IT'S A GREAT PLEASURE TO HAVE ALESSANDRO VINDIGNI HERE WITH US. AND VERY WELL-KNOWN IN THE FIELD OF DNA REPAIR AND WRECK WRECK HELICASES WHERE HE'S WORKED FOR MANY YEARS. PARTICULARLY WITH RECQ1. HE HAS BEEN -- HE'S FROM ITALY. HE WORKED IN TRIAGE FOR MANY YEARS. RECENTLY A COUPLE YEARS AGO, HE WENT TO ST. LOUIS. ST. LOUIS UNIVERSITY. WHERE HE'S NOW A PROFESSIONAL, HAS ALREADY GOTTEN UP AND GOING, EXCITING THINGS COMING FROM THE NEW LABORATORY. IN ADDITION TO THE WORK IN THIS AREA, VERY ELOQUENT WORK DETERMINING [INDISCERNIBLE] FORMATION HE'S WRITTEN A EUNICE KENNEDY SHRIVER CITING PAPER [INDISCERNIBLE] WHERE RECQ 1 SEEMS TO PLAY A VERY IMPORTANT ROLE. WE'RE SWITCHING OVER TO THE DATA STREAM. ALESSANDRO VINDIGNI WILL PRESENT HIS TALK. I HOPE YOU CAN ALL HEAR ME. A PLEASURE FOR ME TO BE BACK HERE. AND ALSO, THANK YOU TO MY TALK IN THIS [INDISCERNIBLE] SERIES. AND [INDISCERNIBLE] TWO YEARS AGO FROM NOW. AND MY LAB HAS BEEN INTERESTED WITH DNA REPAIR AND [INDISCERNIBLE] OF EDUCATION. AND SO MANY OF YOU KNOW, THERE ARE SEVEN [INDISCERNIBLE] [INAUDIBLE] SO FOR EXAMPLE, THERE ARE AGENTS THAT WE USE SUCH AS, FOR EXAMPLE, [INDISCERNIBLE] COMPLETING AGENT. OR AGENT THAT [INDISCERNIBLE] BY DEPRIVING CELLS, LIKE FOR EXAMPLE [INDISCERNIBLE] CELLS HAVE NEEDED THE [INDISCERNIBLE]. PERHAPS NOT SURPRISINGLY, MANY OF THESE MOLECULES BASICALLY FOR [INDISCERNIBLE] TREATMENT IS RATIONAL BEHIND THE USE OF THESE MOLECULES. SINCE THEY TARGET [INDISCERNIBLE]. FOR MANY OF THESE MOLECULES, [INDISCERNIBLE] AND EVEN MORE IMPORTANTLY, THEY START MECHANISM BY WHICH THE EDUCATION MACHINERY RESPONDS TO STRESS. THE FOCUS OF MY TALK TODAY WILL BE [INDISCERNIBLE] THAT RAPIDLY EMERGING FOR REPLICATION STRESS RESPONSE, WE SHOULD [INDISCERNIBLE]. HERE I HAVE A MOVIE TO SORT OF SHOW YOU HOW THESE MECHANISMS WORKS. SO EDUCATION FOR -- FIRST OF ALL, NOT REQUIRED ANY DNA [INDISCERNIBLE] AND WHAT SEEMS TO BE HAPPENING IS THAT WHENEVER THERE IS A LESION AHEAD OF THE REPLICATION FORK, SHOWN HERE, THE REPLICATION FORK CAN REVERSE USING INFORMATION CONTAINING THE LEADING [INDISCERNIBLE]. FORMING A SO-CALLED [INDISCERNIBLE] STRUCTURE. THIS STRUCTURE WILL PREVENT THE COLLISION OF THE REPLICATION FORK AHEAD OF THE FORK. SECONDLY, THE ONLY WAY IT IS PREPARED REPLICATION PRESUMES. ON THE OTHER HAND, WE CAN ALSO IMAGINE THAT [INDISCERNIBLE] FORK, THEN REPLICATION FORK SIMPLY MOVES FORWARD. AND NEEDING TO [INDISCERNIBLE]. SO THE REPLICATION FORK CAN REVERSE WAS REALLY SUGGESTING THAT MORE THAN 35 YEARS AGO, BY A GROUP THAT WAS STUDYING UV DAMAGE [INDISCERNIBLE] CELL. BUT LONG CAN BAITED IN THE FIELD, BECAUSE REALLY, THERE WASN'T ANY DIRECT EVIDENCE THAT THESE FRACTURE MUCHS ARE ACTUALLY PRESENT IN [INDISCERNIBLE] CELLS. AND LAST YEAR, THERE WERE A GROUP OF [INDISCERNIBLE] SHOWS THAT THE REPLICATION FORK REVERSEED. [INDISCERNIBLE] ONE IN ADDITION. THE MAIN TECHNIQUE THAT THE GROUP USED [INDISCERNIBLE] A SAMPLE FORK, DETECTED BY MICECOSCOPY, WHERE THE AND THESE ARE THE TWO ARMS OF THE FORK. SO BY [INDISCERNIBLE] MARKING THE LEVEL TO SHOW THAT REPLICATION FORK REVERSAL [INDISCERNIBLE] AND THERE IS A HIGH FREQUENCY OF FORKS [INDISCERNIBLE] OR