>>I AM FROM THE NIDDK WORKSHOP SUPPORTED BY THE GLYCOSCIENCE COMMON FUND WORKING GROUP. AND WE FEATURE SEVERAL OF THE TOOLS THAT HAVE BEEN DEVELOPED WITHIN THIS INITIATIVE AND ALSO SOME ADDITIONAL OUTSTANDING SPEAKERS. DON'T BE SHY ASKING QUESTIONS IN THE CHAT OR SENDING EMAILS TO THE PANEL OR CONTACTING THE SPEAKERS. FOR SAKE OF TIME WE'LL START WITH THE FIRST SPEAKER MICHAEL GELB. REMINDING THE SPEAKERS YOU HAVE 15 MINUTES FOR YOUR PRESENTATION PLUS FIVE MINUTES FOR QUESTIONS. LET'S ALSO THANK IN PARTICULAR AMANDA THAT HAVE BEEN THE DRIVING FORCE BEHIND THIS VERY POPULAR WORKSHOP SERIES. MUTE YOURSELF IF NOT PRESENTING. >> CAN YOU HEAR ME? >> WE CAN HEAR YOU, MIKE. >> GOOD MORNING, EVERYBODY. THANK YOU. SO I'M NOT A GLYCOBIOLOGIST BUT I ALWAYS SORT OF ANDENNERRED MY WAY INTO -- WANDERED MY WAY INTO THE FIELD FOR MY WORK IN NEWBORN SCREENING. I'M GOING TO COVER THE BAY SIX OF NEWBORN SCREENING SO WE'RE ON THE SAME POINT. I'M GOING TO TALK ABOUT MY CO-POLYSACCHARIDE OSIS. MUCOPOLYSACCHARIDE OSIS AND GLYCANS. WE'LL TALK ABOUT ENZYME MULTIPLEX FOR MDS AND THEN THE KEY IS OPTIMIZING NEWBORN TRAINING NPS. NPS 1 USING SECOND TIER BIOMARKERS WHICH IS THE GLYCOSAMINOGLYCANS. WHICH IS VERY IMPORTANT TO CHEMISTRY AND MEDICINE. I'LL TRY TO GO TO THE NEXT SLIDE. >> IT DOESN'T SEEM TO ADVANCE. CAN YOU GO TO THE NEXT SLIDE. SO BRINGING YOU UP TO SPEED. NEWBORN SCREENING WE TEST ALL BABIES AT BIRTH FOR RARE GENETIC DISEASE BUT NOT ALL THOUSANDS OF DISEASES RETEST FOR ABOUT 50 DISEASE THAT ARE TREATABLE IF WE START EARLY IN LIFE. SO IF WE DON'T SCREEN BABIES BORN, THEY GET SICK WE DON'T KNOW WHAT'S WRONG, SICK MONTHS LATER THEY DIE, BY THEN IT'S TOO LATE TO TREAT THE BABY, SYMPTOM ARE IRREVERSIBLE. OFTEN NO FAMILY HISTORY, THESE ARE RARE DISEASES UNLESS MAYBE THE FIRST SIB WAS BORN WHO DEVELOPED THE DISEASE. IT WOULD BE NICE TO CATCH THE FIRST KID BEFORE -- SO THE SECOND KID DOESN'T HAVE TO BE SACRIFICEED. IN THE UNITED STATES NEWBORN SCREENING IS PART OF STATE PUBLIC HEALTH AND EACH STATE HAS THEIR OWN NEWBORN SCREENING LAB AND SMALLER STATES ARE CHANGING. NEWBORN SCREENING USES FDA APPROVED NEWBORN SCREENING KITS OR THEY DEVELOP LABORATORY DEVELOPED TESTS. CDC AT FEDERAL LEVEL MAINTAINS QUALITIES CONTROL. THIS IS A STATE PROGRAM. THE WAY YOU EXPAND NEWBORN SCREENING, THREE YEARS AGO WE ADDED MPS 1 TO THE PANEL. THERE'S TWO WAYS ONE IS PARENTHOOD ADVOCACY WHICH IS MORE A STATE BY STATE CAMPAIGN GRASSROOTS, THE OTHER IS NIH THROUGH FIRST DIVISION FUNDS SECRETARY ADVISORY COMMITTEE ON HEALTH AND HEARTABLE DISORDERS IN NEWBORN AND CHILDREN. YOU CAN -- ANYBODY CAN NOMINATE A NEW CONDITION FOR CONSIDERATION ADDITION TO THE RECOMMENDED UNIFORM SCREENING PANEL, SO CALLED RUSP. THIS GOES THROUGH EVIDENCE REVIEW PROCESS FOR CONSIDERATION. SO THEN IF IT'S ON THE RUSP ALL STATES ARE ADVISED TO HAVE THE CONDITION BUT NOT MANDATORY, IT'S STILL DEPENDS ON STATE LEGISLATION. MOST STATES IN THE UNITED STATES SCREEN FOR 40 TO 50 CONDITIONS. I WOULD SAY NEWBORN SCREENING IS MORE PROGRESSIVE IN TAIWAN AND LESS PROGRESSIVE IN EUROPE, JAPAN AUSTRALIA AND CHINA AND MOST OTHER COUNTRIES SCREEN LITTLE SO WE'RE MANY BETWEEN TAIWAN AND SAY EUROPE. NEXT SLIDE PLEASE. SO MY LAB HAS BEEN INVOLVED OVER THE LAST DECADE IN THE EXPANSION OF NEWBORN SCREENING TO INCLUDE A SECOND SET OVALISSOMAL STORAGE DISEASES OUGHLIESSOMAL -- LYSESOME A.M. -- LYSOSOMAL STORAGE DISEASE, DUE TO BREAK DOWN OF CELLULAR COMPONENTS IN THE LYSIS. SOME ARE TREATABLE BY ENZYME REPLACEMENT THERAPY. SO THE BIOTECH COMPANIES MAKE RECOMBINANT AND FUNCTIONAL ENZYME WHICH IS ADMINISTERED BY IV EVERY FEW WEEKS. AND THEN TURNS OUT THESE ENZYMES CAN BE SPONTANEOUSLY TAKEN UP IN CELLS BY HETERO CYTOSIS FOLLOWED BY ENDOSOMAL DELIVERY SO THE ENZYME GETS INTO ORGANELLE WITHOUT ACTUALLY PENETRATE THROUGH MEMBRANE. IT WORKS FOR NON-NEUROLOGICAL LSD WHEN THE ENZYME DOESN'T NEED TO GET INTO THE BRAIN CNS. BUT EVEN DISEASES THAT HAVE A BIG CNS COMPONENT WE CAN TREAT SOME OF THEM BY BONE MARROW TRANSPLANTATION SO THE BONE MARROW IS REPLACED WITH DONOR CELLS WHICH CONTAIN FUNCTIONAL ENZYME. THESE REPOPULATE THE BONE MARROW AND SUPPLY HEMOPOIETIC CELLS OF THE BLOOD BUT SOME CELLS SPONTANEOUSLY BUREAU INTO THE BRAIN AND THEN SECRETE LYSOSOMAL ENZYME INTO BRAIN INTERSTITIUM WHERE IT'S TAKEN UP BY RESIDENT CELLS OF THE BRAIN SO THIS IS THE SO CALLED CROSS DIRECTION. THERE ARE MORE THAN 20 ONGOING GENE THERAPY CLINICAL TRIALS RIGHT NOW AND LSDs ARE EXPECTED AMONG THE FIRST DISEASE TREATABLE BY GENE THERAPY. IT'S LOW HANGING FRUIT BECAUSE YOU DON'T HAVE TO EXPRESS ENZYME IN EVERY CELL IN THE BODY BECAUSE THE ENZYME TRAVELS FROM CELL TO CELL AND TISSUE TO TISSUE. IT'S KIND OF AN UNUSUAL SITUATION. SO WE NEED TO DEVELOP TECHNOLOGY, NEWBORN SCREENING TECHNOLOGY TO KEEP UP WITH RAPID EXPANSION OF TREATABLE GENETIC DISEASE. AND THE KEY IS MASS SPECTROMETRY, NOT FLUORESCENCE AND NOT DNA SEQUENCING. NEXT SLIDE. I'LL SKIP THIS. THERE'S A LOT OF DNA SEQUENCING GOING ON BUT IT TURNS OUT IT'S NOT REALLY WELL SUITED FOR NEWBORN SCREENING, STILL TOO SLOW AND EXPENSIVE BUT EVEN MORE SERIOUS, WE OFTEN DO NOT KNOW HOW TO INTERPRET ALL THE GENOTYPES. WE HAVE VARIATIONS OF UNKNOWN SIGNIFICANCE, WE HAVE PARTIALLY PENETRANT MUTATIONS, WE ARE NOT GOOD AT INTEGRATING ALL PARTIALLY PENETRANT MUTATIONS TO MAKE CLEAR PREDICTION OF PROGNOSIS OF DISEASE. MORE WE'LL HAVE CARRIER ANALYSIS IN PLANNED PARENTHOOD BUT FOR NEWBORN SCREENING THIS HAS LIMITED USE RIGHT NOW. IT'S ALL NEWBORN SCREENING IS BASED ON BIOCHEMICAL ANALYSIS. NEXT SLIDE. SO THE -- I'LL TALK MUCOPOLYSACCHARIDE, ONE IN 70,000 BIRTHS. HOE WAS THE PIONEER WHO DEVELOPED REPLACEMENT THERAPY, IT WAS A STORY FEATURED ON OF 0 MINUTES. RECOMBINANT THE ENZYME IS TAKE UNUP CELLS TRAVELS TO LYSESOME, DEVELOPED BY BIOMARINE PHARMACEUTICALS. (INAUDIBLE) IS RYAN'S FATHER, FORMER PRESIDENT OF THE -- SOCIETY, NOW TERRY KLINE HAS THAT ROLE, WE WORKED CLOSE WITH THESE GUYS. NEXT SLIDE. WHAT WE HAVE IN NEWBORN SCREENING IS NO URINE SAMPLES NO OTHER TISSUE SAMPLES, IS AT THE BROADMAN CENTER ON FILLER CART. THE KEY IS 18 YEARS AGO (INAUDIBLE) ARGENTINA SHOWED MANY LYSOSOME LYSOSOMAL ENZYMES ARE STILL ACTIVE IN DRIED BLOOD SPOTS. THEN ABOUT 14 YEARS AGO WE DEVELOPED TANDEM MASS SPEC FOR MULTIPLEX ANALYSIS FOR SEVERAL ENZYMES IN DRIED BLOOD SPOTS. WE CAN ADD A CASSETTE OF ENZYME SUB RATES TO THE DRY BLOOD SPOT EXTRACT, LET INCUBATE, MAKE THE ENZYMATIC PRODUCTS AND ADD INTERNAL STANDARDS WHICH ARE CHEMICALLY IDENTICAL TO PRODUCTS BUT HAVE HYDROGEN REPLACED BY DETIER YUM SO WE MEASURE THE PRODUCT AND THE INTERNAL STANDARD BY TANDEM MASS SPEC. THE MORE PRODUCT YOU HAVE PER UNIT TIME THE MORE ENZYME ACTIVITY. IT'S TRIVIAL TO FIND PRODUCT AND INTERNAL STANDARD AN LITHES IN COMPLEX FLUID BY BLOOD BY TANDEM MASS SPEC. WE CAN DO MANY ENZYMES AT THE SAME TIME. USING A SINGLE MULTIPLEX TECHNIQUE. NEXT SLIDE. THIS IS AN EXAMPLE, FOR MPS 1. WE WOULD HAVE AN IGERONIC ACID AS GLYCOSITIC LINKAGE TO THIS ARTIFICIAL GROUP YOU SEE IN BLACK. THAT WOULD BE A TYPICAL SUBSTRATE. NEXT SLIDE. THIS IS FOR POMPEII BUT INTERNAL STANDARD CHEMICALLY IDENTICAL PRODUCT BUT MAYBE WE HAVE DETIER YUM HERE. THIS PRODUCT IS INFINITELY MULTIPLEXABLE. WE CAN BURY NUMBER OF METH LIEN GROUPS, VARY STRUCTURE OF CARBON. SO WE HAVE AS MANY CHANNELS AS WE WANT, THAT HAVE DIFFERENT MASSES WE CAN SEE ALL THESE ENZYMES AT AT THE SAME TIME. NEXT SLIDE. THE SOME OTHER FEATURES OF THE PRODUCT, HAS DICARBON ALLELE WHICH CAPTURES PROTON VERY NICELY FOR PROTONATION IN THE GAS PHASE FOR MASS SPEC. THEN THIS CARBONATE CLEAVES QUANTITATIVELY BY COLLISION INDUCED DISSOCIATION REGENERATE A SINGLE PRODUCT ION, THAT'S POSITIVELY CHARGED AND DIRECTING EVERYTHING ALONG THE SINGLE REACTION PATHWAY GIVES INCREASE SENSITIVITY. WHILE YOU CAN'T SEE THIS BUT RECENTLY WE PUBLISHED A MULTIPLEX PANEL FOR ALL THE MPS, THERE'S 8 OR 9 MPS DISEASES. YOU ARE MISSING A SINGLE ENZYME INVOLVED IN THE DEGRADATION OF GLYCOSAMINOGLYCANS GLYCOPOLYMERS SOCCER TIN SULFATE, HEPARAN SUP FATE, YOU NEED 8 OR NINE ENZYMES TO BREAK THEM DOWN. YOU HAVE GLYCOHYDROLASE, ACETYLTRANSFERASES, AND SEQUENTIALLY BREAK THESE THINGS DOWN. ON THE NEXT SLIDE. WE HAVE FDA APPROVED KIT WITH -- THAT CAME OUT FIVE YEARS AGO. THIS PAPER CAME OUT IN ANALYTICAL CHEMISTRY DOING ALL MPS AT THE SAME TIME. WE CAN DO MANY MORE ENZYMES THAN THIS, THIS IS JUST AN MPS PLEX FOR ALL AT THE SAME TIME. EACH PRODUCT HAS A UNIQUE RETENTION TIME AND MASS SPEC SIGNATURE AND WE CAN DO