MORE 50% OF THE SEQUENCES ARE DERIVED FROM FIND ELEMENT AND THEREY A FAIR SHARE OF CONTRIBUTION FROM OTHER TYPES OF TRANSPOSONS. SO WE THEN WONDERED THEN, DOES THE MRNA ACTUALLY OCCUR AT THIS TARGET SITE. TO JUST THIS QUESTION, WE SELECTED SEVERAL TYPICAL TARGET AND ANALYZED THEIR DEGRADATION OR EXPRESSION PROFILE DEEP SEQUENCING, FOR EXAMPLE THIS, IS MRNA ENCODE BY THE TDRD ONE GENE, WITH THE SHORT FIVE POINT AND UTI HERE, THE MEETING FRAME HERE AND THE RELATIVELY LONG UTI HERE AND YOU CAN SEE THAT THERE ARE TWO TRANSPOSON ELEMENTS IN THE PRIME UTR THAT BELONG TO THE EARL MENTORSHIP SKILL, ONE CALLED D-ONE AND ONE CALLED D-TWO. WHEN WE DID THE DEEP SEQUENCING OF THE MIDDLE SITES ISOLATED FROM MUTANT, WE FOUND THAT INDEED, AS OF THESE TARGET SITES FOR EXAMPLE, BINDING SITE HERE FOR DONE OR DTWO, ANOTHER REGION I DIDN'T SHOW YOU, THERE'S A CLIFF OF ABUNDANCE WHICH SUGGESTED TO US THAT THE EXTRA INVOLVED IN DEGRADING THIS MRNA AT THE TARGET SITE AND IT'S ABOUT 50% OF THE TOTAL ABOPPEDDANCE OF THE PARTICULAR SPECIES OF MRNA, WHICH SUGGEST TO US EITHER THIS DEGRADATION IS DOSE DEPENDENT ON ME, AND ANOTHER INDEPENDENT MECHANISM THAT TAKE CARE OF THE REST OF DEGRADATION, BUT IN ANY CASE, IF THE PIWI ACTIVITY IS INVOLVED IN THE DEGRADATION AS I SHOWED YOU EARLIER IN THE SLIDE, THESE PROTEINS HAVE RNA, AND IF WE REGULATE THESE GENES OR PROTEINS, DOWN IN VIVO, CAN YOU SEE THE DEGRADATION IS NOT ABOLISHED IF WE OF WHICH INDEED IS THE CASE. SO THESE ANALYSIS ALLOW US TO CONCLUDE THAT THE [INDISCERNIBLE] BINDING TO ITS TARGET MESSENGER RNAs TO THE DEGRADATION OF THE TARGET SITE. WHENEE LINE UP ALL THE [INDISCERNIBLE], THE TARGETING MESSENGER RNAs IN THE CELLS, IT'S GOOD TO KNOW THAT THE DEGRADATION SITE 10 NUCLEOTIDES AWAY FROM THE FIVE PRIME END OF RNA, SO THIS LED US TO FIND A NEW MECHANISM WHICH IS MEDIATED BY COMPLEX AND DEGREATS THE TARGET MESSERG RNA BY THE COMPLETE SEQUENCE COMPUE MENTALITY OF THE PIRNA, IF THIS MODEL IS CORRECT, IT WILL EXPECT THAT IF YOU GENERATE THE TRANSGENIC MICE REPORTER MRNA, COMPENTINE REGIMENNING THE PROTEIN HERE AND IF WE INSERT THE UTR, NOW WE CAN ALSO GENERATE THE SIBLING MOUSE LINE IN WHICH THIS SEQUENCE CAN BE ELIMINATE BOOED THE CREE RECOMBINATION, WITH THESE TWO MODELS WE CAN ASK THE QUESTION, WILL,A SIGNED ELEMENT INDUCE--REDUCE EVERYEXPRESS OF THE TARGET MRNA, IN SPERMATOCYTES IF SO, CAN WE DEGRADE THE MRNA. SO THIS IS RESULT. THIS IS THE MRNA FOR THE PENDING ELEMENT AND HERE'S IDENTICAL MRNA, AND IT'S [INDISCERNIBLE]. NOW WHEN YOU'RE LOOKING TO THE PREMRNA, LEVEL FOR THESE TWO MRNAs, THEY ARE VERY SIMILAR OR IDENTICAL. NOW IF YOU LOOK INTO THE MATURE MRNA, YOU CAN SEE ONCE THE ELEMENT IS REMOVED THE MATURE BECOMES SIGNIFICANT OVER EXPRESSED, WHICH SUGGESTS THAT INSIDE, THIS ELEMENT IS MEDIATING THE DEGRADATION OF THIS MRNA, AND SHOWS THAT ALSO INVOLVE THANKSGIVING PROCESS BECAUSE NOW, IF YOU INTRODUCE THIS TRANSGENE WHICH STILL COMPARE THE ELEMENT INTO THE M IWI MICE, CAN YOU SEE IT'S AGAIN OVEREXPRESSED. SO THIS ANALYSIS, HAVE ALSO FOUND IN VIVO TO THE INDIGENOUS GENES AND WE DELETE NOT THE MULTIPLE ENDOGENOUS GENES AND THE ELEMENT OF UTI WHICH LEADS TO SIMILAR RESULTS. SO THIS ANALYSIS ALLOWS US TO CONCLUDE THAT THE TRANSPOSONS PLAY AN ACTIVE ROLE IN EGGULATING THE EXPRESSION OF--REGULATIN THE EXPRESSION OF THE PiRNA, AND MEAN WHILE WE NICE THAD SOME MRNAs THAT ARE UPREGULATED DO NOT CHANGE TRANSPOSON SEQUENCES SO THEN THE QUESTION BECOMES WHAT PiRNA SEQUEENS IS THERE TO REGULATE THE DEGRADATION FOR EXAMPLE, THIS IS THE STEM PT MRNA, WHICH IS FIVE POINT UTR HERE AND THREE POINT UTR HERE YOU CAN SEE WE CAN MAP OF THESE INTO THIS TRANSCRIPT. HOWEVER THIS IS NOT LOCALIZED, SECONDARILY ARE THEY ARE NOT TRANSPOSED. TSE ARE ACTUALLY DERIVED FROM THE PSEUDOGENE OF THE PG GENE THAT WE CALL THE SSTEM PSEUDOSEQUENCE, NOW IF THIS IS THE CASE, ONE SHOULD BE ABLE TO PREDICT THAT IF YOU KNOCK DOWN THE EXPRESSION OF THE PSEUDOGENE, YOU SHOULD HAVE THIS EXPRESSION, AND KNOCK OUT THE STEM BP EXPRESSION, THESE ARE REDUCED TO BACK ONE LEVEL AND TO CONCLUDE OR PREDICT THAT THE EXPRESSION OF ITS COGNITIVE--MRNA, SHOULD BE GREATER INCREASED AND THAT INDEED IS THE CASE. SOPHISTICATEDY WHEN YOU KNOCK OUT THE PSEUDOGENE, LEADING TO SUGGESTING OF THE COGNIZANT MRNAs AND THIS OVEREXPRESS GAGES IS WITH THE MATURE MRNA LEVEL BECAUSE WHEN YOU LOOK INTO THE PRECURSOR RNA AND EFFECTS RNA FOR STEM, THERE'S NO CHANGE OR INCREASE. SO THIS TYPE OF ANALYSIS ALLOWED US TO FURTHER CONCLUDE THAT MANY SUDO GENES PLAY AN IMPORTANT ROLE IN REGULATING THE EXPRESSION OF THEIR COGNITIVE MRNAs VIA THE PATHWAY. SO ENCOURAGED BY THESE RESULTS WE FURTHER WANTED TO KNOW IF THE PiRNA MEDIATE THE DEGRADATION OF THE LNCRNA, SO THE PROFILING OF LNCRNA IN DIFFERENT TISSUES IN THE MOUSE. AS YOU CAN SEE BONE MARROW, ESL, LORCHGS OF LINCRNA, AND IF YOU ZOOM INTO THE LNCRNA, COME IS SPECIFICALLY EXPRESSING SPERMATOCYTES, THERE