THE TREATMENT WHICH ARE [INDISCERNIBLE] BECAUSE WHAT THEY'RE SHOWING IS THAT [INDISCERNIBLE] REVERSED FORK FROM 33% DOWN TO 4.3%. INDICATING THAT INFORMATION OF THESE STRUCTURES IS HIGHLY [INDISCERNIBLE] THEY CONFIRM THE SAME RESULT USING [INDISCERNIBLE] KNOCKOUT CELL LINES, MASS CELL LINES. PARTICULARLY HERE, [INDISCERNIBLE] YOU SEE AGAIN IN THIS CASE, THE FRACTION OF THE REVERSE FORKS DECREASES FROM 26% TO 10%. YOU MIGHT APPRECIATE THAT MAYBE THE DECREASE IS NOT AS BIG AS THE ONE BY USING [INDISCERNIBLE] BUT THIS IS CAUSE WE KNOW THAT PARP ACTING CELLS. [INDISCERNIBLE] AND WHEN THE PATH [INDISCERNIBLE] TO THIS PARP 1 CELL, DECREASED TO 2.7%. SO DEFINING [INDISCERNIBLE] SUGGESTED AGAIN THAT THE NEW MECHANISM OF REPLICATION, THAT THESE CELLS ARE CHALLENGED. AND THE FORMATION OF THESE REVERSE FORKS IS HOE SO YOU DEPENDENT ON [INDISCERNIBLE]. HOWEVER, [INDISCERNIBLE]. WHY WOULD THIS WOULD BE REQUIRED TO DRIVE THE FORMATION OF THE REVERSE FORKS? AND THIS QUESTION IS PERTINENT BECAUSE THERE WAS A STUDY PUBLISHED BACK IN 2007, SHOWING THAT TREATMENT OF CELLS [INDISCERNIBLE], LESION ALSO [INDISCERNIBLE] AHEAD OF THE REPLICATION FORK. THAT [INDISCERNIBLE] AHEAD OF THE REPLICATION FORK WOULD BE SUFFICIENT TO DRIVE FORK REVERSAL. SO THIS IS THE CASE, WHY WOULD PARP T BE REQUIRED? [INDISCERNIBLE] FROM THE WORK IS IF WE HAVE FORMATION OF REVERSE FORKS, HOW ARE THEY EVENTUALLY STOPPED? IS THERE A FACTOR OR MULTIPLE FACTORS WHICH ARE REQUIRED TO RESTART THESE FORKS ONCE THE LESION IS REPAIRED? THESE ARE THE QUESTIONS WE ADDRESS IN OUR WORK. BEFORE I GO INTO THE ACTUAL DATA, ONE MORE INTRODUCTORY SLIDE ON [INDISCERNIBLE], NOT BEING PRODUCTION FOR THESE [INDISCERNIBLE] BUT ONE SLIDE FOR PEOPLE THAT MIGHT NOT BE FAMILIAR WITH THESE ENZYMES. ENZYMES THAT [INDISCERNIBLE]. THEY ARE VERY WELL CONSERVED. EPITHELIA ALL THE WAY TO [INDISCERNIBLE] BUT THE MAIN DIFFERENCE BETWEEN THE [INDISCERNIBLE] AND HUMANS IN GENERAL, [INDISCERNIBLE] THERE IS ONLY ONE OR TWO [INDISCERNIBLE]. IN HUMAN CELLS THERE ARE FIVE OF THEM. CALLED WRECK RECQ 1, RECQ 5. AND WE KNOW THAT MUTATIONS OF RECQ 4, [INDISCERNIBLE] LEADS TO AN INCREASE IN [INDISCERNIBLE]. I DON'T MIND GOING TO THE THE [INDISCERNIBLE] BECAUSE THAT'S NOT REALLY THE PURPOSE OF THIS TALK BUT I WILL PARTICULARLY MENTION THE DIFFERENCE. SO AS MANY KNOW, [INDISCERNIBLE] ALSO SUGGEST THAT THE COMMUNICATORS, ALTHOUGH THEY ARE IN [INDISCERNIBLE], THEY'RE ALSO LIKELY TO PLAY DISTINCT FUNCTION IN CELLS. THE GOAL WITH THE DISTINCTION FUNCTION OF THESE PROTEINS MIGHT BE. WHEN [INDISCERNIBLE] REACTOR, WE DECIDED TO ESTABLISH A COLLABORATION WITH A GROUP [INDISCERNIBLE] AND THE IDEA WAS BASICALLY, HOPEFULLY, TO FIND PROTEINS THAT INTERACT SPECIFICALLY WITH EACH ONE OF THE FIVE [INDISCERNIBLE] AND SO IN THIS CASE, TO CREATE HER293 CELL LINES THAT EXPRESSES A VERSION OF EACH LINE OF [INDISCERNIBLE]. AND IN THIS CASE, THE GENO [INDISCERNIBLE] IS SPECIFICALLY [INDISCERNIBLE] WHOLE CELL. AND THEN WE [INDISCERNIBLE] EXPRESSION LEVEL OF THIS PROTEIN USING DIFFERENT [INDISCERNIBLE]. SO WE CAN VISUALIZE, ACTED THEN AS MENTIONED BY THE [INDISCERNIBLE] WE GO DEMISE THE EXPRESSION OF THE [INDISCERNIBLE] PROTEIN. SECRETION WHERE THE EXPANSION LEVEL OF THE CELLULAR PROTEIN IS MORE OR LESS EQUIVALENT TO THE EXPRESSION LEVEL OF THE EXXONINOUS PROGRAM. [INDISCERNIBLE] ASSOCIATED WITH OVEREXPRESSION OF THE PROTEIN. THEN WE WENT THROUGH TWO STEPS OF REPLICATION TO IDENTIFY PROTEINS [INDISCERNIBLE] FOR THE PURPOSE OF TODAY'S TALK, TO FOCUS ON [INDISCERNIBLE], AND IN PARTICULAR [INDISCERNIBLE] THAT WE FIND IS THE MOST PROMINENT BINDING PARTNER THAT WE FOUND ON OUR SCREEN WAS 5-1. THIS TABLE SHOWS YOU [INDISCERNIBLE] THAT WE, THEN [INDISCERNIBLE]. SO THEN WE WENT ON TO CONFIRM THAT RECQ 1 [INDISCERNIBLE] AND SO WE COULD SEE THAT, INDEED, BASICALLY RECQ USE BEEN ANTIBODIES. AND THEN [INDISCERNIBLE] REGULATED BY THE REPLICATION OF [INDISCERNIBLE]. AS YOU KNOW, MANY [INDISCERNIBLE] IN PROTEIN ARE INTERACTING ALSO WITH THE POLY ADP RIBOSYL THAT IS FORMED. SO TO PERFORM THIS, YOU USE [INDISCERNIBLE] YOU CAN CLEARLY SEE THAT [INDISCERNIBLE] THERE IS INTERACTION, WHICH IS DECREASING IF YOU USE THE [INDISCERNIBLE]. THEN TO SEE WHETHER THE INTERACTION WITH THOSE REGULATED [INDISCERNIBLE] INDUCTION. AND, INDEED, WE SAW THAT IF WE, FOR EXAMPLE, TAKE THAT [INDISCERNIBLE] WE SEE THAT THE INTERACTION IS SLIGHTLY INCREASED BUT THIS IS AGAIN DECREASING THE [INDISCERNIBLE] AND [INDISCERNIBLE] SAME CONCLUSION USING OTHER DAMAGING AGENTS SUCH AS [INDISCERNIBLE] OXYGEN PEROXIDE. MEANING THAT THE [INDISCERNIBLE] IS INCREASED, DAMAGING ACTION AND DECREASED IN THE PRESENCE OF THE [INDISCERNIBLE] SO THEN WE WENT ON TO CONFIRM THAT ACTUALLY THE TWO PROTEINS, PHYSICALLY INTERACT, USING THE VERSIONS OF WRECK -- RECQ 1. WE CONFIRMED THAT BY USING BOTH [INDISCERNIBLE]. AND USING THE [INDISCERNIBLE] WE ALSO USED RECQ 1 WHICH ALLOWS THE [INDISCERNIBLE] IN DOMAIN OF RECQ 1 WHICH IS IMPORTANT. AND PERHAPS NOT SURPRISINGLY, WHAT WE SAW IS THAT THERE IS ENOUGH [INDISCERNIBLE] BINDING, WHICH CONTAINS THE SO-CALLED DOMAIN OF IT AND I THINK THAT'S NOT VERY SURPRISINGLY, BECAUSE BASICALLY THEY WENT [INDISCERNIBLE] LESS CONSERVED THAN ARE COMMUNICATED AND [INDISCERNIBLE] PROTEIN-PROTEIN INTERACTIONS. AND ALSO, WE WENT ON AND MAPPED THE DOMAINS OF PATHS WHICH WRECK WRECK 1 FINDING -- RECQ 1 PINNING. ONE IS THE [INDISCERNIBLE] ALSO CORRESPONDING TO THE DNA BINDING DOMAIN. THE OTHER IS THE PART WHICH IS [INDISCERNIBLE] AND I SHOULD MENTION, A FEW YEARS AGO ALSO SHOWED THAT [INDISCERNIBLE] FURTHER INTERACTING WITH PARP, INTERACTING WITH THE SAME TWO [INDISCERNIBLE] RECQ 1. WHICH IS INTERESTING. THE OTHER THING WE DID, TEST WHETHER RECQ 1 WAS INTERACTING WITH PARP, WHERE PARP WAS THE [INDISCERNIBLE]. AND BECAUSE AGAIN, IF YOU REMEMBER THE SLIDES [INDISCERNIBLE] THE INTERACTION. SO THE ASSAY DONE IN THE RECQ 1 FAMILY THAT WE USED IN THE [INDISCERNIBLE] ASSAY, AND TOLD THAT YES, RECQ 1 INVOLVING PARP RECOGNITION IS ALSO BINDING [INDISCERNIBLE]. FINALLY, WE ALSO TESTED RECQ 1 BY NOT ONLY [INDISCERNIBLE], SUBSTRATE. AND WHAT WE FOUND THE SAME CENTER OF THE REGION CAN BE [INDISCERNIBLE] BY PARP. AGAIN, THIS IS NOT SURPRISING BECAUSE MANY [INDISCERNIBLE] INTERACT WITH PARP, ALSO KNOWN TO BE [INDISCERNIBLE] BASICALLY, PROGRESSI ONIVE