THIS INSIDE OF 1.5 MINUTES PER NEWBORN WHICH IS ABOUT THE SPEED WE NEED TO SUPPORT HIGH THROUGH PUT WITH NEWBORN SCREENING. NOW WE ARE COMING TO THE GLYCOSAMINOGLYCANS SO THE PROBLEM WITH ENZYME ASSAYS, THE ADVANTAGES IS WE CAN DO IT VERY EASILY, VERY FAST AND CHEAP, IT'S THE METHOD OF CHOICE, STILL IS FOR THE FIRST TIER NEWBORN SCREENING. BUT WE HAVE THIS PROBLEM OF PSEUDODEFICIENCIES SO YOU DON'T NEED 100% ENZYME, 100% OF POPULATION MEAN. MAYBE 20% OF THE ENZYME WOULD BE ENOUGH TO KEEP YOU HEALTHY. SO A NEWBORN SCREENING YOU TYPICALLY HAVE TO SET A CUT OFF SAY THE ENZYMES BELOW 10% YOU WOULD SAY THE NEWBORN IS SCREENED POSITIVE IN THE FIRST ROUND. THERE'S OVERLAP BETWEEN HEALTHY POPULATION AND THE DISEASE POPULATION. THERE WILL BE NORMAL PEOPLE THAT HAVE 10% ENZYME, SOME PATIENTS THAT ARE CLOSE TO 10%. THERE'S NOT A PERFECT SEPARATION. YOU WILL HAVE SO CALLED PSEUDODEFICIENCIES WHERE THE ENZYME READS LOW, IN VITRO BUT IT'S NOT A RESULT OF THE ASSAY, JUST YOU DON'T NEED 100% ENZYME TO BE HEALTHY. THESE DISEASES ARE CONTINUUM, IF YOU HAVE THE LOWER THE ENZYME THE MORE SEVERE DISEASE, THE EARLIER THE ONSET. SO IT'S NOT A BLOCK THING. IN NPS 1 THE PROBLEM IS BREAK DOWN OF HEPARAN SULFATE WHICH HAVE HIDURONIC ACID. THERE'S A SULFATE, THEN THE ACID THAT'S DEFICIENT AND TAKES OFF THE TERMINAL ACID ON THE NON-RELEASE. IF YOU CAN'T DO THAT YOU STALL AND BREAK DOWN OF HELL WRITTEN SULFATE AND SKULL FEW AT A TIME B WHICH HAS HYDRERONIC ACID. WE HAVE NON-REDUCING END OF POLYMER AND REDUCING END THAT'S ATTACHED TO THE PROTEIN CORE. AND IT TURNS OUT IN A TYPICAL STATE WE MIGHT HAVE 20 TO 30 LOW ENZYME SAMPLES PER YEAR AND MOST OF THESE PATIENTS DO NOT HAVE MPS 1 SO WE NEED A SECOND TIER ANALYSIS, THAT TURNS OUT TO BE ANALYZE THE LEVELS OF THE GLYCOSAMINOGLYCANS. SO THE IDEA IS IF YOU CAN'T BREAK DOWN HEPARAN SULFATE AND DERMATIN SULFATE THEY ACCUMULATE AT BIRTH. SO WHY NOT THE FIRST TIER TASK? BECAUSE IT'S TOO SLOW, IT'S NOT AS GOOD AZINE SURING THE ENZYME. BUT TO DO IT ON 20 TO 30 LOW ENZYME SAMPLES PER YEAR IS CERTAINLY EASY TO DO. SO WE DO IT SECOND TIER. ENZYME FIRST BIOMARKER SECOND. NEXT SLIDE. SO WE COMPARED THREE DIFFERENT METHODS FOR THIS GLYCOSAMINOGLYCANS ANALYSIS. ONE IS CALLED INTERNAL DISACCHARIDE METHOD. WE TAKE A 3-MILLIMETER BLOOD SPOT PUNCH, DEGRADE WITH BACTERIAL HEPARINASES AND MEASURE ALL OF THESE INTERNAL DISACCHARIDES BY LCMS MASS SPEC. THE OTHER METHOD WE CALL ENDOGENOUS DISACCHARIDE METHOD, TURNS OUT THERE ARE ENDOGENOUS GLYCOHYDROLASE IN HUMAN TISSUE THAT GENERATE SHORT GLYCOSAMINOGLYCANS FRAGMENTINGS, IDENTITY OF THESE ENZYMES ARE NOT KNOWN. WE LOOK AT THESE FRAGMENTS WITHOUT ANY ENZYMATIC DIGESTION, WE DRIVETIZE REDUCING END OF FRAGMENTS WITH REAGENT CALLED PMP WELL KNOWN IN CARBOHYDRATE CHEMISTRY. THIRD IS CALLED SENSE PRO. WE DIGEST WITH ENZYMES AND WE LOOK AT THE SO CALLED NON-REDUCING END. WHEN THE ENZYMES DIGEST POLYMERS THEY LEAVE A DOUBLE BOND IN THE URONIC ACID BECAUSE THEIR LYASES ARE NOT HYDROLASES. AND THE NON-REDUCING END IS UNIQUE NOT HAVING DOUBLE BOND BECAUSE IT COMES FROM THE LEFT END OF THE POLYMER THAT IS RELEASED WITHOUT A DOUBLE BOND SO WE LOOK AT THAT FRAGMENT SPECIFICALLY. ON THE NEXT SLIDE YOU CAN SEE THAT HERE, WE HAVE A FRAGMENT PORTION OF HEPARAN SULFATE. THE ENZYME ELIMINATES THE HYDROXYL GROUP FROM THE YOUR RONIC ACID BECAUSE YOU HAVE ASITIC PROTON, YOU GET A DOUBLE BOND IN THE SUGAR. SO INTERNAL DISACCHARIDE WE LOOK AT THESE DISACK RIDES THAT HAVE THE UNSATURATED URONIC ACID. IN THE SENSE P PRO WE LOOK AT TERMINAL DISACCHARIDE FROM NON-REDUCE END. IN THE ENDOGENOUS METHOD WE LOOK AT WHATEVER BIOMARKER HAPPENS TO BE PRODUCED BY ENDOGENOUS ENZYMES, SOME SHORT CARBOHYDRATE FRAGMENT. AND IN THE CASE OF MPS 1 I'LL SHOW YOU THE STRUCTURE IN A MINUTE. SO WE LOOK AT A LYASE WE GET THIS UNSATURATED. SO THE IDEA IS IF YOU ARE MISSING ENZYME IN THE DE DEGRADATION OF THIS POLYMER YOU WILL ACCUMULATE THESE GUYS. AND SO WE MEASURE THIS AS A BIOMARKER. THIS IS THE INTERNAL DISACCHARIDE METHOD. WE CARRIED OUT A DETAILED STUDY SO WHAT WE NEED ARE NEWBORN BLOOD SPOTS FROM PATIENTS THAT WENT ON TO DEVELOP MPSs SO HOW DO WE GET NEWBORN, THESE KIDS ARE BORN, THEY ARE DIAGNOSED LATER. SO TURNS OUT LIKE CALIFORNIA AND NEW YORK, A FEW OTHER STATES SORE LEFT OVER DRIED BLOOD SPOTS FOR MANY YEARS. CALIFORNIA FOR ESSENTIALLY INDEFINITELY IN GIGANTIC FREEZER. WE CAN GO TO THE NATIONAL MPS SOCIETY, CONTACT THE FAMILIES, WITH MPS KIDS, AND ASK PARENTS TO REQUEST THE STORED NEWBORN BLOOD SPOT BE SENT TO ME FOR RESEARCH. THIS IS A VERY NICE FEATURE TO DO RETROSPECTIVE ANALYSIS. SO WE GET BECAUSE WE NEED NEWBORN BLOOD SPOTS, WE ARE NOT SO INTERESTED IN LEVELS OF THESE POLYMERS LATER IN LIFE. WHAT'S RELEVANT TO THE NEWBORN SCREENING OF COURSE IS WHAT THEY HAVE AT BIRTH. SO HERE YOU HAVE 30 HEALTHY NEWBORN IN GRAY AND YOU HAVE SEVERE NPS PATIENTS IN BLUE AND YOU HAVE A COUPLE OF PATIENTS THAT HAVE THE ATTENUATED MPS 1 DISEASE. THIS IS THE LESS SEVERE DISEASE WHERE YOU HAVE MORE RESIDUAL ENZYME AND SYMPTOMS START LATER IN LIFE. BUT WE WANT TO CATCH THESE KIDS AS WELL. BECAUSE THEY ARE VERY REITABLE. THIS SHOWS PRETTY GOOD SEPARATION BETWEEN THE -- THIS IS THE TWO MARKERS ON THE LEFT FROM HEPARAN SULFATE, ON THE RIGHT IS DERMATIN MARKER. YOU CAN SEE THE SEPARATION. IT WORKS WELL BUT THERE'S OVERLAP IN THE NORMALS WITH THE ATTENUATED SEPARATION IS NOT GOOD. ON THE NEXT SLIDE, THIS IS SENSE PRO ABOUT -- WORKS ABOUT AS WELL AS INTERNAL DISACCHARIDE MARKER. THIS MARKER IS NOT PARTICULARLY GOOD. I 0S 0 DISACCHARIDE WORKS WELL. ON THE NEXT SLIDE YOU HAVE THE WINNING METHOD. THIS IS THE ENDOGENOUS METHOD THE REASON IT WORKS WELL IS THAT THE ENDOGENOUS -- THIS IS A DISACCHARIDE WE DON'T KNOW THE STRUCTURE, IT'S A URONIC ACID WITH A HEXOSAMINE WITH ONE SULFATE. WE HAVE A GOOD GUESS WHAT IT IS BUT DON'T KNOW EXACTLY WHETHER THE URONIC ACID IS GLYCOONIC OR IDERONIC ACID AND WE DON'T KNOW IF PIT'S GALACTOSE OR GLUCOSE, WE'RE IN THE PROCESS OF FIGURING IT OUT BUT THIS IS A MARKER THAT HAS THE RIGHT MASS TO BE THIS GUY. AND WHAT'S NICE IS THE RANDOM NEWBORNS ARE QUIET, THEY HAVE ESSENTIALLY NO MARKER. YOU SEE THAT THE TWO ATTENUATED PATIENT VERSUS 30 FOLD MORE THAN THE RANDOM AND 89 FOLD MORE ON AVERAGE FOR SEVERE. SO THE SEPARATION BETWEEN MPS 1 PATIENT AND NORMAL REFERENCE RANGES GIGANTIC FOR THIS AND THIS IS THE METHOD OF CHOICE. >> ONE MINUTE. >> I'LL BE DONE IN A SECOND. SHOW YOU PRECISION NOW WE CAN DO NEWBORN SCREENING SO THE MAYO CLINIC IS USING THESE METHODS AND THEY HANDLE IT FOR KENTUCKY. SO MOST STATES THEY MEASURE THE IDERONIDASE THEN GENOTYPING AND IT WORKS A LITTLE BIT BUT THEY ARE LOST IN SOME CASES AS I SAID DNA IS NOT SO GOOD AS PEOPLE THINK. BIOCHEMISTRY WINS. SO OUT OF THESE 20 TO 300 TO 1 HAVE ELEVATED GAGS IN THE DRY BLOOD SPOTS AND THESE TURN OUT TO BE MPS 1 PATIENTS SO WE CAN SCREEN FOR MPS 1 WITH ESSENTIALLY PERFECT PRECISION WHAT ABOUT FALSE NEGATIVES DO WE MISS ANY? WE DON'T KNOW, PROBABLY NOT. FROM BUT THE ONLY WAY TO TELL IS TO WATCH AND SEE IF SOMEBODY TURNS UP LATER WITH THE DISEASE. OVER THREE YEARS WE HAVE NOT HAD THAT PROBLEM. MINNESOTA NOW USES THIS METHOD WE ARE TRYING TO CONVINCE ALL THE OTHER NEWBORN SCREENING LABS TO GO TO A BIOCHEMICAL GLYCOSAMINOGLYCANS MARKER FOR THE SECOND TIER. IF THEY USE DNA THEY ARE MOSTLY LOST SO WE CAN DO VERY HIGH PRECISION. AND THE KEY IS WE SHOULD DO THIS WITH DRY BLOOD SPOT WE SHOULD DO -- CAN YOU IMAGINE CALLING 20 TO 30 PARENTS AND TELLING THEM YOU MIGHT HAVE MPS 1 WE NEED FOLLOW-UP BUT ONLY ONE OF THEM HAS MPS 1. WE LIKE TO DO THIS BEFORE WE CALL -- THAT'S WHAT WE DO IN KENTUCKY. NEXT SLIDE I THINK WE ARE DONE. JUST TO SHOW YOU THAT THIS IS A 25 COMPLEX FOR 25 DIFFERENT DISEASES. JUST TO SHOW MPS 1 IS PART OF A PANEL OF THINGS WE CAN DO BY LC MASS SPEC, PROVIDES UNIVERSAL SOLUTION TO KEEP UP WITH THE EVER-EXPANDING NEED FOR NEWBORN SCREENING OF ADDITIONAL DISEASE. THIS IS PUBLISHED IN GENETICS MEDICINE RECENTLY. NEXT SLIDE. THANKS TO MY GROUP. THIS IS FUNDED BY A LARGE NIH GRANT FROM MY LAB OVER LAST 12 OR 13 YEARS, WE HAVE A LOT OF FUNDING FROM INDUSTRY. THIS IS MY GROUP, THANKS FOR THEM DOING MOST OF THE WORK WE TALKED ABOUT TODAY AND WE WORK WITH PARENT FOUNDATION. I'LL STOP HERE. SEE IF THERE'S ANY QUESTIONS. >> THANKS SO MUCH. IN THE INTEREST OF TIME UNFORTUNATELY WE HAVE TO MOVE LATER FOR QUESTIONS. >> NO PROBLEM. >> SEND EMAIL AND WE'LL FORWARD IT. GO TO NEXT SPEAKER, THAT'S JONATHAN AMSTER. >> JUST WANT