ARE ALL OVER 6000 SUCH LNC RNAs WHICH WE CALL THE METAL RNAs AND WE THEN PROFILE THE EXPRESSION OF THESE LNC RNAs FROM THE WILD-TYPE VERSES THE MUTE ANTS TO SEE WHETHER THE EXPRESSION IS IMPACTED BY THE MUTANT AND TO OUR SURPRISE, MORE THAN 25%, MORE THAN 1500 LNC RNAs ARE SIGNIFICANTLY UPREGULATED FOR THE SPERMAT O CITE, WHICH ALLOW US TO CONCLUDE THAT INDEED THE LNC RNA, AND TRANSPOSON SEQUENCE SITE, BEING THE AND SO NOW WE SHOWED THAT TRANSPOSON SEQUENCES CAN REGULATE EXPRESSION OF LARGE NUMBER OF LINCRNA DURING PATHWAY, SO TO SUMMARIZE MY TALK WHAT I'VE SHOWN YOU TODAY S&P WHAT WE NORMALLY CONSIDER EXPRESSION OF THE GENE, WE THINK ABOUT THE TRANSCRIPTION OF MRNA AND THEN RECOLLECT EVENTS BECOME MATURE MRNA. NOW WE NEED TO BE COGNIZANT OF THE FACT THAT TRANSPOSONS ALSO TRANSCRIBE LOW LEVEL AND THEY WILL GENERATE THE TRANSPOSONS FOR THE [INDISCERNIBLE] WHICH TOGETHER WE HAVE PIWI PROTEINS BIND THE UTR AND TARGETING THE MRs TO INGULATE THE EXPRESSION. AND WE SHOULD ALSO BE COGNIZANT OF THE FACT THAT TWO OF THE JEERPS ARE IN THERE AND TO GENERATE THESE PIWI AND THESE AGAIN TARGET THE CORRESPONDING MRNA TO REGULATE THE EXPRESSION. LUCKILY THAT'S ALSO THE CASE FOR THE REGULATION OF LINC RNA WHERE T TRANSPOSON TRANSCRIBE AND WROTE IN REGULATING THEIR EXPRESSION. SO IF YOU LIKEN THE GENOME AS IT WERE, THEN WE SHALL REMEMBER THAT THE TRADITIONAL GENES ARE REALLY THE OLD WORLD AND WE ALL ALSO REMEMBER THAT IN THE OLD WORLD THERE ARE ALSO SUPERGENES AND TRANSPOSE ON THAT PEOPLE DISCOVERED MANY YEARS AGO. SO SEVERAL YEARS AGO, YOU KNOW MY LAB AND OTHERS FOUND ALSO A NEW WORLD OF GENES, SO TODAY I HOPE I HAVE SHOWN YOU THAT THESE TWO WORLDS ARE CONNECTED. PiRNAYS CAN REGULATE TRADITIONAL GENES AND LNC RNAGENES CAN REGULATE SEED O GENES AND TRANSPOSONS AND THEY CAN ALSO LINK TRADITIONAL GENES AND MOST OF THEM WERE SUPPORTED BY THE COLLEAGUES IN MY LAB. I WOULD WOULD LIKE TO THANK THE NIH FOR THIS WONDERFUL AWARD WHICH ALLOWED US TO DO THIS WORK OTHERWISE IT WOULD BE IMPOSSIBLE TO ACHIEVE AND I HOPE THIS AWARD WILL CONTINUE TO SUPPORT MANY MANY EXCITING DISCOVERIES. THANK YOU. [ APPLAUSE ] >> TIME FOR A FEW QUESTIONS. >> YEAH, THAT'S GREAT WORK. I HAVE A QUESTION ABOUT THE [INDISCERNIBLE] PATHWAY THAT YOU'RE-- >> WHAT PATHWAY. >> SO THERE'S A BATH WAY CALLED [INDISCERNIBLE] WORKS AS FOR AS I KNOW IN SOMATIC CELLS NOT ONLY PRODUCTIVE CELLS ALSO AS FAR I KNOW IT DOESN'T INVOLVE PiRNABUT IT DOES INVOLVE [INDISCERNIBLE]. THE AND THERE ARE COMMONALITIES, ARE, IN,A, BINDING PROTEIN IN COMMON BETWEEN THE [ISCERNIBLE] MEDIATED MRNA DEGRADATION GRATES--GRATUE DITION AND THE MIWI PiRNA MEDIATED DEGRADATION. >> THAT'S A GOOD QUESTION,--WE HAVE NOT DONE THAT WORK AND I WOULD NOT BE SURPRISED IF THE PATHWAY IS INVOLVED, ACTUALLY THE CELLS FEATURE DOMATIC CELLS IS QUITE ROBUST. A GENETIC HAVE BEEN IMPLICATED IN THE PANIC, AND TISSUE FOR OTHER PEOPLE. >> SOMEONE WESTBOUND THING REAL QUICK, I LIKE IT TALK BUT THEN THE MRNA HAVE TO BE COLLECTED AND MAYBE 1 MILLION PiRNAs BUT MANY COLLAPSE BECAUSE THEY'RE ALL THE SAME SEQUENCE [INDISCERNIBLE]. AND MULTIPLE LOCI AND COMING FROM THE LNCRA, LNC ARE NOT CLNAASKS YOU'RE TALKING ABOUT L[INDISCERNIBLE] >> AND ACTUALLY PiRNAASKS MAY BE OTHER FUNCTIONS, THAT TAKES A LOT OF PROPORTION FOR PiRNAs AND WE'VE SHOWN THAT THIS IS VERY IMPORTANT AT THE GENETIC PROGRAMMING AND THESE ARE ONLY-ACTUALLY A NEW FUNCTION WHEN YOU SEE PIWI PROTEIN ON IN THE CYTOPLASM. >> YEAH, MY LAST POINT SO THIS IS TRANSCRIPTION. >> POST TRANSCRIPTION ON, IT'S ONLY THE SET OF [INDISCERNIBLE]. >> SO I HAVE A COUPLE OF QUICK QUESTIONS, ARE THERE ANY CLASSICAL FUNCTIONS THAT ARE UPREGULATED AND WE-- >> WE DID GO ANALYSIS, THEY ARE ENRICHED CAN THE WHAT. >> AND ALSO DO YOU SEE ANY CHANGES IN DNA METHYLATION AT ANY LOCI? >> WE ARE WORKING ON THAT YES. >> OKAY. >> THANKS. >> I HAVE A QUESTION, IT'S, YOU KNOW IT'S WHAT YOU'RE DESCRIBE SUGGEST AN UNDERLYING MODE OF REGULATION, THAT'S GENOME PROTECTION BUT IT'S--REGULATORY, SO WHAT IS IT? IS IT SOMETHING FUNDAMENTAL OF THE PIRNA PATHWAY THAT RESTRICTS IT TO THE GERM LINEAGES BECAUSE LIFE FINDS A WAY OF TAKING APPARATUS AND USING THEM TO THEIR ADVANTAGE IF A VARIETY OF CELLS. >> WELL, ANSWER THANKSGIVING QUICKLY AND PHILOSOPHEICALLY AND WITH THE GERM LINE CENTRIC, AND REALLY IT'S THE MOST IMPORTANT TISSUE TO PROTECT FOR EXAMPLE, ALEXY SAID THE PROTECTING AS A GENETIC IMMUNE SYSTEM AND PRACTICALLING AND NOW FOR PROTECTING OVEREXPRESSION OF THOSE GENES AND A LOT OF THESE REGULATIONS NEED TO BE HIGHER UP AND MECHANISM WHICH MIGHT BE UNIQUE TO THIS LINE QUICKLY FOR THE LINC RNA, RIGHT AT ENDO MEIOSIS, AND REPLACING PINGS AND DURING THAT, THE GENOME IS WAY OPENED AND MY SUSPICION IS A LOT OF THAT MIGHT BE [INDISCERNIBLE] TRANSCRIBED, AND NOT TAKEN CARE OF THAT DEVELOPMENT WE WILL NOT PROCEED, THIS YOU CAN SEE IN REGULAR SOMATIC CELLS, THANK YOU. >> THANK YOU. >> [ APPLAUSE ] >> OUR