PHENOTYPE. THAT ADP IS NOT [INDISCERNIBLE]. [INDISCERNIBLE] IS THAT IF WE STARTED [INDISCERNIBLE], AND SET UP THE [INDISCERNIBLE] INTO THE [INDISCERNIBLE], IN THIS CASE, WE SEE THAT [INDISCERNIBLE] IS STILL ABLE TO PREVENT FOR SLOWING, INDICATING THAT IN THIS CASE, WHEN WE NEED IT FAST, CELLS HAVE STILL [INDISCERNIBLE] WHICH IS IMPORTANT TO THE START OF ADVANCED FORK WHICH IS [INDISCERNIBLE]. SO WE CUE NOT HAVE THIS. SO THEN WE DECIDED TO VALIDATE ALL THESE USING A [INDISCERNIBLE]. WE SEE THAT'S [INDISCERNIBLE] ONLY TECHNIQUE AVAILABLE TO ACTUALLY VISUALIZE THESE. AND IN THIS WAY WHAT WE WANT TO SEE IS THERE THAT FORMATION OF [INDISCERNIBLE]. THIS IS THE REVERSAL OF THE FORK. THAT BECOMES -- [INDISCERNIBLE] TO 30%, IF WE NEED THE PATH, THE FRACTION OF PREVENT FORKS DECREASES TO 86%, CONSISTENT WITH THE NOTION THAT [INDISCERNIBLE] REVERSAL. IF WE USE THE PATH IN THE ABSENCE OF RECQ 1, THE FRACTION OF REVERSE FORKS REMAIN HIGH. HERE AGAIN, THE [INDISCERNIBLE] MISSING TO DRIVEN THE FORKS. AND THESE EXPERIMENTS SHOWN HERE, [INDISCERNIBLE] TREATING CELLS WITH CANCER, THEN REUSING THEM FROM CANCER TREATMENT AND WAITING THREE HOURS AFTER CANCER [INDISCERNIBLE] BEFORE PREVENT FORK. WHAT YOU SEE HERE IS THAT EVEN AFTER THREE HOURS AFTER THE REMOVAL, THERE IS A HIGH FRACTION OF REVERSE FORK IN ABSENCE OF RECQ 1, LEADING THAT RECQ 1 IS THE KEY FACTOR AND NO OTHER FACTORS IN THE CELL CAN ACCOUNT FOR THE ABSENCE OF RECQ 1. THIS IS WHAT WE HAVE PERFORMED, WHERE WE THINK [INDISCERNIBLE], WHICH IS [INAUDIBLE] NOW, THIS HAS ACTUALLY INDICATION BECAUSE YOU KNOW THAT WHEN [INDISCERNIBLE] ALWAYS USING THE CLINIC FOR CANCER TREATMENT. AND ALSO USED IN THE [INDISCERNIBLE] WHEN YOU [INDISCERNIBLE] COMBINATION WITH [INDISCERNIBLE]. SO I THINK HOW WE HAVE A RATIONAL INFORMATION, BECAUSE ONCE IT HAPPENS, IF WE TREAT THE [INDISCERNIBLE] AND PATH INHIBITOR. IN THIS CASE, RECQ 1 WOULD BE FREE TO GO. SO IT WOULD BE SAFE TO [INDISCERNIBLE] FORKS BEFORE THE LESION IN THE [INDISCERNIBLE]. SO RECQ 1 LEADING TO THE [INDISCERNIBLE]. AS YOU KNOW, PATH INHIBITOR KNOWN TO BE PARTICULARLY EFFECTIVE IN A BRCA 1 OR 2 DEFICIENT [INDISCERNIBLE], AND THEY ARE TWO ESSENTIAL PROTEINS, SO THIS WOULD EXPLAIN WHY THE COMBINATION OF THESE TWO DRUGS IS PARTICULARLY EFFECTIVE AGAINST CELLS THAT ARE DEFECT IN [INDISCERNIBLE] GENES. HOWEVER, WITH [INDISCERNIBLE] THAT IF WE NEED A PATH [INDISCERNIBLE], THEN WE WON'T HAVE A [INDISCERNIBLE]. AND THE REASON WHY IS BECAUSE IN THIS CASE, EVEN THOUGH PATH IS INHIBITED, WE ARE [INDISCERNIBLE] WHICH IS IMPORTANT TO [INDISCERNIBLE] ACCUMULATION OF THE REVERSE FORK FACTORS, WHICH THEN MAY BE ESTABLISHED IN A LATER TIME BY [INDISCERNIBLE]. ACTUALLY, THERE IS NO EVIDENCE THAT THIS IS PROBABLY THE CASE. SO IF THIS IS MODEL IS RIGHT, WHAT OUR [INDISCERNIBLE] WAS, CANCER CELLS [INDISCERNIBLE] INHIBITORS WE SHOULD HAVE ACCUMULATION OF [INDISCERNIBLE]. BUT THESE CELLS [INDISCERNIBLE] DEFICIENT BACKGROUND, SHOULD BE [INDISCERNIBLE]. AND WHEN I SAY [INDISCERNIBLE] CELLS I SHOULD MAKE SURE THAT IN THIS CASE WE USED VERY LOW DOSES OF [INDISCERNIBLE] PAUSE OF COURSE IF YOU USE MICRO MOLAR DOSES YOU WILL SEE THAT [INDISCERNIBLE] ACCUMULATION. BUT THAT NANOMOLER DOSES, WE DON'T SEE [INDISCERNIBLE] ACCUMULATION. AND SO WE WANTED TO DEMOING STRAIGHT AND WE USED TWO PARALLEL APPROACHES TO IDENTIFY [INDISCERNIBLE] MEASURE. SO THIS IS A [INDISCERNIBLE] EXPERIMENT. WHERE YOU USE UNITING [INDISCERNIBLE] OF THE CONTROLS, AND AGAIN, YOU SEE THAT YOU TREAT CELLS IN THIS CASE WITH 25 NANOMOLER CANCER PATIENTS, WE HAD NO ACCUMULATION OF [INDISCERNIBLE]. HOWEVER, WITH [INDISCERNIBLE] CONSISTENTLY, WE NEEDED PATHS [INDISCERNIBLE] ACCUMULATION. BUT IF WE NEEDED PATH EFFICIENT BACKGROUND, THEN THE FRACTION OF [INDISCERNIBLE] GOES DOWN. AND THEN WE ALSO VALIDATED THIS USING AN APPROACH [INDISCERNIBLE] AND IN THIS CASE, WHAT WE DID WAS TO LOOK AT THE [INDISCERNIBLE] BETWEEN GAMMA [INDISCERNIBLE] AS A DOWNSTREAM OF [INDISCERNIBLE]. AND [INDISCERNIBLE] IN THIS GRAPH IS SHOWN BY THIS GRAY BOX, WHICH IS SHOWN HERE. AND AGAIN, WHAT WE SEE IS THAT [INDISCERNIBLE] SAME BY USING 25 NANOMOLER, AACCUMULATION OF [INDISCERNIBLE] OUR IF YOU USE [INDISCERNIBLE] GOES DOWN. SO WE THINK THAT THIS REALLY PROVES OUR MODEL THAT WE HAVE BASICALLY, NOW CALL IT AN IMPORTANT MECHANISM OF [INDISCERNIBLE] RESPONSE ACTIVATED AT THE [INDISCERNIBLE] INHIBITION. WE MAY PREDICT WHAT MAY BE [INDISCERNIBLE] BY LOOKING ALSO AT THE [INDISCERNIBLE] GENETIC BACKGROUND. SO [INDISCERNIBLE] JUST TO MAKE SURE THAT THOSE ARE NOT [INDISCERNIBLE], SO THESE ARE BASICALLY THE -- THIS IS A TAKE HOME MESSAGE OF ALL OUR WORK. SO AGAIN, WE THINK WE HAVE IDENTIFIED ANOTHER ROLE FOR THE HUMAN [INDISCERNIBLE] IN THE FORKS THAT REVERSED [INDISCERNIBLE] LESION. ROLE IS MUCH SHARED BY OTHER COMMUNICATIONS, AND WE ALSO EXPLAIN WHAT MIGHT BE THE ROLE OF [INDISCERNIBLE] IN THIS CONTEXT, AGAIN, NOT REALLY TO DRIVE THE FORMATION OF THE ADADVANCED FORKS BUT MOSTLY TO PREVENT RECQ 1 FROM [INDISCERNIBLE]. THIS IS IMPORTANT FOR TWO REASONS, FIRST OF ALL, [INDISCERNIBLE] LEADING TO FORK REVERSAL IN A LARGE [INDISCERNIBLE] OF THE DNA CELLS AND SECOND, BECAUSE NOW I THINK OUR FINDING PROVIDE A NEW RATIONALE FOR DEVELOPMENT OF NOT OBJECTIVE [INDISCERNIBLE] FORK REVERSAL IN [INDISCERNIBLE], TO BASICALLY SENSITIZE US TO LOWER THE DRUGS THAT ARE USED IN THE [INDISCERNIBLE]. AND OF COURSE NOW WE ARE ACTIVELY BEGINNING THIS ASPECT, ALSO SEARCHING FOR A TRADITIONAL FACTORS THAT MIGHT BE INVOLVED IN THIS PROCESS. I'D LIKE TO THINK BY ACKNOWLEDGING THE PEOPLE THAT DID THE WORK, AND SOME ARE NOW WITH ME, IN ST. LOUIS. SOME ARE IN [INDISCERNIBLE]. IN PARTICULAR I WOULD LIKE TO ACKNOWLEDGE [LIST OF NAMES] THAT THE LOCATION OF ST. LOUIS, AND SHE'S [INDISCERNIBLE] MY LAB IN ST. LOUIS. AND [INDISCERNIBLE] KEY COLLABORATORS, WHICH ARE [INDISCERNIBLE] KEY COLLABORATORS FOR THE STUDY [INDISCERNIBLE] OF THE UNIVERSITY OF [INDISCERNIBLE] WHO DID THE [INDISCERNIBLE] WORK. INITIALLY WAS INCREMENTAL [INDISCERNIBLE] THANK YOU FOR YOUR ATTENTION. [APPLAUSE] >> THANK YOU VOUCH FOR AN EXCELLENT TALK. AND NOW WE'RE ALSO ON TIME, WHICH IS APPRECIATED. NOW WE'RE GOING AROUND TO THE DIFFERENT PLACES AND ASK FOR QUESTIONS, AND ONE QUESTION PER CENTER HERE. TO BEGIN WITH, AND WHY DON'T WE START WITH NIHS. ARE YOU