TO THANK YOU FOR THE INVITATION TO TELL EVERYBODY ABOUT THE TECHNOLOGY THAT WE HAVE BEEN DEVELOPING IN MY LABORATORY. PROJECT THAT'S BEEN GOING ON FOR SEVERAL YEARS NOW. THAT THERE ARE MANY DIFFERENT TYPES OF GLYCANS PRESENT AT CELL SURFACE AND CLICK FORWARD, WANT TO HIGHLIGHT THE -- OUR TARGETS ARE PROTEOGLYCANS WITH DECK CHAINS, MORE SPECIFICALLY HEPARAN AND HEPARAN SULFATE, POLYSACCHARIDES AND WE ARE PARTICULARLY INTERESTED IN TRYING TO DETERMINE PATTERN OF MODIFICATION THAT EXIST IN SMALL REGIONS OF THESE CHAINS WHICH LEAD TO SPECIFIC BINDING WITH PROTEINS AND ACTIVATE THOSE PROTEINS IN BIOLOGICAL PROCESS. NEXT SLIDE. SO WE HAVE BEEN -- IN TERMS OF TRYING TO DO STRUCTURAL ANALYSIS ON GLYCAN, IN ONE SENSE IT'S EASIER PROBLEM THAN LOOKING AT END GLYCANS FOR EXAMPLE, BECAUSE THE COMPOSITION, THE TYPES OF SUGARS INVOLVED ARE DETERMINED BY THE FAMILY. CLASS OF GAG WE ARE LOOKING AT AS WELL AS LINKAGE POSITION. BUT WHAT WE ARE INTERESTED IN ARE THESE MODIFICATIONS THAT OCCUR ALONG THE CHAIN BY A HOST OF DIFFERENT ENZYME SANS FER RACES, ACETYLACES AND POLYMER RACES THAT PROVIDE TRANSFERASES. COMBINE STRUCTURE TO THESE CHAINS. BECAUSE IT'S AN ENZYMATIC PROCESS, COMPLETION THERE'S A LOT OF HETEROGENEITY UP AND DOWN A CHAIN WHEN YOU COMPARE DIFFERENT CHAINS TO EACH OTHER. AND IT SEEMS LIKE ALMOST INTRACTABLE PROBLEM BUT WELL KNOWN IN SHORT STRETCHES CHAIN THERE ARE PARTICULAR PATTERN OF MODIFICATION THAT NEED TO SPECIFIC BINDING TO TARGET PROTEINS. AND OF COURSE BEST STUDY ONE OF THESE IS SACCHARIDE SEQUENCE HERE WHICH LEADS TO BINDING (INAUDIBLE) WHICH PLAYS IMPORTANT ROLE IN (INAUDIBLE) PROCESS. SO HOW DOES ONE FIND THESE REGIONS OF MODIFICATION IN CHAINS THAT MAYBE SEVERAL HUNDRED SUGARS IN LENGTH? THAT'S THE PROBLEM WE HAVE BEEN TRYING TO TACKLE. THIS IS WELL STUDIED EXAMPLE, THERE ARE MANY EXAMPLES WHERE PROTEINS THAT ARE KNOWN TO HAVE IMPORTANT INTERACTIONS WITH GLYCOSAMINOGLYCANS BUT THE PATTERN MODIFICATION THAT LEADS TO BINDING (INAUDIBLE) SO THAT'S WHAT WE ARE TRYING TO TACKLE, THIS TECHNOLOGY. NEXT SLIDE. AN EXAMPLE OF SOMETHING THAT INSPIRES THIS DEVELOPMENT IS WORK DONE BY (INAUDIBLE) IN COPE HAY WHO DISCOVERED SEVERAL YEARS AGO THAT BLOOD CELLS INFECTED WITH PARTICULAR STRAIN OF MALARIA EXPRESSES PROTEIN THAT DIRECTS THEM TO THE PLACENTA. AND NOT TO OTHER CELLS IN THE BODY. LATER ON THEY DISCOVERED THIS PROTEIN NOT ONLY TARGETS SULFATE EPITHELIAL CELLS BUT ALSO WIDE VARIETY OF TUMOR CELLS SO IT'S THOUGHT THAT SKULL FACE MAY PLAY A ROLE -- SULFATES MAY PLAY A ROLE. SO WOULD BE NICE TO KNOW WHAT IS PATTERN OF MODIFICATION? STILL NOT KNOWN. THAT'S AN EXAMPLE OF A PROBLEM THAT (INAUDIBLE). THE CHALLENGES ARE THAT SPECIFIC GLYCANS ARE HIGHLY HETEROGENOUS, YOU CAN'T APPLY OTHER TYPES OF ANALYTICAL METHODS KNOWN TO BE SCIENCE INFORMATION. MASS SPECTROMETRY SURROGATE FOR LOOKING AT MIXTURES AND SMALL AMOUNTS OF MATERIALS COUPLE WITH SEPARATION METHODS. SO THESE GLYCAN CHAINS ARE LONG BUT WE CAN USE DEEP POLYMERIZATION METHODS, PARTIAL POLYMERIZATION RELEASE OF INTEREST, BUT THEN NEXT WE REQUIRE SOME METHODOLOGY TO FIND THE REGIONS THAT HAVE PATTERNS THAT WE ARE INTERESTED IN, AND SEPARATE THEM FROM ONES THAT ARE MODIFIED REPRESENT CHEMICAL NORMS. SO IN TERMS OF DOING MASS SPECTROMETRY, THESE SACCHARIDES ARE -- GAG OLIGOSACCHARIDES ARE HIGHLY ACIDIC WHICH IS MOST STABLE IN MASS SPECTROMETRY AS AN IONS, MOST MASS SPEC METHODS ARE SET UP AS PAN AN IONS. THIS IS IS NOT A TREMENDOUS TECHNOLOGICAL PROBLEM BUT DOES REQUIRE THOUGHT ESPECIALLY WHEN IT COMES TO SEPARATION BECAUSE THEY ARE SO IONIC, THEY ARE NOT EASILY SEPARATED BY HPLC WHICH IS STANDARD METHOD FOR ANALYSIS OF MASS SPECTROMETRY. SO WE RESORTED TO CAPILLARY ELECTROPHORESES WHICH WORKS QUITE WELL WITH THAT. AGAIN NEGATIVE MODE IONIZATION FOR MASS SPEC ANALYSIS AND WE HAVE TO ADAPT OUR CCB TO WORK WITH IONS AS WELL. THIS MODIFICATION OF INTEREST, SKULL FAY SHUN, IT HAS TO ADAPT THE METHODS TO DO TANDEM MASS SPECTROMETRY REQUIRED TO SEQUENCE. WE HAVE TO ADAPT IT TO THIS FEATURE. SULFATION. SO THERE'S FOUR PARTS TO THIS FOUR PARTS TO THIS PLATFORM, ONE IS METHOD FOR FINDING STRETCHES, FALSE STRETCHES OF GLYCOSAMINOGLYCANS CHAIN THAT BEHIND SPECIFICALLY WITH A TARGET PROTEIN. -- BIND SPECIFICALLY WITH A TARGET PROTEIN. THEN THERE'S ONLINE SEPARATION, DEVELOPMENT MASS SPEC DEVELOPMENT, WE USE HIGH RESOLUTION MASS SPEC AND WE ALSO USE ADVANCE MASS SPECTROMETRY METHODS TO GET AT BOTH COMPOSITION AND SEQUENCE OF GLYCOSAMINOGLYCANS LIGO SACCHARIDES. FINALLY THIS VERY COMPLEX MASS SPEC GROUP WE GET FOR ANALYSIS REQUIRES SOME SORT OF AUTOMATION FOR THE ANALYSIS AND INTERPRETATION. SO I'LL FORWARD THESE REQUIRE SOME DEVELOPMENT WHICH I'LL TRY TO COVER BRIEFLY REMAINING TIME TALK ABOUT THESE THREE. ANALYSIS AND AUTOMATION BOTH JOSE'S GROUP AND MY GROUP PUBLISHED PAPERS IN INTERPRET GAG SPECTRA SO I'M NOT GOING TO (INAUDIBLE) NEXT SLIDE. SO IN ORDER TO GO FROM CHAIN THAT'S PERHAPS A COUPLE OF HUNDRED SUGARS IN LENGTH, THAT IS SOMETHING THAT'S TRACTABLE. WE CAN USE INCOMPLETE DIGESTION. CHEMICAL OR ENZYMATIC METHODS TO DIGEST THINGS DOWN TO THE THESIS, NEEDS TO BE SEPARATED BY SIZE EXCLUSION. AND THEN TAKE A FRACTION OF THE SIZE TO WORK WITH EACH OF THESE FRACTIONS CONTAIN MANY DIFFERENT DEGREES OF SULFATION AND AYUSOMERIC COMPOSITION SO VERY COMPLICATED MEASURES UNDER EACH THESE PEAKS. WHICH SIZE DO YOU GET AS MUCH THE LONGER THE CHAIN IS, THE HIGHER THE PROBABILITY IS THAT PATTERN MODIFICATION THAT YOU WANT PRESENT BUT MORE ISOMERIC POSSIBILITIES AND ANALYSIS. SHORTER THE EASIER THINGS BECOME BUT HIGHER THE PROBABILITY IS THAT YOU GET CHAIN IN THE MIDDLE OF ANEN INTERESTING PATTERN YOU AREK LOUK AT. SO OUR SWEET SPOT IS IN THIS FOUR TO TEN OR 12 RANGE. THAT'S WHAT WE WORKING ON FOR NUMBER OF YEARS. SO THE QUICKLY TO TALK ABOUT HOW TO PULL DOWN THE CHAINS THAT HAVE INTERESTING PATTERNS, PATTERN YOU ARE LOOKING FOR THAT REACT STRONGLY WITH PART OF THE PROTEIN, THIS WORK IS DONE ON THE LYNN HART LABORATORY AND THEY HAVE TRIED MANY DIFFERENT APPROACHES SOME QUITE SOPHISTICATED INVOLVING MAGNETIC BEAD TECHNOLOGY, -- TARGET PROTEINS. IN THE END TURNS OUT METHOD THAT WORKS BEST FOR ENOUGH MATERIAL TO WORK TO DO MASS SPECTROMETRY WITH THIS VERY SO CALLED CONCEPT OF USING SPIN COLUMN SIZE MEMBRANE. SO THIS IS JUST TO RETAIN PROTEIN THAT IS THE TARKT AND THEN BUT IT'S LARGE ENOUGH FOR TO PASS THROUGH GAG SET OF DIGESTED DOWN TO SIZE SO IF YOU EPIQUEUE BAIT PROTEIN WITH GAGS THEY INTERACT STRONGLY YOU CENTRIFUGE THIS, NON-BINDERS GO THROUGH AND BINDERS AND MOVE THIS AND FINALLY SALT TO RECOVER HIGH AFFINITY SACCHARIDES. NEXT SLIDE, PROOF OF CONCEPT IS SHOWN HERE, THIS IS JUST REALLY SIMPLE EXAMPLE OF USING WELL DEFINED SACCHARIDE (INAUDIBLE) AND THE 58-KILO DALTON THROMBIN PROTEIN. SO THIS SHOWS THAT YOU CAN RETAIN ANTI-THROMBIN, IN INCUBATE WITH -- RETAINED BUT THEN YOU -- COMES THROUGH AND YOU CAN SEE MASS SPEC THE BEAUTY OF THIS METHOD IS THE PROTEIN STAYS ON TOP AND YOU CAN REPEAT THE PROCESS OVER AND OVER AT LEAST TEN TIMES AN BUILD UP LARGE AMOUNT OF THIS MATERIAL. SO NOW LET'S MOVE TO THIS CE MASS SPECTROMETRY PART OF THE PLATFORM. SO THIS IS ACTUALLY -- THERE'S BEEN NO TECHNOLOGY DEVELOPMENTS IN TERMS OF HARDWARE DEVELOPMENTS. WE ARE USING COMMERCIALLY AVAILABLE EQUIPMENT FOR THIS EXPERIMENT. ANY LCE WILL WORK. THIS IS VERY LOW (INAUDIBLE) REMARKABLE TO SEE. MASS SPECTROMETER IS AN ORGAN TRAP SO WE WANT A HIGH PERFORMANCE MASS SPECTROMETER BUT THIS IS NOT THE STATE OF THE ART, THIS IS WIDELY AVAILABLE INSTRUMENT THAT IS PRESENT IN MANY LABORATORIES. I MIGHT BE WORKING ON THIS COUNTER PROBLEM. AND THEN THE THING THAT JOINS THE TWO INTERFACE HERE, THIS IS THE CE MASS SPEC INTERFACE AND THAT ALSO IS A COMMERCIALLY AVAILABLE DEVICE THAT USES (INAUDIBLE) SO ILLUSTRATED ON THE NEXT SLIDE. SO SEPARATION CAPILLARIES SPLIT INSIDE PUSHED UP INSIDE OF THE NANOSPRAY TIP FINE POINT. THIS HAS BEEN ETCHED WITH HF AND PUSHED UP ALL WAY TO THE END, THERE'S A VERY SMALL MIXING VOLUME SO THAT'S AS THINGS MIGRATE OFF THE CE COLUMN, MIXED IN WITH THIS SHEATHE FLOW INTERFACE, MAKES A HUNDRED LITTER VOLUME HERE SO WE DON'T GET (INAUDIBLE) THIS SPRAYS. YOU CAN GO FORWARD. SO THAT'S SHOWING SPRAY. ONE OF THE THINGS THAT WE HAVE TO DEVELOP IS BECAUSE WE ARE LOOKING AT AN IONS, WE HAVE TO USE USE REVERSE POE LAYERTY C -- POLARITY. MASS SPEC ENDS WITH POSITIVE VOLTAGE, AND THE PROBLEM WITH THIS IS ALTHOUGH THIS PUSHES OUR AN IONS TOWARDS MASS SPECTROMETER, IT -- IN