NEXT SPEAKER IS DIANNA LAIRD, AND SHE WILL TALK ABOUT APOPTOSIS DURING FETAL DEVELOPMENT ELIMINATES CLONEALLY RELATED GERM CELLS. >> ALL RIGHT WE WILL TALK ABOUT APOPTOSIS DURING FETAL DEVELOPMENT ELIMINATES CLONEALLY RELATED GERM CELLS. SO THE BROAD QUESTION I'M INTERESTED IN AND HOW THE INFORMATION IS TRANSFERRED BETWEEN GENERATIONS AS SHOWN BY THIS--HOW THE PROCESS OF EMBRYONIC DEVELOPMENT SHAPES THE DEVELOPMENT OF THESE GERM CELLS AND SHAPES THE PULL OF GAMETES THAT ARE AVAILABLE, SO SPECIFICALLY WE'RE INTERESTED IN WHETHER THERE ARE MECHANISMS DURING THIS DEVELOPMENT WHICH ACT AS QUALITY CONTROL MECHANISMS IN THE GERM LINE AND THIS REALLY HAS BEARING NOT ONLY ON FERTILITY WHICH IS IMPORTANT TO MANY OF US, BUT ALSO THE PROCESS OF EVOLUTION ITSELF AND THE TRANSMITTANCE OF DISEASE BETWEEN GENERATIONS AND AND WHICH ARE VERY SIMILAR TO HUMANS WE MOSTLY THINK THE DEVELOPMENT BEGINS WITH FERTILIZATION WITH THE FUSION OF THE GAMETES, THE EGG AND SPERM AND SHORTLY AFTER THE FUSION OF THIS, FIVE AND HALF DAYS IN MICE AND ABOUT APPROXIMATELY FIVE WEEKS IN HUMANS, WE SEE THE REAPPEARANCE OF THE GERM LINE WHICH IS SPECIFIED AT THE SMALL NUMBER OF STEM CELLS, FROM THE EPIBLASTS AND THESE GERM LINE STEM CELLS MIGRATE, THEY PROLIFERATE WHILE THEY'RE MIGRATING AND THEY CONVERGE UPON THE RIDGES AT THE STATION IN MICE AND HUMANS AND DEVELOPMENT OF THE GERM CELLS OCCURS SIMILARLY IN THESE DIPLOID CELLS IN MALE AND FEMALE UNTIL [INDISCERNIBLE] AFTER WHICH POINT THE DIFFERENTIATION OF THE FETAL GONADS, OVARIES AND INTESTIS INDUCES DEFERENTIATION OF THE STEM CELLS, SO HERE WE SEE DIFFERENTIATION OF THE DIFFERENTIATION OF THE OOCYTES BEGINS WITH THE ENTRY INTO MEIOSIS ONE AND ARRESTS DURING MIRROR IMAGEOTIC PROPHASE WRASSE IN THE MALES THERE'S A BIFORT OF PROLIFERATION FOLLOWED BY MITOTIC ARREST AND SPERMAT O GENESIS RESUMES FROM THE STEM CELLS IN THE POSTNATAL PERIOD. NOW A REALLY NOTABLE FEATURE OF GERM CELL DEVELOPMENT IS THAT IT'S WASTABLE AND IT'S KNOWN IN THE FEMALE GERM LINE THAT SOMETHING AROUND 90% OF THE GERM CELLS THAT ARE PRODUCED ARE ACTUALLY GOING TO DIE. LET THIS IS A LITTLE BIT HARDER TO GET A HANDLE ON IN THE MALE BECAUSE THERE'S SO MUCH PROLIFERATION IN THE POSTNATAL PERIOD BUT SUFFICE FIES IT TO SAY THAT MALE AND FETAL COMPARTMENTINALLY THERE ARE WHAT APPEAR TO BE SPECIFIC WINDOWS OF PROGRAM CELL DEATH. ONE IN THE FETAL PERIOD WHICH SENTORS APPROXIMATELY AROUND 13.5 IN MICE AND ANOTHER PERIOD OF APOPTOSIS IN THE POSTNATAL PERIOD OF BOTH MALES AND FEMALES AND IT'S KNOWN THAT THIS APOPTOSIS IS BOTH OF THESE APOPTOTIC WAVES APPEAR TO BE SOMEWHAT DEVELOPMENTALLY PROGRAMMED BY THEIR TIMING AND PARTICULARLY IN MALES, IF YOU DON'T HAVE THIS PERIOD OF APOPTOTIC AND APOPTOSIS, THEN YOU'RE STERILE AND THIS IS KNOWN BY A NUMBER OF DIFFERENT MUTE ANTS AND SPECIFICALLILET BACKS MUTE ANT AND THE WAY WE CHOSEN TO ENTER THIS QUESTION IS TO ASK WHAT IS THE PURPOSE OF OF THIS APOPTOTIC WAVE, AND CHOSEN TO FOCUS ON THE MALE, PARTICULARLY BECAUSE AT THE REQUIREMENT FOR THIS APOPTOTIC WAVE, EITHER THE FETAL OR THE POSTNATAL WAVE FOR FERTILITY IN MALES. SO IT WAS KNOWN THAT THE SCHEDULE A WAS NECESSARY FOR FERTILITY BUT THE WAVE OF THE FETAL PERIOD WAS NOT WELL CHARACTERIZED AND SO, WE ADDED TO SOME OF THE KNOWN MARKERS, WHICH WERE BY IN FIVE WITH SORTING AND AS WELL AS LOOKING BY MORPHOLOGY IN ELECTRON MICROSCOPY AND WE'VE ADDED TO THIS WITH THE LATE APOPTOTIC MARKER WITH THE INTRINSIC PATHWAY, SO THE MALE FETAL GERM IS AT A 10 FOLD INCREASE IN THE SURROUNDING SOMATIC CELLS BUT ONE IMPORTANT THICK WE WANT TO LOOK AT IS WHAT IS THE ACTUAL 3D LAY OUT OF THIS APOPTOSIS IN HAD THE GERM LINE AND ONE THING THAT WE DEVELOPED IS 3D IMAGING OF THE INTACT GONAD USING CONFOCAL MICROSCOPY. SO HERE YOU SEE A MEWY OF THE FETAL TESTIS SHOWN HERE WHICH IS SETTING ON TOP OF THE MUCIN EFROSU, AND YOU SEE THE GERM CELLS WITH THE MARKER GCNA SO CAN YOU SEE IT COMES ON AND THOSE ARE CONDENSED INTO CORDS AND THEN USING THE MARKER CLEAVED P A RP, CAN YOU SEE THAT'S GOING TO TURN ON IN THE SUBSET OF GERM CELLS IN A SUBSET OF GERM CELLS HERE AND CAN YOU SEE THAT THESE POSITIVE CELLS ARE NOT RANDOMLY DISPURSED THROUGHOUT THE GONAD AND NOT THROUGHOUT THE CHORDS BUT THEY'RE CLUSTERED IN THESE LITTLE GROUPS OF CELLS AND THIS WAS ASHES PARENT NOT ONLY AT A 13.5 WHICH WAS THE PEEK OF APOPTOSIS BUT NEAR THE BEGINNING OF THE APOPTOTIC WAVE AT E12.5 AS WELL AS THROUGH THE CONCLUSION OF E13.5. SO WE THOUGHT THERE WAS A REASON THAT THESE GERM CELLS ARE CLUSTERED, WE WANTED TO SHOW MATHEMATICALLY THAT THE APOPTOTIC GERM CELLS WERE NOT OCCURRING IN A DIFFERENT FASHION AND WE EMPLOYED AN ALEGORITHMS CALLED RIPLEY'S K-FUNCTION WHICH ASKS FOR EACH GIVEN EVENT WHICH OUR CASE IS A GERM CELL AND WE EXTRACT THOSE COORDINATES FROM THE PICTURE I SHOWED YOU AND THAT'S ARRAY INDEED MATT LAB SO FOR EACH OF THESE EFFECT WHAT IS IS THE PROBABILITY OF A SIMILAR OBJECT BEING