THERE? >> I WILL, WE ARE HERE. WE CAN'T SEE OURSELVES. THANK YOU FOR THE TALK. IT WAS EXCELLENT. I WAS WONDERING IF IN YOUR SYSTEM OR OTHER SYSTEMS, IF OVER EXPRESSION OF RECQ 1 OR PARP IS WOULD INCREASE DOUBLE STRAND BREAKS? >> OVEREXPRESSION? >> IS THIS ANYTHING THAT YOU COULD DO, ANY FORM OF GENO TOXIC THAT STALLS THE FORK AND PROMOTES FORK REVERSAL, IF YOU OVEREXPRESSED THE PROTEINS NECESSARY FOR REVERSING SUCH FORKS, WOULD YOU -- SORRY. I GUESS YOU WOULD HAVE TO INHIBIT THEM. MY MISTAKE. CAN YOU GET MORE GENO TOXICITY OR CYTOTOXICITY FROM INHIBITING THE FUNCTION OF THESE PROTEINS TOGETHER? >> YEAH, I THINK THAT'S A GOOD POINT. ACTUALLY, WE DIDN'T TEST THAT. BUT -- SORRY [INDISCERNIBLE] CAN YOU SEE ME? >> YES. >> SO WE DIDN'T TEST THAT, BUT [INDISCERNIBLE] THAT THAT'S THE CASE, BECAUSE WHEN YOU ACTUALLY DID THE [INDISCERNIBLE] EXPRESSING THE RECQ 1, WE COULD SEE THERE WAS MORE TOXICITY WITH THE INCREASED LEVEL OF RECQ 1. BUT WE DIDN'T TEST IT DIRECTLY. >> THANK YOU. INTERESTING. >> OKAY, WE CAN'T REALLY SEE YOU RIGHT NOW, THE PEOPLE THAT ASK QUESTIONS, SO PLEASE IDENTIFY YOURSELF. LET'S GO TO OREGON. >> AT THIS POINT WE DON'T HAVE ANY QUESTIONS. THANK YOU VERY MUCH. >> OKAY. PITTSBURGH? >> NICE WORK. WE DON'T HAVE ANY QUESTIONS AT THE MOMENT. >> [INDISCERNIBLE] MICHIGAN? >> YES HAVE THANK YOU VERY MUCH FOR GREAT TALK. IF [INDISCERNIBLE] IS REMOVED WOULD YOU THE FORKS START UP AGAIN WITH THE SAME SPEED THAT THEY DID BEFORE YOU INHIBITED. THE OP1? -- TOP 1? >> SO YOU'RE ASKING IF [INDISCERNIBLE], IF FORK NORMALLY? >> YES. >> REMOVAL OF [INDISCERNIBLE], THEN YOU WILL SEE THAT FORK CAN REVIEW NORMALLY, IF RECQ 1 IS PRESENT, OF COURSE. >> OKAY. THANK YOU. >> SORRY. TO BE MORE PRECISE, KEEP IN MIND THIS IS THROUGH WHEN YOU USE [INDISCERNIBLE], RIGHT? SO WE'RE TALKING ABOUT [INDISCERNIBLE] UNDER THESE CIRCUMSTANCES. OKAY. THANK YOU VERY MUCH. NICE TALK. >> OKAY. STONEBURG, BRUCE? ARE YOU THERE? >> YES. BRUCE HERE. WE DO HAVE A QUESTION. NAMELY, DO YOU SEE THE SAME PROTEINS AND MECHANISMS INVOLVED FOR OTHER REPLICATION FORK INHIBITORS? I'M THINKING OF HYDROXY ARREARA, WHERE YOU DON'T GET PROTEIN D.N.A. CROSS LINKS. >> THAT'S AN EXCELLENT QUESTION. THANK YOU FOR THAT. THAT'S WHAT WE ARE STUDYING RIGHT NOW. AND I CAN TELL YOU THAT YES, WE DO HAVE PRELIMINARY EVIDENCE THAT THE FORK REVERSAL IS A MORE GENERAL TRANSDUCTION THAT TAKES PLACE ALSO WHEN [INDISCERNIBLE] CHALLENGED. AND [INDISCERNIBLE] INCLUDING [INDISCERNIBLE]. ACTUALLY QUITE INTERESTING, I WOULD SAY, BECAUSE THE [INDISCERNIBLE] -- WHAT WE ARE WILLING TO PROPOSE IS THAT THE FORK REVERSAL IS [INDISCERNIBLE] WHENEVER THERE ARE OBSTACLES BASICALLY ON THE REPLICATION FORK. NOW THE FACT THAT WE MIGHT SEE FORK REVERSAL WHEN CHALLENGING [INDISCERNIBLE], ALSO SUGGESTIONS THAT MAKE FORK REVERSAL IS CERTAIN INDICATIONS IS DRIVEN BY [INDISCERNIBLE], IN THE [INDISCERNIBLE] INHIBITOR AT THE BEGINNING OF MY TALK, YOU MIGHT HAVE ACCUMULATION OF [INDISCERNIBLE] AHEAD OF THE FORK THAT MAY BE RESPONSIBLES TOIOUSLY DRIVING [INDISCERNIBLE]. RIGHT? WHEN YOU USE THE [INDISCERNIBLE], OF COURSE YOU ARE NOT CREATING DAMAGE AHEAD OF THE WORK. THE QUESTION WOULD