A STANDARD -- OPPOSES THIS, MIGHT ACTUALLY BE LARGER THAN THIS. MOVING OUT SO WE WORK WITH COVALENT MODIFICATIONS TO NEUTRALIZE THE POSITIVE CHARGE HERE SO WE CAN GET RID OF -- MAKE IT GO SAME DIRECTION AS MASS SPECTROMETER. SO THAT'S A SMALL DEVELOPMENT THAT WAS REQUIRED TO MAKE THIS WORK. SO JUST SHOW COUPLE OF EXAMPLES, THESE ARE JUST SIMPLE BINARY MIXTURES WHERE YOU HAVE -- THESE ARE HEPARAN SULFATE TETRAMERS, THE ONLY DIFFERENT BETWEEN THE SULFATE AND URONIC ACID CELL SEPARATION INDUCING IN NEEDED SUGAR. AND SEPARATION, NEXT SLIDE IS EVEN A MORE DEMANDING SEPARATION. THE ONLY DIFFERENCE BETWEEN THESE TWO COMPOUNDS, IDURONIC ACID HERE, GLYCORONNIC ACID HERE AND YOU CAN SEE THE SEPARATION. BINARY MIXTURES AREN'T THE KIND OF MIXTURES WE ARE GOING TO BE FACING, LOOKING AT MORE COMPLEX MIXTURES LIKE THAT ON THE NEXT SLIDE. ANOXOPARIN WHICH IS A LOCAL HEPARAN INDUCED BY CHEMICAL DIGESTION OF HEPARAN. THERE'S MAYBE DOZENS OR MAYBE HUNDRED COMPONENTS PRESENT FROM DP 3 UP TO DP 20 ROUGHLY. SO YOU CAN SEE THAT THERE'S SOME COMPONENTS THAT ARE PRESENT HIGH ABUNDANCE SOME LOWER ABUNDANCE, NOT EVERYTHING IS WELL SEPARATED BUT WE ARE GETTING DECENT SEPARATION OF HIGHLY COMPLEX MIXTURE. MASS SPECTROMETRY AT HIGH RESOLUTION GIVES US COMPOSITION OF DIFFERENT COMPONENTS SHOWN ON THE NEXT SLIDE. THIS IS JUST TO EMPHASIZE ALL WE GET OUT OF HERE ARE COMPOSITION SO WE GET CHAIN LINK DEGREE OF SULFATION AND DEGREE OF -- BUT IN ORDER TO GET STRUCTURE WE WILL HAVE TO GET (INAUDIBLE). NEXT SLIDE. SO IN ORDER TO BE ABLE TO SEQUENCE, YOU HAVE CHOICE OF MANY DIFFERENT METHODS OF ION ACTIVATION. IF YOU ARE NOT MASS SPECTROMOTIST YOU PROBABLY HAVEN'T HEART OF THESE, -- HEARD OF THESE, MOST PEOPLE WHO HEARD OF TAN WOMEN MASS SPECTROMETRY ARE FAMILIAR WITH COLLISION INDUCED DECOMPOSITION, THAT DOESN'T WORK WELL FOR GAG. WE SPENT PROBABLY A DECADE LOOKING AT THIS METHOD ELECTRON DETACHMENT DISSOCIATION. NEXT SLIDE. THIS SHOWS WORK DONE ACTUALLY IN A TRANSFORM MASS SPECTROMETER. TO SHOW YOU COMPARE RESULTS BETWEEN COLLISION INDUCED DISSOCIATION AND MOLECULAR DETACHMENT DISSOCIATION WHICH OCCURS BY FIRING BEAM OF ELECTRONS AT SOME IONS THAT ARE STORED AND ELECTROMAGNETIC FIELD WITH ICR (INAUDIBLE). AND EVEN IF YOU ARE NOT A MASS SPECTROMOTIST YOU CAN SEE THERE'S A LOT MORE ESPECIALLY DOWN LOWER, THERE'S MORE -- THERE ARE MORE PEAKS HERE, HIGH DENSITY PEAKS AND INTERPRETATION OF THIS LEADS TO THESE FRAGMENTATIONS SO YOU CAN SEE MOSTLY YOU GET GLYCOSITIC CLEAVAGE ICID BUT WITH ELECTRON DETACHMENT ASSOCIATION YOU GET CROSS CROSS LINK CLEAVAGE WHICH IS IMPORTANT TO FIND WHERE MODIFICATIONS EXIST WITHIN A SUGAR. SO THIS WORK WAS DONE ON AN ICR BUT WE ARE TRYING -- WE HAVE NOW GOING THIS OVER TO ORBITRAP AN INSTRUMENT MORE WIDELY AVAILABLE TO THE BIOLOGICAL RESEARCHERS. SO THIS REQUIRES A DIFFERENT ION ACTIVATION METHOD BECAUSE THIS METHOD HERE ONLY WORKS WITH ICR. SOMETHING THAT WOULD WORK IN AN TRANSFER DISSOCIATION. SO THAT IS AN ION ION REACTION THAT OCCURS GAG POLYANION AND REAGENT CATION THAT LEADS TO ELECTRON TRANSFER CREATING A RADICAL SITE IN THE SUGAR THAT LEADS TO VERY INTERESTING FRAGMENTATION. THAT HAPPENS IN ION INSTRUMENT AND THEN TRANSFERRED TO HIGH RESOLUTION (INAUDIBLE). SO THIS SHOWS YOU WITH THE SEPARATION WE HAVE BEEN ABLE TO NOW GO IN ON SOME OF THESE GET SEQUENCE INFORMATION. THIS GIVES YOU A FALSE SENSE HOW WELL WE HAVE BEEN ABLE TO SEQUENCE. WE ARE ONLY GETTING GLYCOSITIC CLEAVAGE WITH THESE AT THIS POINT SO WE KNOW THE COMPOSITION IS SUGAR. SO WE ARE CONFIDENT ABOUT 20 AND SULFATION. THIS IS A 6S, COULD ALSO BE 3S SULFATION IN AMINO SUGAR, IT'S MODIFICATION BUT IT IN FACT OCCURS IN HEPARAN AND THAT'S WHAT LEADS TO ITS ACTIVITY. SO -- >> YOU NEED TO WRAP UP. >> I ONLY HAVE ONE MORE SLIDE AFTER THIS. SO ANYWAY, THE PROBLEM HAS TO DO WITH LOW CHARGE STATES WE ARE GETTING, THAT'S A PROBLEM OF CCB. I TOLD YOU ABOUT METHODS FOR ISOLATING THE INTERESTING BINDING REGIONS THAT PRODUCE SCAFFOLDS SUITABLE FOR OUR TECHNOLOGY USING CZE FOR SEPARATING THINGS, OPTIMIZING ACTIVATION METHODS SO THAT YOU GET GOOD SEQUENCING. THAT'S AN IMPORTANT PART OF THE WHOLE PLATFORM. RANDOM HURDLES HAS TO DO WITH LOW CHARGE PROBLEM WHEN WE COMBINE CCB WITH -- CZE WITH MASS SPEC SO WE ARE WORKING ON METHODS TO INCREASE CHARGE ON GAG EMPLOYING OTHER ION ACTIVATION METHODS THAT ARE NOT (INAUDIBLE). FINALLY THE LAST SLIDE IS ACKNOWLEDGES. >> BE GLAD TO ENTERTAIN QUESTIONS. >> THANKS SO MUCH, BECAUSE OF LACK OF TIME POST YOUR QUESTIONS IN THE CHAT AND MAKE SURE THAT JOHN WOULD BE HAPPY TO ANSWER THEM AT THE END OF THE DISCUSSION. NEXT SPEAKER IS GEERT JAN BOONS. >> I ALSO WOULD LIKE TO THANK PAMELA AND (INAUDIBLE) AND NIH FOR PUTTING THIS MEETING TOGETHER. AND GIVING ME AN OPPORTUNITY TO SPEAK. I CAN'T -- OKAY. SO IT'S WELL APPRECIATED THAT HUNDREDS OF PROTEINS REQUIRE HEPARAN SULFATE FOR BINDING AND BIOLOGICAL ACTIVITY. ONE OF THE BEST STUDIED CASES IS -- BINDING TO HEPARAN SULFATE IMMEDIATE YATES A CONFIRMATIONAL CONTROL. THERE BY MEDIATES. THERE BY ACTIVATING THE PROTEASE ACTIVITY AND BEING ABLE TO ACTIVATETROPIN MEDIATING COAGULATION PATHWAY. IT REQUIRES A SPECIFIC PEN TA SACCHARIDE WHICH IS SHOWN HERE. REMOVAL OF A SPECIFIC SULFATE LEADS TO A DRAMATIC DECREASE IN BINDING AND BIOLOGICAL ACTIVITY. MULTI-THROMBIN IS FEW CASES WHICH IT'S KNOWN REQUIREMENT. STILL AN IMPORTANT QUESTION OUT THERE. THAT IS DO SPECIFIC HEPARAN SULFATE GLYCAN SEQUENCE CONFIRM SELECTIVE PROTEIN BINDING PROPERTIES? IT IS DIFFICULT TO DETERMINE LIGAND REQUIREMENTS AS JOHN ALREADY OUTLINED. BECAUSE OF THE STRUCTURAL DIVERSITY OF HEPARAN SULFATE. HEPARAN SULFATE STARTS AS A RELATIVELY SIMPLE POLYSACCHARIDE BUT ACETYLGLUCOSAMINES LINKED TO GLYCORONNIC ACID. A NUMBER OF ENZYMATIC MODIFICATIONS. FIRST THE (INAUDIBLE) IS REPLACED BY SULFATE. THE GLUCO RONIC ACID IS POLYMER IZED AND C 2 AND C 6 POSITIONS CAN BE EFFECTED. THESE MODIFICATIONS ARE OFTEN INCOMPLETE. AS MANY AS 20 DIFFERENT DISACCHARIDES HAVE BEEN IDENTIFIED. THAT DIFFER IN URIC ACID COMPOSITION AND SULFATION. CAN BE PUT TOGETHER IN MANY WAYS CREATING ENORMOUS STRUCTURAL DIVERSITY. TO PROBE LIGAND HEPARAN SULFATE BINDING PROTEINS, WE ARE DEVELOPING A MULTI-LEVEL APPROACH THAT PROVIDE LARGE SECTIONS OF HEPARAN SULFATE POLYSACCHARIDES. EVIDENCE BASED ON A NOTION THAT IN HEPARAN A LIMITED NUMBER OF DISACCHARIDES APPEAR. SO WE HAVE DESIGNED A COLLECTION OF 10 DISACCHARIDES THAT RESEMBLE DIFFERENT PATTERN AND BEING URONIC ACID OR IDURONIC ACID, THE BUILDING BLOCKS OF HEPARAN SULFATE. THE CENTER IN C 4 POSITION ARE MODIFIED BY A TBS ETHER. CAN BE REMOVED EITHER BY BASE OR NEUTRA FILLIC BASE. POSITIONS THAT NEED TO BE SULFATED ARE MODIFIED BY A LEFT ETHER AND THAT CAN BE REMOVED BY (INAUDIBLE) WITH ACETATE. HERE IS MODULAR SYNTHETIC APPROACH WORKS. IF I WERE TO ASSEMBLE AN OLIGOSACCHARIDE THAT HAS A GLYCORONNIC ACID WITH A SULFATE, ON THE C 2 POSITION WE USE A BUILDING BLOCK IN WHICH THE SULFATE IS MASKED BY (INAUDIBLE) POLYSACCHARIDE ASSEMBLY IS LEFT REMOVED AND CHEMICALLY SULFATED. HERE IS ANOTHER MODULE, IF OLIGOSACCHARIDE CONTAINS THIS OLIGOSACCHARIDE, WE USE HYDRONIC ACID, HAVING LESS IN THE FUTURE WHERE SULFATE NEEDS TO BE PRESENTED SO THESE BUILDING BLOCKS CAN BE USED OVER AND OVER AGAIN. TO MAKE MANY OLIGOSACCHARIDES. AND TO DATE WE HAVE CREATED A LIBRARY OF AS MANY AS 120 DIFFERENT SULFATE OLIGOSACCHARIDES, THAT DIFFER IN BACKGROUND COMPOSITION AND SULFATION PATTERN AND HAVE SIZES RANGING FROM TETRA TO OPTO SACCHARIDES. THESE OLIGOSACCHARIDES HAVE BEEN PRINTED TO PROBE LIGAND REQUIREMENTS OF HEPARAN SULFATE BINDING PROTEINS. HERE IS AN EXAMPLE OF FGF. FGF 2 RECOGNIZES LYSESOME HERE YOU CAN SEE HOW WE ORGANIZED THE MOLECULES OF THE HEPARAN SULFATE SO THEY ARE ORGANIZED FROM LEFT TO RIGHT BASED ON AN INCREASE IN NUMBER OF SULFATES. SO ONE, TWO, THREE, FOUR, FIVE OR SIX SULFATES AND THEY DIFFER IN COMPOSITION AND POSITIONS WHERE SULFATES ARE PRESENT. MOST HIGHLY SULFATED MOLECULES ARE BEING -- BY FGF 2. WE HOPE YOU OBSERVE THAT IN SACCHARIDES OF INTERMEDIATE SULFATION LEVEL, THESE ARE SIGNIFICANT, WE RECEIVE HIGH SELECTIVITY. JUST TO GIVE YOU A COUPLE OF FEATURES OF FGF 2 BINDING, WHEN AN IDURONIC ACID IS PRESENT ON SULFATE WITH C 2 WE SEE POTENT BINDING. WITH THIS SULFATE IS MOVE TO C 6 POSITION AS IN THIS COMPOUND 27, BINDING IS LOST. HERE IS ANOTHER EXAMPLE IDURONIC ACID WIH A TWO SULFATE, BOTH RECOGNIZED ON A 6 POSITION IT'S NOT. FURTHER ANALYSIS OF THE DATA SHOWS THAT TYPE OF URONIC ACID