WITHIN A PARTICULAR RADIUS WHICH WE DENOTE AS EITHER R OR T. SO WE COMPARE THE OBSERVATIONED DISTRIBUTION WHICH IS HERE, KT AND WE OBSERVE THAT AND COMPARE THIS TO AN EXPECTED DISTRIBUTION AND SO IF THERE'S NO DIFFERENCE BETWEEN THESE THEN WE WOULD EXPECT AS WE GO FROM ZERO TO OVER20-MICRONS OF RADIUS WE EXPECT A FLAT LINE AND SO WHEN WE LOOK AT THE OBJECTS THE GERM CELLS THEMSELVES, WE SEE THERE'S A SMALL DEVIATION AS WE GO OUT BY RADIUS WHERE WHEN WE COMPARE THIS TO THE SUBSET OF DYING GERM CELLS WE SEE THAT THERE'S A SIGNIFICANT DEVIATION FROM RANDOMNESS SHOWING THE MATHEMATICALLY THIS APOPTOTIC GERM CELLS ARE CLUSTERED AND INTERESTINGLY WHEN WE LOOK AT THE RATIO OF THE EXPECTED, OBSERVED DISTRIBUTION AT E12.5, AND 14.5, WE SEE THE MAXIMUM DISTANCE BETWEEN GERM CELLS IN WHICH WE SEE THIS CLUSTERING ISHT ACTION TO CORRESPONDS TO A SINGLE CELL LENGTH WHICH IS QUITE CONSISTENT AT ALL THESE DIFFERENT STAGES. SO, THIS CLUSTERED APOPTOSIS IN OUR MINDS COULD BE CAUSED BY THREE DIFFERENT REASONS. THE FIRST COULD BE THAT IT COULD BE LOCAL, DUE TO LOCAL INTERACTIONS, IN OTHER WORDS, THE GERM CELLS THAT ARE DYING COULD BE IN A POOR NEIGHBORHOOD, A LACK OF SIGNIFICANCEINALS AND I WON'T SHOW THE DATA, BUT WE LOOKED WITH A NUMBER OF DIFFERENT--NUMBER OF DIFFERENT WAYS WE LOOKED AT THE RATIO OF THE GERM CELL TO THE SURROUNDING SUPPORT CELLS AND WE LOOKED ALSO AT THE DISTRIBUTION OF THE CUSTERS THEMSELVES AND ESSENTIALLY WE DON'T FIND EVIDENCE FOR THIS. SECOND REASON COULD BE THAT THE GERM CELLS ARE IN PHYSICAL CONNECTION THROUGHINIQUE STRUCTURE CALLED CYTOPLAYS MIC BRIDGES, AND THE THIRD REASON COULD BE THAT THERE COULD BE CELL INTRINSIC DEFECTS IN THE GERM CELLS: SO A WORD ABOUT THESE INTRACELLULAR BRIDGES BEFORE I GO THROUGH THE THIRD, AND THESE BRIDGES HAVE BEEN DESCRIBED BOTH IN THE FETAL PERIOD OF GERM CELLS AS WELL AS POSTNATAL PERIOD AND HIGHLY CONSERVED STRUCTURES AND IN THE FEE FETAL PERIOD OF MAMMALS AND THEN THEY BREAK UP, THEY'RE FORMED BY INCOMPLETE CYTOKINEESIS AND KNOWN THROUGH MUTE ANT ANALYSIS THAT THEY'RE REQUIREDDED FOR MALE FUTILITY AND SO ONE POSSIBILITY IS PERHAPS THROUGH THESE CONNECTIONS, THE SIGNALS ARE TRANSMITTED SO ADDRESS THIS WE LOOK AT A MUTANT IN TEXT 14 AND THIS MUTANT LOOKS THE EXTRA CELLULAR BRIDGES SO SHOWING YOU WHOLE MOUNTS OF RETRO ZYGOUS LITTER MATES VERSES THE MUTANT, CAN YOU SEE THE GERM CELLS IN THE ACCORDS AND I WILL TELL YOU THAT THERE'S A VERY SIMILAR LEVEL OF APOPTO SURPRISINGLY, IN THIS MUTE ANT WHEN WOO LOOK AT THE ALEGORITHMS WE SAW THE CLUSTERING WAS SIGNIFICANTLY LESS, AND IMPORTANTLY, WE ALSO MEASURED THE DISTANCE BETWEEN THE APOPTOTIC OBJECTS AND WE SAW THAT SO COMPARED TO A WILD-TYPE HERE AND HERE WE EXPRESSED THIS IN CELL LENGTH. WE SEE THAT THE--AT APPROXIMATELY ONE CELL DISTANCE, ONE CELL DISCIPLINARY AMTERIS THE MAXIMUM DISTANCE OF INTERACTIONS BETWEEN THE APOP TO THE ICOBJECTS IN THE WILD-TYPE WHEREAS IN THE MUTANT WHERE YOU HAVE APOPTOSISIS, THIS SEEMS TO BE SLIGHTLY GREATER IMPLYING THAT THE APOPTOSIS IS STILL CLUSTERED BUT THE GERM CELLS ARE DYING ARE A BIT FARTHER APART. SO IN OUR MINDS THIS SUGGESTED THAT THE BRIDGES ARE NOT NECESSARILY THE CAUSE OF THIS GERM CELL CLUSTERING, SO THESE EVENTS ARE OCCURRING INDEPENDENTLY, IN CLONEALLY RELATED CELLS. SO OUR NEXT QUESTION WAS TO LOOK AT THE CLONAL RELATIONS OF THESE CELLS AND TO DO A CLONAL LABELING SO WE TOOK A STRATEGY USING TWO DIFFERENT MULTIALCOHOL CORED REPORTER MICE, THAT ARE AVAILABLE, ONE IS CONFETTI AND ONE IS RAINBOW AND WE CROSS SAID THESE TO A DRUG INDUCED CREE, WHICH IS THE OXFOUR CREE ER AND SO BY GIVING THESE TO THE MICE AT DAY 10.5 WHICH IS JUST AT THE CONCLUSION OF GERM CELLS MIGRATION AS THEY ENTER THE GONADS THEN WE'RE ABLE TO TERMINENTLY LABEL THE GERM CELLS IN THE RESULTING TESTIS WITH ONE OF FOUR DIFFERENT RANDOM COLORS. AND AND THEN WE CAN ALLOW THEM TO LOOK AT THE TIME POINTS AND THE DISTRIBUTION AND PARAMETERS CHANGE IN RESPONSE TO APOPTOSIS, SO THE FIRST THING WE NOTICED WAS THAT IT HAD BEEN REPORTED IN A FUNCTION THAT HAD BEEN SPECULATED FOR THESE INTRACELLULAR BRIDGES WAS THAT THEY SINGER RONNIZE CELL DIVISION SO THAT'S PRECISELY WHAT WE SUE WITH OUR CLONES SO HERE YOU SLEEP APNEA AND OBESITY IN PINK, THE POSITIVE GERM CELLS AND YOU CAN SEE THEY'RE RESTRICTED TO THIS MORANGE CLONE AND IN THIS PARTICULAR MOUSE THIS, IS THE RAINBOW, GFP IS CONSITTATIVE AND THE EXCISION IN THE GERM LINEAGE EXCIDES THE GFP OR THE MCHERRY RANDOMLY, THIS TOLD US THAT OUR CLONAL LABELING WAS WORKING AND APPEARED TO BE RESTRICTED TO CLONES. ALL RIGHT THAT PLAY AGAIN, WHAT INTERESTINGLY YOU CAN SEE THAT THE CLONES ARE--THAT THIS ORANGE CLONE IS ENTIRELY EXPRESSING FOSTER NURSED FOCUSED ON HISTONE H-THREE AND THESE OTHER ORANGE CELLS ARE EITHER AG METROPOLITANNED VERSION OF THE REST OF THE CLONE OR SEPARATELY ARISING CLONE ARE NOT EXPRESSING PHOSPHO HTHREE. SO THEN IMPORTANTLY WE WANT TO LOOK AT