BE HOW DO YOU REVERSE THE FORKS? WE THINK IN THAT CASE, THERE MIGHT BE FACTORS THAT ARE ACTUALLY INVOLVED IN THE FORMATION OF [INDISCERNIBLE]. VERY PRELIMINARY DATA. WE THINK, YES. A GENERAL [INDISCERNIBLE] >> OKAY. THANK YOU. >> THANK YOU. BRUCE AND KENTUCKY? DAVE, KENTUCKY? [INDISCERNIBLE] ISLAND? >> HELLO, THIS IS PHIL AT NORTH CAROLINA. I HOPE YOU'RE HEARING. >> YES. >> THANK YOU. I'M GOING TO CONTINUE ON WITH A QUESTION ALONG THE LINES THAT BRUCE POSED. AND THAT IS, OTHER KINDS OF LESIONS WHERE YOU'RE GETTING THIS KIND OF FORK REVERSAL, WHAT DO YOU THINK IS GOING ON WITH THE CMG HELICASE, WHICH IS REALLY THE DOMINANT HELICASE DRIVING FORK PROGRESSION. HOW DO YOU KNOW THAT HELICASE OFF TO ENABLE THE FORK TO REGRESS IN THE FIRST PLACE? >> OKAY. SO YOU MEAN WITH [INDISCERNIBLE] NDISCERNIBLE] >> YES. >> SO YEAH. SO WHAT WE DID [INDISCERNIBLE] EVERYBODY ELSE MUTE? OKAY. SO CAN YOU HEAR ME NOW? >> YES. >> SO [INDISCERNIBLE] WE DID A [INDISCERNIBLE] TO ALLOW THE FORMATION OF REVERSE FORKS. THAT WAS [INDISCERNIBLE] STRUCTURES, AND [INDISCERNIBLE] HOW DO THEY EVENTUALLY REASSOCIATE TO GET INTO THE [INDISCERNIBLE] OF THAT REPLICATION FORK. THIS IS SOMETHING WE'RE INVESTIGATING. AT FIRST THEY WON'T NEED TO DEASSOCIATION. AND WE HAVE [INDISCERNIBLE] TO THIS IS THE CASE. >> THANK YOU. >> OKAY. AND LET'S TAKE -- SO DO WE HAVE A QUESTION FROM HERE. ONE QUESTION AT THIS POINT. >> I WAS WONDERING ABOUT THE LEVELS OF RECQ 1 IN CANCERS. IS IT INCREASED. >> YES. HIGH INCREASE, YES. IN MOST CANCERS, YES. EXPRESSION LEVELS. RIGHT? YES. >> SO JOHN AT BETHESDA, DO YOU HAVE ANY QUESTIONS? >> WE ENJOYED THE TALK BUT DON'T HAVE ANY QUESTIONS. >> OKAY. I THINK I GOT THROUGH SO RIGHT NOW WE'LL -- IT'S OPEN FOR QUESTIONS FROM ANYWHERE, INCLUDING HERE. SO MICHAEL, YOU CAN ANSWER NOW. >> IN THE FIBER ASSAYS, CAN YOU DISTINGUISH A REVERSAL FROM STAGNATION OF THE RECENTLY INCORPORATED [INDISCERNIBLE] >> NO. FROM THE FIBER ASSAY, WHAT WE CAN SAY, ALL WE CAN SAY IS THAT THERE IS REPLICATION FOR [INDISCERNIBLE]. THAT'S WHY WE NEED TO COMBINE THE FIBER EXPERIMENTS WITH THE NECROPSCY EXPERIMENTS TO REALLY [INDISCERNIBLE] USED IN FORMATION OF REVERSE FORKS. WE ARE INTERESTED IN LOOKING AT DEGRADATION. THAT IS SOMETHING THAT COULD HAPPEN AT THE LEVEL OF REVERSE FORK BUT THAT WOULD REQUIRE DIFFERENT TEAM SO IN ORDER TO LOOK AT THE DEGRADATION OF FORKS WHAT WE DO IS DO WHAT [INDISCERNIBLE], AND THEN TREATMENT. SO IN THAT WAY, THE FORKS WOULD BE READ AND GREENE AND IF THERE IS DEGRADATION, THEN THEY SHOULD BECOME SHORT. THAT ACTUALLY THE SAME APPROACH THAT WAS USED BY [INDISCERNIBLE] TEAM TO LOOK AT [INDISCERNIBLE] DEGRADATION FORKS. FROM THE EXPERIMENT, WE CAN SEE THAT THERE IS REPLICATION FORKS [INDISCERNIBLE]. TO ASSOCIATE THAT IN THE [INDISCERNIBLE] REVERSE FORKS, WE THEY'D TO COMBINE THE DATA TO [INDISCERNIBLE]. >> OKAY. >> DO WE HAVE A QUESTION FROM STONEY BROOK? HELLO? >> [INDISCERNIBLE] [INAUDIBLE] >> THIS IS RACHEL. AND WHAT? OKAY. MY QUESTION IS ABOUT THE CHICKEN FOOT STRUCTURE THAT YOU CALLED IT. THERE IS LIKE THE REVERSED PIECE. WHAT IS THAT WORKING OFF OF? HOW IS THAT BUILDING ITSELF? >> THE REVERSE