IS IMPORTANT. SO AGAIN, SULFATE IDURONIC ACID RECOGNIZE THE SAME COMPOUND NOW AS GLUCO RONIC ACID IS (INAUDIBLE). USING THIS TECHNOLOGY WE HAVE MAPPED LIGAND REQUIREMENTS OF MANY HEPARAN SULFATE BINDING PROTEINS. NOT ONLY ENDOGENOUS PROTEINS BUT ALSO MANY EXOGENOUS PROTEINS. SPECIFICALLY FROM MICROBE HEPARAN SULFATE FOR BINDING AND CELL ENTRY. A RECENT EXAMPLE IS THAT OF SARS COV-2 IS NOW QUITE A BIT OF DATA AVAILABLE THAT SARS COV-2 EMPLOYS HEPARAN FOR CELL ATTACHMENT AND ENTRY AND HEPARAN MAY BE A POTENTIAL VIRAL AGENT. WE EMPLOY OUR -- TO PROBE LIGANDS REQUIREMENTS OF THE RBD OF SARS COV-2. AS YOU CAN SEE IS IT EXHIBITS SELECTIVITY FOR SPECIFIC SEQUENCE. IT REQUIRES AT LEAST HEXA SACCHARIDE FOR POTENT BINDING THAT IS POLYSILL -- HIGHLY SULFATED. SULFATE ON C 2 AND C 6 AND VARIOUS REPEATING UNITS ARE REQUIRED FOR BINDING. REMOVAL OF ONE OF THESE SULFATES RESULTS IN A SUBSTANTIAL REDUCTION IN BINDING WHICH WE VALIDATED BY MORE DETAILED BINDING STATUS. RECENTLY WE HAVE STARTED ANOTHER MODIFICATION OF HEPARAN SULFATE, NAMELY 3-0 SULFATION SO GLUCO RONIC ACID CAN BE SULFATED AT C 3 POSITION SHOWN HERE. THIS IS ACTUALLY A RAMIFICATION THAT OCCURS AS LAST STEP IN MODIFICATION. INTERESTINGLY, THE ENZYMES THAT INSTALL SULFATE IS THE LARGEST GROUP OF I SEW ENZYMES AND SEVERAL IDE -- ISOENZYMES AND SEVERAL IDENTIFIED SO FAR. THAT ARE EXPECTED TO HAVE DIFFERENT SUBSTRATES SPECIFICITIES. 3-0 SULFATIOM IS IMCOLLATED IN MANY BIOLOGICAL PROCESSES. HOWEVER IT'S DIFFICULT BECAUSE OF RARE TO IDENTIFY AND DETERMINE LIGAND REQUIREMENTS OF PROTEINS THAT RECOGNIZE THIS STRUCTURE. SO WE HAVE EXPANDED MODULAR SYNTHETIC APPROACH TO INCLUDE (INAUDIBLE) THAT HAVE THREEFOLD SUP FATE. WE TARGETED HEXA SACCHARIDES BECAUSE OUR STUDIES SO FAR LEARNED THAT HEXA SACCHARIDES ARE EVENTUALLY A GOOD SIZE TO PROBE LIGANDS REQUIREMENT. THERE ARE LARGE NUMBER OF MOLECULES POSSIBLE SO CAREFUL TARGET SELECTION IS IMPORTANT. SO WE SURVEYED THE LITERATURE TO IDENTIFY STRUCTURE IN WHICH GLUCOSAMINE IS SULFATED AT THE C 3 POSITION. WE NOTICE THAT THIS CAN BE FLAMED BY GLUCO RONIC ACID AS WELL AS IARONIC ACID. THAT CAN BE FURTHER SULFATED AT THE 2 POSITION AND ALSO THE 6 POSITION OF 2 POSITION OF IDURONIC ACID AND 6 POSITION OF THE GLUCOSAMINE. SO BASED ON THIS WE DESIGNED A SEQUENCE CENTRAL TRISACCHARIDE MODIFIED WHERE GLCNAC IS -- IDURONIC ACID OR GLUCO RONIC ACID, WHERE C 2 POSITION IS ARE EITHER HYDROXYL OR SULFATE AND SAME IS CASE FOR THE C 6 POSITIONS. WE KEPT THE FLANKING REGIONS CONSTANT. SO BASED ON THIS CONSIDERATION WE HAVE 27 DISACCHARIDE, HEXA SACCHARIDES. TO MAKE THESE MOLECULES WE EXPANDED THE CAPABILITIES OF OUR APPROACH TO INCLUDE ANOTHER ORTHOGONAL GROUP FOR C 3 POSITION. NOT ETHER, WHICH CAN BE SELECTIVELY REMOVED BY (INAUDIBLE) PDQ. SO THE TDS CAN BE REMOVED AND THIS MOLECULE CAN THEN BE CONVERTED INTO A LIGAND GROUP. (INAUDIBLE) CAN BE CLEAVED AS ACCEPTERS THAT CAN BE EMPLOYED IN OLIGOSACCHARIDE ASSEMBLY GIVING QUICK ACCESS TO MANY DIFFERENT OLIGOSACCHARIDES. SO THIS ARE THE BUILDING BLOCKS TO ASSEMBLE THE LIBRARY OF SACCHARIDES AND THEY HAVE DIFFERENT PATTERN OF (INAUDIBLE), SULFATES NEED TO BE INSTALLED. AND THEY HAVE SACCHARIDE FORMATION TO ALLOW MOLECULE TO BE EXTENDED INTO A HEXA SACCHARIDE. HERE YOU SEE HOW IT WORKS, WE HAVE THIS GLYCOSYLIC SECOND TORE AN AN OMERIC LINKER THAT PROVIDES PROTECTION TO THE AMEAN FOR IMMOBILIZATION OF ARRAY. FIRST GLYCOSYLATION REMOVE THE F, PERFORM A SECOND GLYCOSYLATION THAT GIVES A HEXA SACCHARIDE. THEN -- CAN BE REMOVED AN SULFATES INTRODUCED. AND NOW WE HAVE TWO OPTIONS. NEGATIVELY ENOUGH ETHER IS CLEAVED AND SULFATE TO -- OR THE MOLECULE WILL UNDERGO HYDROGEN NATION AND TOGETHER WITH BENZYL ETHER, ENOUGH IS REMOVED AND WE HAVE THE PARENT COMPOUNDS LACKING A C 3 SULFATE. SO OLIGOSACCHARIDES WERE PRINTED AS A MICROARRAY SO THE NUMBERS 1, 2 INDICATE DIFFERENT BACKGROUND STRUCTURE. COMPOUNDS LABELED AS A C 3 AND 6 SULFATE, B 3 SULFATE, AND C ONLY A 6 SULFATE. SO WE CAN PROBE THE IMPORTANCE OF BACKGROUND STRUCTURE AND MODIFICATION OF CENTRAL GLUCOSAMINE. FIRST WE PROBED THE ARRAY WITH (INAUDIBLE) CHARACTERIZE PROTEIN. AND AS ANTICIPATED, THIS HEXA SACCHARIDE WHICH -- PREVIOUSLY IDENTIFIED FOR 83 BINDING GAVE THE STRONGEST RESPONSIVENESS. REMOVAL OF THE 6 SULFATE ON THE CENTRAL GLUCOSAMINE LED TO SMALL REDUCTION IN ACTIVITY. ALSO MODIFICATION OF TOLERATED. FOR EXAMPLE, WHEN THIS BACKBONE STRUCTURE WAS EMPLOYED, ALSO GOOD ACTIVITY WAS MEASURED. WE ALSO DETERMINED DIFFERENT ACTIVITY AND AS YOU CAN SEE CORRELATES REALLY WELL WITH ARRAY DATA, VALIDATING THIS IS AN ATTRACTIVE APPROACH FOR LIGANDS IDENTIFICATION. NEXT WE EXAMINED A NUMBER OF PROTEINS FOR WHICH DATA IS AVAILABLE THAT THEY BIND TO TWO OR THREE SULFATED STRUCTURE. FGFR EXAMPLE, IT'S A TYROSINE KINASE. PERFORMS A COMPLEX WITH VEGF SULFATE THERE BY MEDIATING MANY BIOLOGICAL ACTIVITIES. YOU CAN SEE THAT ONCE A STRUCTURE THAT HAVE C 3 SULFATE AND SPECIFIC BACKBONE THE 3 SULFATE AND 6 SULFATE REMOVED DRAMATIC REDUCTION IN -- IS OBSERVED. BASICALLY, THIS PROTEIN RECOGNIZES TWO DIFFERENT BACKBONE STRUCTURE WHERE THIS IS EITHER GLYCORONNIC ACID OR TWO SULFATE IDURONIC ACID. WE EXAMINED MANY PATTERN PROTEINS. THIS IS NEUROPHILLIN 1 WHICH IS ALSO TYROSINE KINASE THAT CAN FORM A COMPLEX WITH HEPARAN AND FGF WITH VEGF FOR SIMILARPORIN MEDIATING BUY LOGICAL PROCESSES SUCH AS AXON GUIDANCE AND YOU ALSO SEE THAT BASICALLY A SAME BACKBONE STRUCTURE ARE BEING RECOGNIZED. BUT WITH DIFFERENT DEPENDENCIES ON THE 6 SULFATE. AND 3 SULFATE. HERE IS LESS IMPORTANCE. WE SCREENED MANY PROTEINS AND HERE YOU SEE A COLLECTION. QUITE INTERESTINGLY, WHAT WE ALSO OBSERVE IS THAT THE SAME STRUCTURE OF 3 COMPOUNDS 8 ARE BEING RECOGNIZED, MANY OF THESE PROTON -- PROTEINS HAVE IN COMMON BUT THEY HAVE DIFFERENT DEPENDENCIES OF 6 SULFATE AND TWO SULFATE. THINK IS QUITE INTRIGUING. WE ALSO EXAMINED THE INVOLVEMENT OF HEPARAN SULFATE IN HERPES SIMPLEX VIRUS, ONE INFECTIVITY TO HSV BINDS TO HEPARAN SULFATE THROUGH TWO GLYCOPROTEINS, TB AND TC, THEN SHUTTLES TO THE CELL SURFACE TO BIND TO ANOTHER PROTEIN, TD, AND THEN TOGETHER WITH THIS HVEM, IT CAUSES MEMBRANE FUSION AND ENTRY OF CELLS. FURTHER STUDIES INDICATED GD BINDS TO HEPARAN SULFATE HAVING A 3 SULFATE. SO WE PERFORMED IN COLLABORATION WITH DR. SHUKLA WHO AN EXPERT IN THIS FIELD, THE POTENTIAL OF SYNTHETIC OLIGOSACCHARIDES TO INHIBIT CELL ENTRY AND CELL SUPPRESSION. HERE IS THE DATA, IT'S QUITE INTERESTING. SO AGAIN WE SEE COMPOUND 7 AND 9 BEING INHIBITORS. BUT LEVEL OF INVOLVEMENT OF SULFATE IS QUITE INTERESTING. SO THE THREE SULFATE AND SIX SULFATE MOLECULE, POTENT INHIBITION. AND THE 6 SULFATE IS BEING REMOVED. INHIBITION IS LOST. HOWEVER, THE 3 SULFATE IS REMOVED, INHIBITION IS MUTED. WE ALSO OBSERVED THAT MODIFICATIONS OF THE BACKBONE ARE TOLERATED. SO CLEARLY THE STRUCTURE DON'T HAVE HAVE A DEPENDENCE ON 3 SULFATION. WHAT WE PROPOSE IS THESE MOLECULES DON'T ACT BY INHIBITING G, THEY ACTUALLY INTERFERE WITH GB AND GD. SO THEY INTERFERE WITH INITIAL ATTACHMENT. I I THINK MY TIME IS UP. SO I WILL SKIP THE LAST PART. ACKNOWLEDGE MY CO-WORKERS. (INAUDIBLE) PERFORM MOST SYNTHETIC WORK ASSISTED BY (INAUDIBLE). MANY OF THE STUDIES AND -- PERFORMED FURTHER STUDIES. A NUMBER OF THESE STUDIES WERE PERFORMED WITH THE GROUP OF JOSIA AT THE UNIVERSITY OF ILLINOIS AND BOSTON UNIVERSITY AND RESEARCH WAS SUPPORTED BY NIH. THANK YOU FOR YOUR ATTENTION. >> LET'S MOVE DUE TO LACK OF TIME TO NEXT SPEAKER. LINDA H S I E H WILSON. SO SUMMARIZE AND SEND BY EMAIL, MAYBE TAKE A COUPLE OF MINUTES FROM THE BREAK TO ADDRESS A COUPLE OF THEM. THANK YOU. >> I WOULD LIKE TO THANK -- CAN EVERYONE HEAR ME? I WOULD LIKE TO THANK DR. (INAUDIBLE) AND NIH FOR ORGANIZING THIS WORKSHOP. IT'S A PLEASURE TO HAVE A CHANCE TO TELL YOU ABOUT SOME OF OUR RESEARCH. MY LAB HAS BEEN STUDYING GAGS FOR NUMBER OF YEARS NOW. MAINLY IN THE CONTEXT OF NEUROBIOLOGY. CAN WE GO BACK. THANK YOU. AND IN THE PROCESS WE HAVE DEVELOPED THE TOOLS THAT HAVE OPENED UP OUR ABILITY TO STUDY GAGS. I'M GOING TO TELL YOU ABOUT SOME OF THOSE TOOLS TODAY AND HOPEFULLY THEY WILL BE USEFUL TO OTHERS IN THE FIELD. WE ARE ALWAYS INTERESTED IN COLLABORATION AND APPLYING TOOLS IN DIFFERENT BIOLOGICAL DON TEXT. CONTEXT. GAGS AS YOU KNOW ARE FRONT LINE IN TERMS OF MODULATING PROTEINS AN SIGNALING EVENTS AT THE CELL SURFACE. AND AS CHEMISTS WHAT FASCINATES US ABOUT GAGS ARE THEIR DIVERSE PATTERNS OF SULFATION WHICH FACILITATE INTERACTION WITH HUNDREDS OF PROTEINS. TO