APOPTOSIS IN THE CLONEALLY MARKED MICE. SO HERE YOU CAN SEE, THE LATE APOPTOTIC MARKER, AND YOU CAN SEE IT IN SECTIONS THAT WE'RE ABLE TO VISUALIZE CLUSTERS WHICH ARE DEFINED BY CELLS WITHIN 50- MICRONS OF ONE ANOTHER AND SO THEN HERE YOU SEE THAT THE EGFP HAS GONE OFF AND THESE ARE ALL EXPRESSING IN MSERRULLIAN AND WE'VE SEEN NOW ACROSS A NUMBER OF DIFFERENT EMBRYOS AND A NUMBER OF DIFFERENT EXAMPLES THAT WE HAVE FAILED TO SEE THAT THERE IS A CLUSTER, OF APOPTOTIC GERM CELLS WHICH IS MORE THAN ONE COLOR. SCOW TO SHOW YOU A MEWY TO ILLUSTRATE THE MEASURE WANTS WE HAVE MADE, THIS IS THAT SAME SECTION, SO THIS IS IN THE TESTIS CORD AND YOU SEE THE CELLS AND THESE TWO ARE IN OPPOSITION SO THEY'RE ACTUALLY TOUCHING WHEREAS THIS APOPTOTIC GERM CELL IS NOT TOUCHING THE OTHER TWO AND WE CAN TURN THEM AROUND AND WE CAN MEASURE THE DISTANCE BETWEEN THE TWO TOUCHING APOPTOTIC CELLS AND AROUND 11-MICRONS AND THEN THE TWO NOT UNCONNECTED GERM CELLS ARE MORE LIKE 17-MICRONS APART SO JUST TO SUMMARIZE THE STATISTICS WE'VE SEEN SO FAR, WE FAILED TO SEE ANY APOPTOTIC CLUSTERS WHICH ARE MULTICOLORED OR POLYCLONAL IF YOU WILL AND AMONG THOSE WE'VE MEASURED, WE'VE SEEN SINGLE COLORS BUT PRIMARILY CONNECTED AND A FEW DISCONNECTED GERM CELLS AND THE FACT THAT THESE CELLS ARE NOT CONNECTED REINFORCES THE BRIDGES THAT THEY ARE NOT TRANSMITTING THE APOPTOTIC SIGNALINAL AND WE SEE THE SAME RATIO OF COLORS, AND ALTHOUGH A BIT LESS IN THE MCHERRY CHANNEL PERHAPS BECAUSE THIS IS NOT AS BRIGHT AS THE OTHER AND MORE STATISTICS. SO JUST TO SUMMARIZE, OUR MODELS, OUR QUESTION HAS BEEN TO LOOK AT THE EFFECT OF THE APOPTOTIC WAVEOT DIVERSITY OF GERM CELL CLONES. AND WE'VE SHOWN WITH THE TEXT 14 MUTANT THAT THE BRIDGES IN TEXT 14 MAY COORDINATE DEVELOPMENT PARTICULARLY IN THE FEMALE AND SINCE WE DON'T HAVE A SNAPSHOT OF APOPTOSIG AT CLEAVE P A RP, WE CAN'T SAY WHAT HAPPENS OVER TIME BUT WE BELIEVE THAT THE INTERCELLULAR COORDINATE BRIDGES OF APOPITOSE ESTIMATE THAD. HOWEVER, THE WAVE APPEARS TO BE ACTING INDEPENDENTLY AMONG SEPARATE GERM CELLS SO IT'S MORE A READ OUT OF THE INTRINSIC PROPERTIES RATHER THAN PHYSICAL CONNECTION. SO REALLY THE TAKE AWAY IS THAT THE APOPTOSIS IN APPROXIMATE THE GERM LINE OCCURS IN CLONEALLY RELATED CELLS AND THAT THIS IS REALLY SUGGESTIVE OF A MECHANISM FOR QUALITY CONTROL WHICH IS ACTING SEPARATELY ON EACH GERM CELL AND THE QUESTIONS THAT I THINK ARISE FROM THIS ARE FIRST OF ALL WHAT'S THE BASIS FOR THIS ELEM NATION, IS THERE A PARTICULAR TRIGGER? WHY DO THE GERM CELLS NOT GET ELIM NATTED EARLIER? WHY DID THEY PROLIFERATE AND GROW TO A CLONE OF A CERTAIN SIZE FIRST AND WHAT WE CAN ONLY INFER BUT HAVEN'T BEEN ABLE TO SEE YET IS WHETHER APOPTOSIS IS ACTUALLY ELIMINATING ENTIRE CLONES OF GERM CELLS AND THIS--THE FACT THAT OUR CLONE SIZE IS SIM LAWYER BEFORE AND AFTER THE APOPTOTIC WAVE WITH OUR LABELING METHOD, AND ALLANSPREADLING SHOW WIDE A SINGLE COLORED LOW FREQUENCY METHOD THAT THE CLONE SIZE REMAINS SIMILAR OVER THE PERIOD OF FETAL DEVELOPMENT, SUGGESTS THAT IS THE APOPTOTIC WAVE IS ELIMINATING ENTIRE CLONES. WOE ALSO CAN'T SAY THAT PROLIFICATION IS GIVEN A SHARP LIVE THAT COULD BE PROLIFERATION OF THESE CLONES, SO I WOULD WOULD LIKE TO ACKNOWLEDGE THE PEOPLE WHO DID THE WORK. THIS WORK WAS ENTIRELY THE PROGGENT OF MY FIRST Ph.D. STUDENT DAN NG UYEN, PICTURED HERE AND WE WOULD WOULD LIKE TO ACKNOWLEDGE EVERYONE PICTURED HERE AND THE NIH INNOVATOR PROGRAM. >> DO YOU KNOW IF YOU PUSH DEVELOPMENT INSTEAD OF IN VIVO, INVITRO, DOES THE SAME PROCESS C MOVING THE EMBRYOS OUT? >> SO DOES THE SAME PROCESS OF PROGRAM APOPTOSIS OCCUR IN-- >> IF YOU TAKE THE EMBRYOS AND PUSH THEM FROM 10.5 TO 12 INVITRO. >> SO, WE HAVE NOT DONE A LOT OF ONE CAN CULTURE THE FETAL GONADS DURING THIS PERIOD, WE HAVE NOT DONE A LOT OF THAT IN WHAT WE HAVE DONE WHICH IS WE HAVE ACTUALLY SEEN THAT THERE'S A LOT OF APOPTOSIS, LIKE ABNORMAL LEVELS OF APOPTOSIS AND SOMETHING LIKE 30%. AND SO WE TOOK THIS TO MEP THAT OUR CULTURE CONDITIONS WERE NOT OPTIMIZED AND THAT THIS NOT REPLICATING THE IN VIVO SITUATION AND THEN THE OTHER WAY IS THAT PEOPLE WILL DERIVE GERMS EITHER TAKE THE GERM CELLS OUT OF THE EMBRYO OR DERIVE THEM. >> TO MASTER THE WHOLE EMBRYO AS WELL IN THAT TIME PERIOD, WE CAN TALK LATER. >> YOU ARE--BEYOND ABOUT E9.5, THEY JUST TAKE THE FEE FETAL GONAD LATER. >> WE'LL TALK LATER. >> SECOND QUESTION IS WHY DOES CLEAVE CASP A STHREE NOT CLEAVE-- >> THAT'S MORE OF A TECHNICAL QUESTION, WE HAVE NOT SO CASP A S THREE DOES NOT PENETRATE AND LABEL IN THE CONDITIONS WE USE FOR WHOLE MOUNT SO IT WOULD BE A GREAT MARKER IF WE COULD GET IT TO WORK AND IF PEOPLE HAVE OTHER ANTIBODIES THEY LIKE FOR INTRINSIC CELL DEATH, I WOULDED LOVE TO HEAR ABOUT THEM. >> BEAUTIFUL IMAGES, I HAVE A QUESTION ABOUT THE CLONALITY, IF YOU ANALYZE SAY A HUNDRED GONADS AND YOU OVERLAID THE GEOGRAPHIES OF WHERE YOU SEE THE PHENOMENA, WOULD YOU FIND THERE TO BE