FORK? >> YES. >> YES. WOULD IT BE POSSIBLE TO HAVE A LARGE [INDISCERNIBLE]. >> I DON'T KNOW IF I CAN FIGURE THAT OUT. >> I'LL EXPLAIN, SO BASICALLY, THAT CAN BE A [INDISCERNIBLE] BECAUSE YOU HAVE THE [INDISCERNIBLE] WHICH ARE COMPLEMENTARY. AND THEY CAN JUST STAY TOGETHER; RIGHT? THAT WOULD GIVE THE CHICKEN STRUCTURE. A REPLICATION FORK, IN MY SECOND SLIDE, YOU HAVE THE DATA AND LEADING [INDISCERNIBLE] WHICH ARE COMPLIMENTARY, AND THOSE TWO CAN [INDISCERNIBLE] AND TOGETHER TO FORM A CHICKEN FOOT FRACTURE. THAT'S EASY TO FORM, AND IS NOT VERY [INDISCERNIBLE] UNFAVORABLE PROCESS, BECAUSE YOU WILL SEE [INDISCERNIBLE], RIGHT INSO YOU CONTINUE NEED ANY NEW DNA [INDISCERNIBLE]. DID I ANSWER YOUR QUESTION? >> YES. THANK YOU. FINAL QUESTION OR COMMENT. >> A QUESTION HERE IN CHAPEL HILL. >> ONE MORE. >> I HOPE YOU CAN HEAR ME. I CANNOT SEE YOU. >> [INDISCERNIBLE] CHAPEL HILL. >> HI. >> MY QUESTION HAS TO DO WHETHER -- HAVE YOU DONE ANYTHING TO AVOID ANY FORK MOVEMENT IN SOLUTION? IN OTHER WORDS? HAVE YOU BEEN ABLE TO [INDISCERNIBLE] THE DNA, TO BE SURE THAT THE CHICKEN FOOT STRUCTURE THAT YOU SEE WERE FORMED DURING YOUR EXPENSAL POSITION OR WERE FORMED IN SOLUTION PRIOR TO THE [INDISCERNIBLE]? >> WELL, WE [INDISCERNIBLE] WE HAVE CONTROL FOR THAT. BECAUSE, FIRST OF ALL, WHAT WE SEE IS THAT IF WE DON'T ADD [INDISCERNIBLE] WE CONTINUE HAVE ANY REVERSE FORK STRUCTURE. OKAY? SO IN THE ABSENCE OF 25 NANOMOLER [INAUDIBLE] SORRY. IN THE ABSENCE OF [INDISCERNIBLE], WE DON'T SEE ACCUMULATION OF REVERSE FORKS. AS SOON AS AS WE ADD [INDISCERNIBLE] AND WE DO HAVE ONE HOUR TREATMENT, A SHORT TREATMENT, WE SEE THAT 30% OF FORK UNDERGO FORK REVERSAL. SO THAT'S THE [INDISCERNIBLE] TREATMENT, ALSO BECAUSE OF THE [INDISCERNIBLE] AND WE SEE -- WE ATTEMPT REVERSE FORK EVEN WHEN YOU TREAT THE [INDISCERNIBLE] WITH 30 [INDISCERNIBLE]. SO WHICH MEANS AGAIN, AS SOON AS WE ARE [INDISCERNIBLE], WE START [INDISCERNIBLE] STRUCTURES. NOW, WE [INDISCERNIBLE] OTHER AGENTS, WE MIGHT HAVE REVERSE FORKS THAT MIGHT ORIGINATE BECAUSE OF THE TREATMENT [INDISCERNIBLE] >> DOESN'T SURPRISE YOU THAT NANOMOLER CONCENTRATION, 30% OF FORKS REGRESSING? >> NO. ACTUALLY, I THINK THAT'S THE KEY POINT OF YOUR WORK. [INDISCERNIBLE] FIRST PART OF MY TALK INITIALLY THAT THE [INDISCERNIBLE] WERE DONE BY THE GROUP OF [INDISCERNIBLE]. I MEAN I WOULD SAY IF 30 NANOMOLER COMPOSITION IS [INDISCERNIBLE], EFFICIENT TO USE SIGNIFICANT FORK REVERSAL. BUT AGAIN IS NOT IN USE FORMATION OF [INDISCERNIBLE]. SO WHEN YOU INCREASE THE [INDISCERNIBLE] 1 MICRO MOLAR, WHICH ARE KNOWN TO BE SAY A BUILT MORE TOXIC, IN THAT CASE, YOU ALSO [INDISCERNIBLE] NOW, HAS DONE, LOW DOSES. THERE IS A SIGNIFICANT FRACTION OF FORKS THAT UNDERGO [INDISCERNIBLE] TO BASICALLY PREVENT ANY TYPES OF TOXICITY WITH LOW TREATMENT OF -- WITH TREATMENT OF CELL [INDISCERNIBLE] DOSES. WE TREAT THE DOSE OF [INDISCERNIBLE] WE ALSO SEESON. MORE LESIONS, FORK REVERSAL WOULD NOT BE ABLE TO COMPENSATE FOR THE [INDISCERNIBLE] ON THE DNA. >> THANK YOU. >> THANK YOU. I THINK WE'LL STOP THE SESSION NOW AND WE WILL ASK YOU A FEW MORE QUESTIONS. THANK YOU EVERY ONE, FOR ATTENDING. I THINK IT WORKED PRETTY WELL. >> BYE