US THIS WAS A BEAUTIFUL EXAMPLE OF MOLECULAR RECOGNITION. IF WE COULD UNDERSTAND THE SPECIFICITY OF THOSE INTERACTIONS AND PERHAPS UNLOCK THE SO CALLED SULFATION CODE WE COULD GAIN NEW INSIGHTS INTO MANY FUNDAMENTAL PHYSIOLOGICAL AND PATHOLOGICAL PROCESSES. MANY OF YOU I'M SURE ARE FAMILIAR WITH THE CHALLENGES ASSOCIATED WITH STUDYING GAGS. ONE CHALLENGE IS ENORMOUS STRUCTURAL COMPLEXITY. FOR INSTANCE, A SINGLE HEPARAN SULFATE TETRA SACCHARIDE HAS POTENTIAL TO DISPLAY MORE ON THIS A THOUSAND SULFATION SEQUENCES. ANOTHER CHALLENGE IS THE DIFFICULTY OF PURIFYING WELL DEFINED MOLECULES FROM NATURAL SOURCES. AS A RESULT THE FIELD HAS BEEN LIMITED LARGELY TO WORKING WITH COMPLEX HETEROGENEITY QUESTIONS. THESE CHALLENGES HAVE MADE IT DIFFICULT TO STUDY GAG AND RELATE THEIR PATTERNS TO SPECIFIC PHYSIOLOGICAL FUNCTION. FORTUNATELY, CHEMISTRY PROVIDES A SOLUTION TO SOME OF THESE CHALLENGES. WE HAVE USED ORGANIC CHEMISTRY TO SYNTHESIZE MOLECULES WITH WELL DEFINED SULFATION SEQUENCES AND ALLOWS US TO LOOK SYSTEM SYSTEMATICALLY AND IDENTIFYING NEW FUNCTIONS FOR SPECIFIC MOTIF. GAGS ARE DIFFICULT TO SYNTHESIZE, ONLY A HANDFUL OF LABS HAVE EXPERTISE TO SYNTHESIZE THEM. SO WE HAVE BEEN WORKING TO SOLVE SOME OF THE MAJOR ROAD BLOCKS AND MAKE COMPOUNDS MORE BROADLY ACCESSIBLE. FOR INSTANCE WE HAVE DESIGNED UNIVERSAL BUILDING BLOCKS FOR GAGS PRODUCED IN HALF THE NUMBER OF CHEMICAL STEPS PREVIOUSLY REQUIRED. THIS ACCELERATES THE SYNTHESIS OF GAG AND IS ENABLING US TO GENERATE LARGE COMPREHENSIVE LIBRARIES OF GAG OLIGOSACCHARIDES FOR THE FIRST TIME. TOGETHER WITH (INAUDIBLE) AND JIAN LIU'S LAB WE SYNTHESIZED HUNDREDS OF SACCHARIDES AND WORKING TO AUTOMATE THE PROCESS. THIS PROJECT WAS FUNDED BY THE COMMON FUND UO 1 GRANT AND NOW SBII GRANT WITH GLYCAN THERAPEUTIC. THESE COLLECTIONS OF COMPOUNDS WILL BECOME COMMERCIALLY AVAILABLE AND PROVIDE GREAT PRECISION AND UNDERSTANDING STRUCTURE FUNCTION RELATIONSHIPS ALLOWING FOR SYMPTOMATIC COMPARISONS HOW CHANGES IN THE ARRANGEMENT OF THE SULFATE GROUPS IMPACT PROTEIN BINDING AND FUNCTION. ALSO INTERESTED IN USING THESE LIBRARIES TO EXPLORE THERAPEUTIC POTENTIAL OF GAGS. WE ARE NOT LIMITED TO NATURALLY AVAILABLE STRUCTURE. IN ADDITION TO NATURAL SEQUENCES WE HAVE MADE NON-NATURAL ANALOGS IN SCHOOLS SUCH AS AFFINITY REAGENTS FOR CAPTURING GAG BINDING PROTEINS. IMAGING AGENTS AND GLYCOPOLYMERS. THESE POLYMERS ARE EXCELLENT GAG AND WE CAN CONTROL SULFATION PATTERN AND CHAIN LINKS. WE FUNCTIONALLIZE SURFACE WITH COMPOUNDS GENERATING GLYCAN MICROARRAYS AN BIOSENSOR CHIPS TO INTERROGATE PROTEIN BINDING. WE HAVE DEVELOPED METHODS TO ENGINEER CELL SURFACES WITH SPECIFIC GAG STRUCTURE BOTH NATURAL AND NON-NATURAL. BY PRESENTK SPECIFIC SURFATION MOTIFS ON CELL SURFACE WE CAN CONTROL CELLULAR PROCESSES SUCH AS NEURITE OUTGROWTH AND STEM CELL SULFATE. THESE ENGINEERING PROCESSES GAVE US A GAIN OF FUNCTION APPROACH THAT COMPLIMENTS TRADITIONAL LOSS OF FUNCTION TECHNIQUES SUCH AS HAPPEN RAN PASTE OR GENETIC KNOCK OUTS. WE ENGINEER CELLS IN VIVO WITH SPECIFIC GAG STRUCTURE IN THIS CASE ZEBRAFISH TO PROBE THE ROLE OF SPECIFIC MOTIFS IN ANGIOGENESIS AND TO RESCUE DEVELOPMENTAL DEFECTS. IN COLLABORATION WITH BILL'S GOB AT CAL TECH WE ARE DEVELOPING COMPUTATIONAL METHODS TO PREDICT GAG BINDING SITES IN PROTEINS AND UNDERSTAND HOW THE POLYSACCHARIDES ASSEMBLE MULTI-MERIC SIGNALING COMPLEXES. WE HAVE ALSO USED SYNTHETIC COMPOUNDS TO RAISE MONOCLONAL ANTIBODIES. THESE ARE POWERFUL TOOLS FOR IMAGING SPECIFIC MOTIFS OR BLOCKING THEIR FUNCTIONS. FINALLY WE HAVE BEEN DEVELOPING TRANSFERASE INHIBITORS AND USING COMPOUNDS TO MODULATE THE SULFATION PATTERNS OF GAGS IN CELLS IN VIVO TO PREVENT AXON REGENERATION. SO THIS CHEMICAL PLATFORM PROVIDES A DIVERSE SET OF TOOLS TO ADVANCE A MOLECULAR LEVEL OF UNDERSTANDING OF GAGS. WITH THAT BRIEF INTRODUCTION TO OUR TOOLBOX I WANT TO FOCUS TODAY ON HOW WE HAVE BEEN APPLYING THESE CHEMICAL APPROACHES TO GAIN INSIGHT INTO THE BIOLOGY OF GAGS AND IN PARTICULAR THEIR ROLE IN VASCULAR DEVELOPMENT. AT THE OUTSET, GAGS ARE A BEAUTIFUL EXAMPLE OF THE MOLECULAR RECOGNITION. WHAT WE NOTICED WAS THAT GAGS ARE INTERACT WITH A LARGE NUMBER OF RECEPTOR TYROSINE KINASES. RTK AS YOU KNOW ARE CELL SURFACE RECEPTORS FOR GROWTH FACTORS HORMONES AND CYTOKINES, THEY ARE KEY REGULATORS OF CELLULAR PROCESSES, SUCH AS GROWTH AND DEVELOPMENT AND MUTATIONS IN RECEPTORS OFTEN CONTRIBUTE TO HUMAN DISEASE SUCH AS CANCER. ALL RTK INDICATED CATTEDDED WITH A -- HEPARAN SULFATE, FGF AND ITS RECEPTOR, FIBROBLAST GROWTH RECEPTOR. WE WONDER WHETHER HEPARAN SULFATE INTERACT WITH SOME OF THESE OTHER RTKs, PERHAPS INTERACTIONS REMAINED UNDISCOVERED THAT COULD GIVE INSIGHT INTO THESE IMPORTANT SIGNALING PATHWAYS. SO A GRAD SUNT IN MY LAB AT THE TIME STARTED LOOKING INTO THESE OTHER RE RECEPTORS AND THE ONE THAT CAUGHT HIS ATTENTION WAS THE TIE RECEPTOR. TIE RECEPTORS INTERACT WITH ANGIOPOIETIN LIGANDS, IT'S CRITICAL FOR MATURATION, STABILIZATION AND REMODELING OF THE VASCULATURE. THE SYSTEM OF BLOOD VESSELS AND LYMPHATIC VESSELS THAT CARRY NUTRIENT AND OXYGEN TO CELLS IN THE BOD. THIS IS AN IMPORTANT PATHWAY TO UNDERSTAND AND MODULATE FROM A THERAPEUTIC PERSPECTIVE. UNLIKE VEGF AND OTHER PATHWAYS INVOLVED IN EARLY STAGE OF ANGIOGENIC SPROUTING, THE TIE PATHWAY IS INVOLVED IN LATER STAGE OF BLOOD VESSEL HOMEOSTASIS AND REMODELING. NEW DRUGS TARGETING THE PATHWAY ENTERED CLINICAL TRIALS FOR DISEASE SUCH AS MACULAR DEGENERATION, SEPSIS AND CANCER. SIGNALING THROUGH THIS PATHWAY IS MEDIATED BY TIE 2 RECEPTORS. BINDING OF N 1 OR ANG 4 TO THE TIE 2 RESTORE LEADS TO PHOSPHORYLATION AND ACTIVATION OF DOWNSTREAM SIGNALING. THE -- ON THE OTHER HAND ANG 2 SERVES TO ANTAGONIZE OR INHIBIT THE PATHWAY. THE TIE 1 RECEPTOR HAS BEEN SOMEWHAT OF A MYSTERY. THOUGH IT WAS DISCOVERED MORE THAN 25 YEARS AGO, IT REMAINS AN ORPHAN RECEPTOR. THE CONSENSUS IS THAT TIE 1 HELPS SUSTAIN TIE 2 SIGNALING BY FORMING HETERODIMERS WITH TIE 2. REGULATING ITS TRAFFICKING AT THE CELL SURFACE. WE ASKED WHERE HEPARAN SULFATE INTERACTSES WITH ANY LIGANDS, SURE ENOUGH FOUND THAT ANG 1 AND 4 BIND WITH NANOMOLAR ATIN FINTY TO HEPARAN SULFATE DISPLAYING THE TRISULFATE MOTIF. IN CONTRAST WE SAW NO BINDING OF HEPARAN SULFATE TO ANG 2, ANING THE TAG -- ANTAGONIST. IT BINDS ONLY TO LIGANDS THAT ACTIVATE THE PATHWAY. WE NEXTVATED WHETHER HEPARAN SULFATE FORM A COMPLEX BETWEEN THE ANG LIGAND AND RECEPTORS. SIMILAR TO THE WAY HEPARAN SULFATE FORMS COMPLEX WITH FGF AND ITS RECEPTOR. YEARS AGO WE DEVELOPED A GAG MICROARRAY PLATFORM TO LOOK AT COMPLEXES AND USING THIS METHOD ALEX A GRAD STUDENT IN MY LAB FOUND THAT TIE 2 BY ITSELF SHOW LITTLE BINDING TO THE GAG STRUCTURES ON THE MICROARRAY, SHOWN IN THE GRAY BARS. HOWEVER, WHEN HE INQUEUE INCUBATED TIE 2 IN PRESENCE OF ANG 1 OR 4, HE SAW ROBUST LOCALIZATION OF TIE 2 TO THE ARRAY. NOT SHOWN IN THE READ AND BLUE BARS. SO THIS SUGGESTS THESE ANG LIGANDS RECRUIT TIE 2 RECEPTORS TO THE ARRAY AND FORM A TERTIARY GAG LIGAND RECEPTOR COMPLEX. INTERESTINGLY, THIS COMPLEX FORMS ONL IN THE PRESENCE OF TRISULFATED HS REMOVAL OF THE SULFATE GROUPS AT ANY NITROGEN OR OXYGEN POSITIONS ABOLISHED THE INTERACTION. INDICATING THAT THIS COMPLEX REQUIRES A SPECIFIC SULFATION SEQUENCE. WE ARE CURRENTLY USING THE LARGE COLLECTION OF TETRA SACCHARIDES I SHOWED YOU EARLIER TO IDENTIFY THIS SPECIFIC BINDING SEQUENCE WHICH MAY BE USEFUL FOR SELECTIVELY MODULATING THIS PATHWAY. SO THESE RESULTS SUGGEST HEPARAN SULFATE RECRUIT END LIGANDS TO ENDOTHELIAL CELL SURFACE AND ACTIVATE SIGNALING THROUGH FORMATION OF TERNARY COMPLEXES. TO CONFIRM THIS MECHANISM WE USED A VARIETY OF APPROACHES. HERE FOR EXAMPLE WE APPLIED OUR GAG ENGINEERING METHOD TO DISPLAY TRISULFATED HS OR DESULFATED HS ON THE SURFACE OF ENDOTHELIAL CELLS. THAT FIRST EXPRESSED A HELO TYPE PROTEIN ON THE CELLS, ON THE SURFACE OF THE CELLS, THAT HALO TYPE PROTEIN FORMS A COVALENT LINKAGE WITH LIGANDS WHICH ALLOWS US TO INSTALL DEFINE GAG STRUCTURE ON THE CELL SURFACE. SIMILAR TO THE WAY THAT GAGS ARE PRESENTED ON PROTEOGLYCANS. WHAT MATT FOUND IS WHEN HE DISPLAYED TRISULFATED HS ON ENDOTHELIAL CELLS, HE OBSERVED AN INCREASE IN PATHWAY ACTIVATION. COMPARED TO WHEN HE DISPLAYED DESULFATED HS. THIS INCREASE IN PATHWAY ACTIVATION -- SORRY, DIDN'T MEAN TO GO FORWARD. WAS I DON'T KNOW IF YOU CAN PUT IT BACK ONE SLIDE PLEASE. THANK YOU. WAS OBSERVED ONLY FOR ANG 1 AND 4, NOT FOR ANG 2 WHICH YOU RECALL DOESN'T BIND HS. SO WE FIND USING THIS GAIN OF FUNCTION