OVERLAP? IS IT SPACIALLY SORT OF STOCHASTIC OR SPACIALLY INVARIANT. >> SO YOU'RE ASKING HOW STEREOTYPED IS THE SPLAYSMENT OF CELL DEATH. MYES. >> MAYBE WE HAVE ALMOST DONE A HUNDRED GO GONADS AT THIS POINT THEY'RE LABOR INTENSIVE AND COMPUTATIONALLY INTENSIVE BUT WHEN WE LOOK AT THE PLACEMENT OF EACH CLUSTER, THE CLUSTER PLACEMENT SEEMS TO BE A RANDOM DISTRIBUTION, SO THE CLUSTERS THEMSELVES ARE NOT CLUSTERED OR DISPURSED, SO AND--AND WHAT I DIDN'T HAVE TIME TO SHOW SURVEYS THAT THEY DON'T OCCUR OUTSIDE OF THE CORD SAYS THEMSELVES. SO WITHIN THE CORDS THEY APPEAR TO BE PRETTY REGALLY SPACED SO WE DON'T--REGULARLY SPACED SO WE DON'T HAVE ANY REASON TO BELIEVE THERE'S ANY PATTERNING OF APOPTOSIS, AND WITH RESPECT TO BLOOD VESSELS AND AS I SAID WITH RESPECT TO SUPPORT CELLS. BUT THAT WAS AN INTERESTING QUESTION. >> THANK YOU. >> THANK YOU. >> [ APPLAUSE ] >> OUR NEXT SPEAKER IS HARA LEVY, AND SHE WILL TALK ABOUT IDENTIFICATION OF MOLECULAR SIGNATURE IN CYSTIC FIBROSIS. >> SO I'M A PEDIATRIC DOCTOR AND I'VE WITHIN INTERESTED IN ADDRESSING WITH PEOPLE WITH THE SAME DISEASE AND SAME MUTATIONS AS DEFINED HAVE MARKEDLY DIFFERENT CLINICAL COURSES AND I WONDERED IF WE CAN PAIK A PERSONALIZED APPROACH USING CUREPT ADVANCES IN GENOMICS. SO CF IS INHERITED AND LIFE THREATENING MULTISYSTEM DISEASE FOR PROGRESSIVE DETERIORATION IN LUNG FUNCTION AND ATTRIBUTED TO FUNCTION OF THE GENES ENCODING FIBROSIS REG AUTOOR MEMBRANE GENE CALLED CFTR, WITH THE DISCOVERY OF THE GENE, IT WAS HOPED THAT THAT MING OF A PATIENT'S GENO TYPE COULD PREDICT CLINICAL COURSE AND DISEASE SEVERITY, BUT THIS IF FACT IS NOT THE CASE AND THERE'S A WIDE SPECTRUM OF DECEIVE SEVERITY IN PATIENTS IN CF AND O CONSISTENT GENO TYPE, PHENOTIME CORRELATION, PERHAPS DUE TO PROTEIN ACTIVITY, LEVELS, MODIFIER GENES AND SYSTEM DYNAMICS SO USING ADVANCES IN GENOMIC TECHNOLOGIES, EPIDEMIOLOGY, STATISTICAL ANALYSIS, WE SOUGHT TO USE MICROARRAYS TO TO HAVE A MORE PERSONALIZED APPROACH TO DIAGNOSIS, PROGNOSIS AND PERHAPS THERAPEUTIC RESPON. SO IN CYSTIC FIBROSIS, MUTATIONS IN TED GENE WHICH IS A CHLORIDE CHANNEL EFFECT THE COMPOSITION OF THE MUCUS LAYER LINING THE EPITHELIAL WALL OF THE BRONCH I, IN THIS PATIENTS IN THE CLINIC UPPER WE SEE THE UNEFFECTED PERSON WHICH IS NICE AND CLEAR AS OPPOSE TO THE PERSON WITH CF AND THERE'S MUCUS PLUGGING IN THE CHLORIDE TRANSPORT AND THAL EFFECTS BREATHING MECHANICS. AND ULTIMATELY, PATIENTS DIE DUE TO THE MORBIDITY AND MORTALITY IN THE IMPACT ON THE LUNG, SO THE PULMONARY DISEASE IS THE MOST CHALLENGING PROBLEM IN THE MANAGEMENT OF CF AND THE MAJOR DETERMINANT OF LIFE SPAN AND QUALITY OF LIFE IN INFECTED INDIVIDUALS. SO CURRENTLY WE USE ARE SYMPTOMS, CLINICAL EXAM, IMAGING AND PULMONARY FUNCTION TESTING AND IT'S A BIT LIKE A PILOT FLYING BLIND NOW LUNG VERSES AGE, AND AND CYSTIC FIBROSIS IS A PROGRESSIVE LUNG SO OVERTIME YOU EXPECT THE PERSON TO DO WORSE WHICH IS IN FACT WHAT WE SEE IN MOST OF THE CASES. BUT HERE WE SEE AN INDIVIDUAL WHO'S EIGHT YEARS OF AGE HAVING POOR LUNG FUNCTION AND A PERSON WHO'S 38 YEARS HAVING ASYMPTOMATIC AND PATIENTS WITH THE SAME MUTATION, DEMENTIA 508 IS ONE OF TED MOST COMMON, SO ISSUE IS COULD WE MOLECULARLY GET A BETTER HANDLE ON THIS CLINICAL DIVERSITY. SO USING MICROARRAYS WE ARE ABLE TO INTEGRATE CHANGES OVER TIME SO HERE AS A BASE WE HAVE CLINICAL DESCRIPTORS AND WE ARE TRYING TO INCORPORATE ENVIRONMENTAL COMPONENT WITH GENE EXPRESSION TAKE INTOG ACCOUNT THAT THERE ARE ALREADY KNOWN GENETIC MODIFIERS SO THERE ARE SOME GENE, TGF BETA AS AN EXAMPLE, EGF, THAT HAVE ALREADY BEEN FOUND DO YOU TO CANDIDATE GENE ARCSINAL SIS, AND GENOME WIDE ASSOCIATION STUDIES THAT HAVE BEEN SHOWN TO IMPACT DISEASE SEVERITY BUT THESE ARE ONE GENE AND ONE POINT IN TIME SO WE'RE TRYING TO EXPAND OVER SEVERAL TIME FRAME TAKING AGING INTO ACCOUNT. SO AS BACKGROUND WE'RE INCORPORATING PATIENT HISTORY, EXAM, SYMPTOM QUESTIONNAIRE, MEDICATIONS, PULMONARY SPUTUM TESTS CULTURES INCORPORATING THAT INTO OUR ANALYSIS WHERE WE'RE USING PATIENT SERUM, PLASMA AND DNA. SO TO OVERCOME THE LIMITATIONS AND REPORT IN THE DIRECT PROFILING OF PBMCs WHICH MAY NOT ACCURATELY REFLECT THEACKIST IT THE LUNG AND THE PARTICULARLY THOSE WITH BREATHING DIFFICULTIES CHARACTERISTIC OF CF, WE UTILIZED A NOVEL ASSAY WHERE THE SUBJECT PLASMA IS USED TO INDUCE JEEP EXPRESSION IN HEM HEALTHY PBMCs POPULATION. THIS APPROACH WHERE THE CELLS ARE USED AS REPORTERS THAT SENSITIVELY RESPOND TO SOLUBLE DISEASE FACTORS PRESENT IN THE PERIPHERY HAVE BEEN USED WELL FOR THE STATE AND RHEUMATOID DISEASES AS WELL AS TYPE ONE DIABETES AND OUR LAB AS RELATED TO CF AND IN OUR PRESENT WORK WE ANALYZE PLASMA INDUCED TRANSCRIPTIONAL PROTILES AND SORTING SIGNALSALATE THEM WITHOUT COMMON OUTCOME MEASURES WITH PANCREATIC PULMONARY FUNCTION STATUS AND AGE MATCH CONTROLS. THE SERUM ASSAY WE'VE DEVELOPED GETS A ROBUST SIGNATURE RATHER THAN THE PBMCs TO OBTAIN PATHOLOGIC SPECIMENS AND THIS FAR THE COHORT WE'VE BEEN ABLE TO AC, WE HAVE 400 PATIENTS OF WHICH 139 ARE PROBANDS WE HAVE 17 COMPLETE SET OF TRIS ON WHICH WE USE FOR ANALYSIS WHICH I WILL SHOW, AND WE DID MULTIPLE, WE PERFORM MULTIPLE SETS DIFFERENT TIMES SO THAT WE COULD SO THAT WE COULD LOOK AT WHETHER THERE WAS ANY BATCH EFFECT MEANING TIME IN THE SAMPLE IN THE FREEZER OR AGE OF THE SAMPLE ITSELF, WE CAN SEE THE CLEAR DEMARCATION OF THE CF PATIENTS VERSES THE HEALTHY CONTROLS. EACH COLLUMIN A PATIENT IN EACH ROW SDIFFERENTIALLY EXPRESSED, GENE RED IS UPREGULATED AND GREEN IS DOWN REGULATED TO COMPARED TO HEALTH CONTROLS AND WE MIXED UP THOSE DIFFERENT BATCHES IN BOTH AND YOU CAN SEE A CLEAR DEMARCATION HERE. HERE WE SEE THE GENES SO WE SEE 200 EXPRESSED GENES HERE WHICH I SHOW APPROXIMATELY 23 OF THOSE AND MANY OF THEM ARE RELATED TO IMMUNE FUNCTION. UNSUPER VISED COMPONENT ANALYSIS, WE CAN HIGHER CLUSTERING USING UNFILTERED DATA AND REVEAL THAT THE CF COMPONENT HERE ARE CLEARLY CLUSTER DIFFERENTLY THAN THE HEALTHY CONTROLS, THE FIRST COMPONENT EXPLAINS 16.5 WHILE THE SECOND AND COMPONENTS EXPLAIN FIVE AND THREE OF THE VARIANTS RESPECTIVELY. RESULTS WERE NOT SIGNIFICANTLY EFFECTED BY SAMPLE, COLLECTION DATE OR TIME. HIERARCHICAL CLUSTERING OVER A THOUSAND GENES WERE SIGNIFICANTLY DIFFERENTIALLY EXPRESSED FAR CF IS CONTROLS AND 224 AND 51 SHOW WHAT IS NOT COMMON AND DIFFERENTIATING PANCREATIC SUFFICIENT PATIENTS AND THESE POTENTIALLY WOULD BE GENES WE WOULD THEN LOOK INTO PATHWAYS AND FURTHER KEEP OUT. HERE'S OUR HIERARCHICAL CLUSTERING WHERE WE SEE HEALTHY CONTROLS AND THE PANCREATIC SUFFICIENT PATIENTS WHICH ARE ACTUALLY HEALTHIER CLUSTERING CONTROLS AND WHOLE SPECTRUM OF PANCREATIC INSUFFICIENT PATIENTS, OF VARYING CLINICAL SEVERITY SO YOU SEE A NICE DEMARCATION HERE IN SPECTRUM. WHICH IS SOMETHING THAT WE'RE REALLY NOT ABLE TO TEASE OUT CURRENTLY WITH SYMPTOM HISTORY, BLOOD WORK OR PULMONARY FUNCTION TESTING. THEN WE ASKED WELL MAYBE IS INUST JUST AN INFECTION SIGNATURE SO WOO BEING LOAMACY AT CF PATIENTS THAT WERE ACTUALLY INFECTED IN THEIR AIR WAY WITH THE PSEUDOMOAN ISOTOPE, COMPARED TO HEALTHY CONTROLS, PATIENTS AGE MATCH WITH VIRAL INFECTION AND THOSE WITH PNEUMONIA. AND THESE ARE THE INDIVIDUALS PATIENT SAMPLES AND YOU CAN SEE THAT THERE IS A DIFFERENT HERE SO THAT THE UCF IS UNIQUE AND NOT NECESSARILY INFECTION SIGNATURE AND THIS IS THE MEAN JUST HELPING TO SHOW CLEARLY THE DIFFERENTIATION. WE THEN WANT TO LOOK AT LONGITUDINAL EXPRESSION OVER TIME SO THESE ARE THREE INDIVIDUAL PATIENTS WITH SAMPLES. DIFFERENT TIME POINTS, SHOWING DIFFERENT DEGREES OF INFECTION AND INENTIAL LIE NONINVESTIGATION AND THEN BECOMING INFECTED, THIS PATIENT NONAND THEN BECOMING INFECTED OVER TIME AND SAME THING WITH THIS PATIENT AND WE CAN SEE HERE'S DIFFERENTIAL EXPRESSED GENES AND WHEN WE LOOK AT THESE, THEY'RE ALL--MOST OF THEM ARE RELATIVE TO IMMUNE FUNCTION AND REGULATION. NEXT WE LOOKED WE SAID WELL DOES THE MUTATION CLASS ITSELF IMPACT THE EXPRESSION AND HERE WE SEE HEALTHY CONTROLS IN THE DIFFERENT CLASSES OF CFTR MUSEUMITATIONS FOR MOST OF THE SEVERE TO LESS SEVERE. LET AND THEY ARE DIFFERING CLUSTERING AND THEN WE CAN SEE THE COMMON AND THEN THE UNIQUE GENES. WE ALSO SEE INTERESTINGLY THAT THE PATIENTS WHO ONLY HAVE ONE COPY OF THE MUTATION VERSES THE PROBAND WHICH IS HAVE TWO COPIES OF MUTATION SOMETIMES GENES AND COMMON BUT ALSO GENES THAT ARE NOT COMMON, AND THE PARENTS OBVIOUSLY ARE NOT CLINICALLY APPARENT OR SYMPTOMATIC WHERE THE PROBANDS ARE. SO WE THINK THAT CF IS ACTUALLY A SPECTRUM OF CFTR OPERATING GLOBALLYATHYS, AND CRMS AND THE BEING THE LEAST SEVERE AND CLASSICAL BEING THE MOST SEVERE AND WE THINK THAT OUR WORK CAN BETTER SEIZE THIS OUT. SO IN CONCLUSION WHAT WE HAVE THEN SINCE THIS IS 1990 YEAR AFTER THE CFTR GENE WAS DISCOVERED, WE USE MOLL MONITORARY FUNCTION TEST AND CLINICAL EXAM AND SYMPTOMS TO TRY TO DIFFERENTIATE THE PATIENT AND YOU CAN SEE A HUGE CLINICAL HETEROGENEITY AND WE THINK THAT OUR ARRAY WORK AND PCA WILL HELP BETTER DEFINE THE CLINICAL HETEROGENEITY LEADING TO BETTER BIOMARKERS, IMPROVED WAY OF PREDICTION AND RESPONSE TO THERAPIES. SO WE THINK THE PREDICTIONS AND THERAPEUTIC RESPONSE PROVIDE A WAY FOR CLINICIANS TO HAVE A WAY TO FIGURE OUT WHEN TO PULL THE LEVERS, WHEN AND OW BECAUSE RIGHT NOW, WE'RE USING A ONE-SIZE-FITS-ALL. I'M CURRENTLY AT NORTHWESTERN UNIVERSITY AT CHILDREN'S HOSPITAL, I MOVED FROM MEDICAL COLLEGE OF WISCONSIN AND I WOULD LIKE TO THANK MY CURRENT COLLABORATORS, ANN HARRIS, SUZANNEA AND THOSE WHO HELP WIDE THE ARRAY WORK AS WELL AS OTHERS. THANK YOU. [ APPLAUSE ] >> SO THAT WAS A GREAT TALK, CF IS LIKE A POSTER CHILD, THE JEEP WAS DISCOVERED ABOUT 25 YEARS AGO AND YOU KNOW THE WANTED ERA OF PROPOSED GENOMIC MEDICINE AND THERAPEUTIC WE STILL HAVE APPARENTLY ABSOLUTELY NO IDEA THAT ONE SIPLE MUTATION AND ONE GENE CAUSES WHAT IT DOES, SO ME QUESTION FOR YOU ARE AS