APPROACH, THAT TRISULFATE HS CAN PROMOTE ACTIVATION OF THE ANG TIE PATHWAY AND ONE MECHANISM IS THROUGH RECRUITMENT OF ANG LIGANDS TO THE CELL SURFACE. ANOTHER MECHANISM IS THROUGH DIRECT INTERACTION OF HEPARAN SULFATE WITH TIE 1 RE RECEPTORS. WE FOUND THE ORPHAN RECEPTOR TIE 1 BINDS WITH NANOMOLAR AFFINITY TO TRISULFATED HS. USING COMPUTATIONAL METHOD THAT I ALLUDED TO EARLIER WITH SITE DIRECTED MUTAGENESIS, WE IDENTIFIED THE HS BINDING SITE ON TIE 1. HEPARAN SULFATE BINDS TO A STRONG ELECTROPOSITIVE REGION IN THE END TERMINAL IMMUNOGLOBULIN DOMAIN OF TIE 1. AND NOT SURPRISINGLY THIS REGION IS LINED BY SIX POSITIVELY CHARGED ARGININE AND LYSINE RESIDUES FOUR WHICH ARE ENTIRELY CONSERVED ACROSS MAMMALS. WHEN WE MUTATE OR IMPORTANTLY WHEN WE MUTATE THESE TWO CONSERVED ARGININE RESIDUES IN BINDING SITE WE CAN ABOLISH BINDING HEPARAN SULFATE TO TIE 1 SHOWN HERE. I'M GOING TO COME BACK TO THIS MUTANT IN A MOMENT. IF WE LOOK MORE CLOSE AT AT THE HEPARAN SULFATE BINDING SITE IT'S ADJACENT TO ONE OF THE TWO TIE 2 BINDING INTERFACES. THIS SUGGESTED THAT HEPARAN SULFATE MODULATE THE INTERACTION OF TIE 1 WITH TIE 2. HETERODIMERS OF TIE 1 AND 2 ARE TRANSIENT AND DIFFICULT TO DETECT. HOWEVER, THE COMPLEX CAN BE IMMUNOPRECIPITATED FROM CELLS USING CROSS LINKING AGENTS. SO YOU CAN SEE HERE THAT IN THE PRESENCE OF THE CROSS LINKING AGENT DTSSP, WE PULL DOWN THE TIE 21 AND 2 COMPLEX. WHEN WE REMOVE HEPARAN SULFATE FROM CELLS USING HEPARINASE, WE SEE A REDUCTION IN THE BILL DOWN. SUGGESTING THAT HEPARAN SULFATE PROMOTES OR STABILIZES TIE 1 TIE 2 HETERO DIMERS. TO CONFIRM THESE RESULTS MATH DEVELOPED PROXIMITY LIGATION ASSAY TO PRESERVE HETERO DIMERS DIRECTLY AND WE OBSERVE DECREASE IN HETERODIMER FORMATION UPON HEPARINASE TREATMENT. THOSE THESE EFFECTS ARE MODEST THEY ARE CONSISTENT WITH FACT THAT THERE ARE TWO BINDING INTERFACES INVOLVED IN TIE 1, TIE 2 INTERACTION AND HEPARAN SULFATE AFFECTS ONE OF TWO INTERFACES. THE KEY QUESTION IS WHAT ARE THE FUNCTIONAL CONSEQUENCES DISRUPTING TIE 1 AND 2 INTERACTIONS? I WON'T TAKE YOU THROUGH ALL THE SLIDES THAT MATT PERFORMED, JUST GOING TO SKIP TO THE KEY IN VIVO EXPERIMENTS. YOU RECALL THAT WHEN WE MUTATE TWO OF THE ARGININE RESIDUES IN THE HS BINDING SITE, WE ABOLISH BINDING OF TIE 1 TO HEPARAN SULFATE. USING CRISPER CAS 9 TECHNOLOGY IN MICE WE REPLACE THE ENDOGENOUS TIE 1 GENE WITH THIS TIE 1 DOUBLE MUTANT. THIS SELECTIVELY DISRUPTS THE HS TIE 1 INTERATION IN VIVO AND WE CAN OBSERVE THE EFFECTS OF THAT SINGLE GLYCAN PROTEIN INTERACTION IN VIVO ON VASCULAR DEVELOPMENT AND SIGNAL. WE OBSERVE SIGNIFICANT VASCULAR DEFECT, RETINAS OF THE MICE SHOW INCREASES IN RADIAL GROWTH, VESSEL AREA AND BRANCHING. WE ALSO EXAMINE LUNG TISSUE FROM ADULT ANIMALS BECAUSE THE LUNG IS HIGHLY VASCULARIZED. AND AGAIN, WE OBSERVE SIGNIFICANT DIFFERENCES SPECIFICALLY LOWER LEVELS OF TIE 1 PROTEIN EXPRESSION. AND A SIGNIFICANT DECREASE IN TIE 2 PHOSPHORYLATION AND AKT PHOSPHORYLATION. SO THESE STUDIES INDICATE DISRUPTING HEPARAN SULFATE TIE 1 INTERACTION IN VIVO LEADS TO DECREASED TIE PATHWAY ACTIVATION. REDUCED SURVIVAL SIGNALING AND VASCULAR DEFECTS. IN CONCLUSION WE HAVE SHOWN HEPARAN SULFATE GAGS ARE KEY REGULATORS OF THE ANG TIE PATHWAY IN VIVO THROUGH DUAL MECHANISMS HEPARAN SULFATE SERVES AS A STAT TO REGULATE SIGNALING THROUGH THIS CRITICAL PATHWAY. THESE FINDINGS SUGGEST EXPANDED ROLES FOR HEPARAN SULFATE IN REGULATING THE VASCULAR SYSTEM, THOUGH HS WAS KNOWN TO CONTRIBUTE TO EARLY STAGES OF BLOOD VESSEL SPROUTING THROUGH ANGIOGENIC GROWTH FACTORS SUCH AS FGF AND VEGF. THIS IS THE FIRST TIME TO MY KNOWLEDGE THAT HEPARAN SULFATE HAS BEEN SHOWN TO BE IMPORTANT FROM LATE STAGES OF BLOOD VESSEL MATURATION, HOMEOSTASIS AN REMODELING. THIS HAS IMPORTANT THERAPEUTIC IMPLICATIONS FOR INFLAMMATION, TUMOR ANGIOGENESIS AND METASTASIS, DIABETES AND OTHER DISEASES. MORE BROADLY OUR RESULTS HIGHLIGHT THE DIVERSE MECHANISMS BY WHICH GAG IS CONTROL CELLULAR SIGNALING, THEIR ABILITY TO RECRUIT LIGANDS CELL SURFACE INFORMATIVE TERNARY COMPLEXES RECEPTORS CLASSIC MECHANISM SHARED BY MANY GROWTH FACTORS. HOWEVER, WE THINK THAT ANOTHER IMPORTANT MECHANISM THAT APPEARS TO BE EMERGING IS THE ABILITY OF GAG TO ENGAGE CELL SURFACE RECEPTORS DIRECTLY AND CONTROL THE POPULATION OF RECEPTOR COMPLEXES AT THE CELL SURFACE. WE HAVE SEEN THIS MECHANISM WITH SULFATE AND INTERACTION WITH NEURAL RECEPTORS SUCH AS PKPC. FINALLY MOST IMPORTANTLY I HOPE I HAVE SHOWN POTENTIAL FOR CHEMICAL APPROACHES TO REVEAL NEW FUNCTIONS FOR GAG AND OPEN UP THERAPEUTIC OPPORTUNITIES. LAST I WOULD LIKE TO THANK ALL THE PEOPLE IN MY LAB, MOST OF THE WORK DONE BY A REALLY TALENTED GRADUATE STUDENT IN MY GROUP, MATT GRIFFIN, HE WAS HELPED BY ALEX SOROM WHO DID MICROARRAY STUDIES AND GREG MILLER WHO WORKED ON ON THE COMPUTATIONAL STUDIES. LAE AND TAKUYA HAVE BEEN WORKING HARD ON DEVELOPING THE LIBRARY SYNTHESIS. ALSO THANKS TO COLLABORATORS, AS WELL AS NIH FOR MAKING THESE STUDIES POSSIBLE. THANKS FOR YOUR ATTENTION. >> THANK YOU SO MUCH FOR KEEPING THE TALK ON TIME. IF YOU HAVE ANY SPECIFIC QUESTION IF YOU WANT TO ADDRESS TO LINDA, PLEASE FEEL FREE TO DO SO. GIVE A MINUTE, SOME NOTES CAN BE PUT IN THE CHAT. OTHERWISE MEANTIME I'M GOING TO START TAKING AWAY FIVE MINUTE THE BREAK TO ASK SOME QUESTIONS THAT WERE POSED IN THE CHAT BECAUSE THIS ARE VISIBLE TO THE PEOPLE CONNECTED VIA VIDEO CAST. SO I WOULD ASK YOU TO BE VERY BRIEF. I'M GOING TO START WITH SOME OF THE QUESTIONS IN THE CHAT, ONE MORE MIKE, SOME DISCUSSION ABOUT PRETTY MUCH HOW THIS ASSAYS THAT YOU HAVE DISCUSSED ARE IMPLEMENTED IN THE STATE NEWBORN SCREENING. PLEASE BE BRIEF. MPS ONE IS ON THE RUSP AND MAYBE 30 TO 40 STATES STARTED TO SCREEN FOR IT. THREE OR FOUR OF THESE STATES USE GAGS NOW. FOR SECOND TIER PARTS OF NEWBORN SCREENING. KEY IS TO MAKE IT PART OF NEWBORN SCREENING SO THAT PARENTS DON'T HAVE TO BE CALLED FOR FALSE ALARMS. BUT I DON'T KNOW, STILL 60% OF THE STATES FOR GAGS RELY ON DNA. THIS IS FOR HISTORICAL REASONS BUT DNA IS TURNING OUT TO BE PROBLEMATIC. THERE'S A GENERAL OPTIMISM THAT DNA WILL SOLVE PROBLEMS BUT FOR LSD IT'S A BIG MESS BECAUSE OF VARIATIONS OF NON-SIGNIFICANCE AND INABILITY TO PREDICT GENOTYPE PHENOTYPE CORRELATIONS VERY WELL. GAGS CORRELATE REALLY WELL WITH DISEASE SEVERITY AND IS THE METHOD OF CHOICE, THE DATA IS CLEAR. AND I THINK EVENTUALLY IN THE NEXT YEAR OR TWO ALL LABS WILL BE INCORPORATING GAGS AS PART OFFER MU BORN SCREENING. -- NEWBORN SCREENING. THEY'LL STRUGGLE WITH PHENOTYPES AND REGRET IT. THAT'S ALL I CAN SAY. SO THESE ARE THINGS THAT ARING BUSINESS -- THAT ARE BEING DISCUSSED BUT NO QUESTION SHOWING GAGS TRUMP THE DNA ANALYSIS FOR SECOND TIER. THERE'S NO QUESTION. >> THANKS SO MUCH. I HAVE A QUESTION ALSO FOR GEERT. POSTED ON THE CHAT. IS THERE ANY STRUCTURAL INFORMATION ABOUT THE DEPENDENCE OF THIS SPECIFIC SULFATE FOR BINDING VIRUS PROTEINS? WONDERING WHAT IS KNOWN ABOUT ANY CONFIRMATIONAL ADJUSTMENTS WITH REMOVAL OF WHAT SPECIFIC SULFATE. PLEASE BE CONCISE. >> THERE WAS LIMITED STRUCTURAL DATA FOR PROTEINS SUCH AS FGFR 1, COMPLEX WITH FTF 2 X-RAY CRYSTAL STRUCTURE DATA, NOT ALL DATA AGREES. WE KNOW IDURONIC ACID HAS A (INAUDIBLE) AND CAN ADJUST ITS CONFIRMATION TO A PROTEIN. NOW THAT WE KNOW SPECIFIC -- WITH THESE PROTEINS, OPPORTUNITIES ARE ARISING TO DETAILS STRUCTURAL STUDIES. THAT SOMETHING THAT WE ARE DOING FOR SURE. >> THANK YOU. ONE QUESTION FOR LINDA. WHO IS COMMERCIALIZING THESE LIBRARY AND WHEN WILL INVESTIGATORS BE ABLE TO PURCHASE? >> GLYCAN THERAPEUTICS IS WORKING WITH US TO COMMERCIALIZE THE LIBRARIES. THE INITIAL PLAN IS TO PUT THEM ON MICROARRAYS AND MAKE THEM ACCESSIBLE. THAT WAY ALTHOUGH THERE IS -- THERE IS A LONG TERM PLAN TO PROVIDE COMPOUNDS WE HAVE FIVE OR TEN MGs OF EACH COMPOUND THAT WE HAVE MADE SO FAR. WE WANT TO START WITH MICROARRAYS AND MAKE THEM COMMERCIALLY AVAILABLE. MAYBE JOHN YOU CAN COMMENT ON THE TIME LINE, I THINK THE TIME LINE WAS PRETTY RELATIVELY SOON. THEY -- ROLE THRIVE SOON, THEY STARTED TO MAKE MICROARRAYS WITH SOME OF THESE WERE IN THE PROCESS OF VALIDATING MICROARRAYS. JOHN, DO YOU HAVE ANYTHING TO ABOUT THE TIMING? >> YES. ACTUALLY SOME OF THE OLIGOSACCHARIDES ARE ALREADY AVAILABLE NOW FROM GLYCAN'S WEBSITE. ACTUALLY THEN -- SO THE CHEMICAL SYNTHESIS COMPOUND WILL TAKE A LONGER TIME. WE ARE IN THE PROCESSING TO FIRST PUT ON ARRAY AND THEN EVENTUALLY WILL PUT ON MARKET. IT WILL BE SOON. >> I HAVE ONE QUESTION THAT (INAUDIBLE) OR ANYBODY THAT CAN ANSWER IT. ASKED BY