FOLLOWS: FIRST OF ALL, THE SERUM BIOMARKER PROFILING WITH THE PC ANALYSIS, AND THE CENTERPIECE OF YOUR METHOD, HAS THAT RESULTED IN ACTUAL IDENTIFYING NEW MOLECULAR MECHANISMS NOT MARKERS NOT DIFFERENTIATED BUT MECHANISMS THAT HELP US UNDERSTAND WHY DIFFERENT PATIENTS WITH THE SAME SET OF MUTATIONS HAVE PHENOTYPES AND DIFFERENT DISEASE SEVERITY. SO I WOULD LIKE TO THINK THAT ANY GOOD SERIES OF WORK POSES ADDITIONAL QUESTIONS SO THESE GENES ARE BEING FURTHER ANALYZED FUNCTIONALLY AND INGENUITY PATHWAY AND YOU CAN SEE LOOKING AT THE TRAP, LOOKING AT THOSE ACTIVE RN As AND LOOKING AT THERAPEUTIC RESPONSE BOTH IN CULTURE AND IN PATIENTS SO I DO THINK THAT THIS IS A WAY OF GETTING AT THINGS MECHANISTICALLY. >> BUT YOU DON'T VERMEN INFECTED SPECIFIC EXAMPLES SO FAR WHERE THAT HAS BEEN-- >> THAT'S THE NEXT GRANT APPLICATION. >> WELL, SURE. >> LASTLY, I THINK IT'S REALLY CHARMING THAT YOU MICROARRAYS, ALTHOUGH I DO TOO, AT TRYING TO THE NEW INNOVATOR AWARD IS USING OLD FASHIONED METHODS, MY QUESTION IS WHAT'S ON YOUR MICROARRAY, EXACTLY, IS IT PROTEIN CODING GENES OR ALL OF THE 60,000 GENES OF THE CONSORTIUM HAVE COUNTED. >> IT'S 50,000, IT'S THE ASSAY U13. >> OH IT'S THE A+ B. >> OKAY [INDISCERNIBLE] CODING AND NONCODING. >> NO. >> THANKS. >> OKAY. >> SO I HAVE GENERAL QUESTION ABOUT CF, THEN QUESTION ABOUT QUALITY OF REVIEW WORK. >> IN CF PATIENTS OFTEN HAVE TO GO THROUGH A LUNG TRANSPLANT, SO THE RECEIVING LUNG SHOULD NOT HAVE THE MUTATION BUT STILL THESE PATIENTS HAVE RELAPSE, ESPECIALLY'VE SUED MOANIS, AS WELL AS CHRONIC INFECTION IS THAT RIGHT AND WHY IS THAT? >> SO CAVEATS NOW WITH THE IMPLEMENTATION OF NEW BORN SCREENING THE STORY ABOUT LUNG TRANSPLANTS IS NOT THAT STRAIGHT FORWARD, SO MANY PATIENTS ARE LIVING LONGER. STILL THEIR 40S OR 50S YOU GO THE MEAN AGE OF DEATH IS STILL IN THE 20S. THE SO THAT'S ONE. BUT IT APPEARS THAT THE NEW BORN SCREEN SUGGEST SHIFTING THAT OVER. AS I SAID IT'S A MULTIORGAN DISEASE CROSS LINKING IT'S IN THE EPITHELIUM THROUGHOUT THE BODY SO EACH THOUGH YOU TRANSPLANT THE LUNGS YOU STILL HAVE THE ABNORMAL GENE AND THE OTHER ORGAN. >> PLUS YOU ESSENTIALLY SWITCH ONE SET OF ISSUES FOR THE ISSUE OF THE IMMUNE COMPROMISE WITH THE TRANSPLANT. >> OKAY, AND THE OTHER QUESTION IS, YOU'VE SHOWN THAT CLEARLY YOUR MARKERS ARE NOT JUST A FUNCTIONALITY OF INFECTION, DID YOU HAVE TO COMPARE WITH A NONCF PATIENT THAT HAD ALSO SUMO MONITOROSEIS IN INFECTION IN THE LUNG. >> DISCIPLINARY NOT, MANY OF THOSE PATIENTS WOULD BE TRACH EVENT PATIENTS SO THERE WOULD BE A WHOLE ISSUE OF ADDITIONAL CONFOUNDING VARIABLES. >> YOU THINK BY ANY CHANCE THERE COULD BE A RESPONSE TO SPECIFC TO HAVING LUNGS COLONIZED BY SUIT O MOAN ISOTOPE. >> GOOD THING I WILL LOOK INTO, THANK YOU. >> SO I CLEARLY EXCITING TO LOOK AT THE RELATIONSHIP BETWEEN GENE EXPRESSION, EXTRA GENES THAT ARE INVOFFED IN THE DISORDER AND THEY RELATE TAKEN--THEY CLINICAL OUTCOMES IN IN DISORDER ABOUT YOU THIS DISORDER IS ALSO VERY SENSITIVE, TO THE CLINICAL INTERVENTIO ITSELF, AND OUTCOMES MEASURED BY DEATH, ARE VERY DIFFERENT, IN DIFFERENT TREATMENT CENTERS, SO, THAT SHOULD ALSO BE CONSIDERED AS A SOURCE OF VARIANTS IN TRYING TO RELATE THE GENE EXPRESSION TO THE OUTCOME AND ALSO RAISES THE POSSIBILITY OF INTERESTING INTERACTIONS BETWEEN THE DIFFERENT GENE EXPRESSIONS OR SUBTYPES AND THE NATURE OF THE TREATMENT BUT IN ANY CASE WE DON'T WANT TO FORGET ABOUT THE VARIATION AND THE TREATMENT DELIVERY TAP IS ALSO IMPACTING YOUR DEPENDENT MEASURE OF O >> RIGHT. MENT THIS WAS A SINGLE CENTER STUDY BUT WE DIDN'T KNOW, THE TREATMENT THAT OTHER PATIENTS WE LOOKED AT WAS CONSISTENT. >> OKAY. >> YEAH, THAT'S A GOOD POINT WITHIN THIS, BUT THEN YOU DO HAVING INTERESTING ADDITIONAL SOURCES OF VARIANTS GIVEN WHAT SEEMED TO BE DRAMATIC DIFFERENCES AND OUTCOMES OF TREATMENT CENTERS. >> LAST QUESTION. >> THANK YOU FOR THE INTERESTING TALK. SO I I'M SOMEONE WHO RUNS A CLINICAL LAB MYSELF, AT THE ARRAYS AND TECHNOLOGIES PROVEN DIFFICULT TO IMPLEMENT FOR AS REAL CLINICAL DIAGNOSTICS, SO DO HAVE YOU A PLAN FOR HOW YOU TURN YOUR FINDINGS INTO A PRACTICAL CLINICAL DIAGNOSTIC TOOL? >> WE DO HAVE A RECENT PAPER PUBLISHED IN THE NEW ENGLAND JOURNAL OF MEDICINE, THE LUNG CANCER PATIENT WITH THIS SO THEY DID USE ACTUALLY AN EPARCHS RAY, AS A BY O MARKER FOR LUNG CANCER SO I THINK THERE'S SOME RELATIVELY NEW PRECEDENT OUT THERE, WHERE ARRAY CAN BE USED AS A CLINICAL DIAGNOSTIC. >> [ APPLAUSE ] >> THANK YOU. VERY MUCH. THANK ALL THE SPEAKERS TODAY, REALLY TERRIFIC SERIES OF TALKS. WE HAVE AN EXCITING SET OF POSTERS UPSTAIRS, THE POSTER SESSION LASTS UNTIL ABOUT FIVE P.M. WE WILL RECONVENE HERE TOMORROW AT 8:30 AMENJOY THE POSTER SESSION. FOR THE EARLY INDEPENDENT AWARD SEES, YOU ACTUALLY HAVE LATE INDEPENDENCE TODAY BECAUSE WE HAVE THE EVENING SESSION FROM FIVE TO EIGHT, IT'S IN ROOM F-1, F2 JUST ACROSS THE LOWER LOBBY. SO ENJOY THE POSTER SESSION AND SEE YOU TOMORROW.