MIKE. ARE DISEASES ASSOCIATED WITH LOSS OF PRESERVATION OF GAGS? >> MULTIPLE DISEASES HAVE BEEN IMPLICATED, INCLUDING CANCER. (INAUDIBLE) MANY PIECES OF THE PUZZLE STILL NEED TO BE PUT TOGETHER. ONE IS STRUCTURE (INAUDIBLE) SO NICELY. WE NEED TO KNOW WHICH PROTEINS ARE INVOLVED AND WHAT GLYCANS TO POINT TO. (INDISCERNIBLE) FUTURE LOOKS BRIGHT WITH ALL THESE TECHNOLOGIES COMING ONLINE. AND CLEAR CORRELATIONS WILL BE MADE BETWEEN DISEASE AND MODIFICATION. I KNOW ABOUT GENETIC DISORDERS. >> IT'S INTERRING THAT WE HAVE A TYPE OF MPS FOR ALL KNOWN MODIFICATIONS OF THESE THINGS EXCEPT FOR THREE SULFATION, I'M WONDERING IF LOSS OF IT IS LETHAL. WE HAVE MPS ORDERS FOR EVERYTHING EXCEPT 3 SULFATION AND LOSS OF THIAZOL FATASE, I DON'T KNOW WHAT'S GOING ON WITH THAT. MAYBE IT'S MATTER OF TIME BEFORE WE FIND DISEASES OR MAYBE NOT TOLERATED. >> OKAY. I THINK WE WILL -- (OVERLAPPING SPEAKERS) ENZYMES REDUNDANCY. >> THAT'S A GOOD POINT. YEAH. >> THIS IS A VERY INTERESTING DISCUSSION. IT HATE TO CUT IT OFF BUT PROBABLY SHOULD GIVE AT LEAST TEN MINUTES FOR A BATHROOM BREAK TO EVERYBODY SO WE'LL SEE YOU BACK AT 10:40. AMANDA, PLEASE MAKE ANNOUNCEMENT IF YOU HAVE TO. OR PAM. >> EVERYTHING IS GOING WELL. THANK YOU. >> OKAY. BACK AT 10:40. PLEASE ANSWER IN THE CHAT OR ASK QUESTIONS THERE, DISCUSSION CAN CONTINUE. THANK YOU. >> THANK YOU. I WOULD REMIND PARTICIPANTS THEY SHOULD CONTACT THE SPEAKERS DIRECTLY IF THEY HAVE INTEREST IN THEIR TOOLS OR THEIR WORK. SEVERAL SPEAKERS ARE FROM THE NIH COMMON FUND. AND IT'S INCUMBENT ON THEM TO LOOK FOR PEOPLE TO BETA TEST THEIR TOOLS. YOU ARE WELCOME TO CHAT WITH THEM ABOUT USING THE TOOLS. THANK YOU. >> I WOULD LIKE TO WELCOME YOU BACK TO THE SECOND HALF OF THE FIRST SESSION. DR. JIAN LU WILL TALK ABOUT OLIGOSACK RISE IN GAG SEPARATION. DR. L IU, TAKE IT A I WAY. >> THANKS TO PAM AND ORGANIZERS TO GIVE ME THE OPPORTUNITY TO TALK ABOUT OUR RESEARCH. -- I'M PROFESSOR AT THE SCHOOL PHARMACY UNC AND ALSO A FOUNDER AND CHIEF SCIENTIFIC OFFICER FOR GLYCAN THERAPEUTIC WHICH IS LOCATED IN NORTH CAROLINA. THIS IS MY FINANCIAL INTEREST DISCLOSURE. I THINK IT'S TO THIS AUDIENCE I DON'T HAVE TO SAY TOO MUCH ABOUT THE IMPORTANCE OR ESSENTIAL PART OF THE HEPARAN SULFATE FOR BIOLOGY AND PRESENT ON THE CELL SURFACE INTERACT WITH FGF AND THE INDUCED FGF SIGNAL PATHWAY. AND CONTROL THE GENE ACTIVATION. CHONDROITIN AND IT COMBINES TO CHEMOKINES AND THEN TO ATTRACT GLYCOCYTE TO INDUCE INFLAMMATORY CELLS AND HEPARAN SULFATE ALSO SERVE AS A INITIAL BINDER FOR MANY VIRUS INFECTIONS AND INCLUDING HERPES INFECTION LIKE WE HEARD FROM GEERT JAN BOONS TALK EARLIER. THESE FUNCTIONS ARE ESSENTIAL BECAUSE FOR THE STRUCTURE, THE DETAIL STRUCTURE OF HEPARAN SULFATE AND CHONDROITIN SULFATE. THOSE SUGAR HAVE A VERY SPECIFIC SULFATION PATTERN. CONTAINING IDURONIC ACID FOR HEPARAN SULFATE AND FOR CHONDROITIN SULFATE THEY HAVE DIFFERENT SULFATIONS AND THESE SULFATIONS PLAY KEY ROLES DETERMINING STRUCTURAL SELECTIVITY OF THE GAG. SO FOR US, OUR GOAL IS THAT WE TRY TO PRODUCE THE CHEMICAL REAGENTS AND TOOLS TO STUDY GLYCOSAMINOGLYCANS. SO WE ARE TRYING TO CONSTRUCT WIDE RANGE OF HEPARAN SULFATE AND CHONDROITIN SULFATE OLIGOSACCHARIDE LIBRARIES. WITHIN TWO APPROACHES. ONE APPROACH IS IN COLLABORATION WITH THE JEFFREY HUANG AND DR. LINDA HSIEH-WILSON, THESE GROUP WORKING TO USING CHEMICA APPROACH TO SYNTHESIZE SHORT OLIGOSACCHARIDE AND THIS WAS INITIALLY FUNDED BY COMMON FUND NOW SUPPORTED BY NIH SBIR GRANT. IN MY LABORATORY AND ALSO IN THE GLYCAN THERAPEUTICS IN COLLABORATION WITH DR. LYNNHART WE ARE USING A ENZYMATIC APPROACH SYNTHESIZE DIFFERENT KIND OF GAGS AND THESE SYNTHESIS WERE MAINLY FOCUS ON LARGER SUGARS LIKE FROM SIX MERS TO 18. THOSE ARE -- IT'S A PRIMARY FOCUS IN THE LAB. TO THE ENZYMATIC SYNTHESIS IS THAT ITS A MULTI-ENZYME PROCESS. TO SYNTHESIZE HEPARAN IT'S INVOLVED SKULL FILL TRANSFERASE TO TRANSFER SULFATE TO 6 POSITION OF GLUCOSAMINE AND THERE'S A THREE SULFATE TRANSFERASE RACE IN 3 POSITION AND E PEMERASES TO IN SULFATION, SO THOSE ENZYMES ARE REQUIRED TO SYNTHESIZE HEPARAN SULFATE OR HEPARAN. FOR THE CHONDROITIN SYNTHESIS, WE ALSO NEED SULFUR TRANSFERASE FOR THOSE DIFFERENT ENZYMES WITH DIFFERENT AMINO ACID SEQUENCES. SO THOSE ENZYME INCLUDING THE FULL SULFUR TRANSFER RATION TO TRANSFER SULFATION TO GALACTOSAMINE OR GLCNAC SULFUR TRANSFERASE TO THE 6 POSITION OF THE GALACTOSAMINE. AND ALSO 6 SULFUR TRANSFER SULFATE TO THE GLYCOSAMINE AS WELL. THOSE TWO GROUPS OF ENZYME ALLOW US TO SYNTHESIZE HEPARAN SULFATE OLIGOSACCHARIDE AS WE AS CHONDROITIN SULFATE OLIGOSACCHARIDE. SO THE KEY FOR THESE SUCCESS IN THESE ENZYMATIC SYNTHESIS IS THAT WE HAVE TO BE ABLE TO OBTAIN LARGE AMOUNT OF ENZYMES AND ALSO OBTAIN RELATIVELY INEXPENSIVE CO-FACTOR TO CARRY OUT ENZYMATIC SYNTHESIS. NOW THESE PROCESS ARE GENERALLY FIXED AND IT'S BEEN WELL DEVELOPED IN THE LABS AS WELL AS IN GLYCAN THERAPEUTICS. SO HERE I'M TRYING TO GIVE YOU SEVERAL EXAMPLES WHERE WE SYNTHESIZE THESE OLIGOSACCHARIDE OR DEVELOP THIS ENZYMATIC METHOD, WHAT IS APPLICATION, FIRST IS WE TRY TO USE ENZYMATIC APPROACH TO MODERNIZE HEPARAN DRUG COMMONLY USED ANTI-COAGULANT DRUG. CURRENTLY IS ANIMAL SOURCE MATERIAL. THE RELIABILITY AND THE PURITY IS DIFFICULT TO CONTROL. SO HERE WE ARE USING THE ENZYMA THE I CAN APPROACH, TEOSINTE SIZE HEPARAN THAT IMPROVE SAFETY AND MAINTAIN RELIABILITY OF HEPARAN SUPPLY CHAIN. THEN WE USE HEMOENZYMATIC APPROACH WE CAN SYNTHESIZE TYPE OF HEPARAN CONSTRUCT. FOR EXAMPLE ONE PRIMARY CONSTRUCT THAT DEVELOP UNDER THE DEVELOPMENT OF GLYCAN THERAPEUTICS IS THAT WE SYNTHESIZE 12 MER COMPOUND. THIS 12 MER COMPOUND HAS SIMILAR PHARMACOKINETIC PROPERTIES, COMPARED TO CURRENTLY MARKETED HEPARAN. SO THEREFORE WAS THIS BECOME A DRUG THE DOCTOR CAN BE READILY SWAP WEAN THE 12 MER -- BETWEEN THE 12 MER PRODUCT AND THE LOW HEPARAN CURRENTLY USING. ANOTHER COMPOUND IS A 6 MER PRODUCT. THIS COMPOUND MAJOR ADVANTAGE OF 6 MER COMPOUND IS INEXPENSIVE TO SYNTHESIZE. BASED ON CURRENT ESTIMATION, THE PRODUCTION COSTS FOR SIX MERS IS MUCH LOWER THAN FUNDED PARADOCKS. THE THIRD IS A 8 MER PRODUCT. THIS IS A UNIQUE STRUCTURE THAT HAS ANTI-COAGULANT ACTIVITY. THE PROPERTY OF THIS COMPOUN IS THAT IT'S ME TABBIZED VERY FAST -- METABOLIZED VERY FAST SO USEFUL FOR THOSE PATIENT WHOSE VULNERABLE FOR BLEEDING SIDE EFFECTS SO THOSE GIVE A DIFFERENT DEPENDS ON THE STRUCTURE, WE CAN CONTROL THE PHARMACO PROPERTY OF THESE DRUGS AND THEREFORE DRUG DISCOVERY DEVELOPMENT. WE SYNTHESIZE VARIETY OF THESE HEPARAN STRUCTURE, WE CAN PUT ON ARRAY, IN EVIDENCE PREVIOUS SESSION THERE'S ONE OF THE AUDIENCE ASK WHETHER OR NOT COMPOUNDS ARE AVAILABLE RIGHT NOW TO MAKE THESE IN THE GLYCAN YOU CAN DO THE COMMERCIAL MICROARRAY ANALYSIS AS WELL AS ACTUALLY ALL OF THESE COMPOUNDS LISTED HERE ARE COMMERCIALLY AVAILABLE, YOU CAN OBTAIN FROM GLYCAN THERAPEUTICS WEBSITE. WE HAVE A TOTAL OF CLOSE TO A HUNDRED TO 120 RESEARCHERS CAN PURCHASE IN THE REASONABLY PRICE WHICH IS COMPARE -- IT'S ABOUT $100 TO $1,000 DEPENDING ON COMPLEXITY OF THE STRUCTURE. WE USE THESE ENZYMATIC APPROACH WE SYNTHESIZE THESE LABELED DISACCHARIDE STANDARDS. FOR THOSE PEOPLE WHO IS DOING THESE HEPARAN SULFATE DISACCHARIDE ANALYSIS YOU SEE THOSE ARE AUTHENTIC STRUCTURE. BY USING THESE C 13 LABEL DISACCHARIDE STANDARDS WE CAN CONDUCT QUANTITATIVE ANALYSIS OF HEPARAN FROM BIOLOGICAL SOURCES. WITH THE SENSITIVITY AND THE NANOGRAM SCALE. AND THIS ARE AUTHENTIC STRUCTURE, WE CAN CANCEL OUT ALL THESE VARIATIONS FROM BIOLOGICAL SAMPLES HERE IN THE ANALYSIS OR CHEMICAL COUPLING REACTIONS YOU CAN USE THE C 13 LABEL DISACCHARIDE STANDARDS TO CALIBRATE THE ANALYSIS AND IMPROVE THE ACCURACY FOR THE HEPARAN ANALYSIS. THEN THE REMAINING TIME I WANT TO TALK ABOUT HOW WE USE THESE HEPARAN OLIGOSACCHARIDE TO DESIGN ANTI-INFLAMMATORY PROPERTIES. THOSE HEPARAN SULFATE AND CHONDROITIN SULFATE. THERE'S TWO PROJECTS THAT I WANT TO TALK ABOUT, ONE IS THE COLLABORATION WITH PROFESSOR (INAUDIBLE) FROM BUFFALO. ANOTHER ONE FROM -- FROM UNC. THE FIRST EXAMPLES WERE DEALING WITH THE OVERDOSE LIVER INJURY. SO PROBABLY YOU KNOW THAT ASEAT MINUTE -- ASEAT MINUTE FIN IS COMMON LIE USED IN THE U.S., IN EUROPE AND ALSO IN OTHER PART OF THE WORLD. HOWEVER, THE OVERDOSE OF ACETAMINOPHEN CAUSE SEVERE, LIFE THREATENING ACUTE LIVER INJURY. AND IN U.S. IS ABOUT 80,000 HOSPITAL VISIT PER YEAR BECAUSE OF THE APAP OVERDOSE. SO IT'S RIGHT NOW THERE'S A METHOD TO TREAT THESE APAP OVERDOSE USING A SISTINE HOWEVER, THIS HAS TO BE CAPTURED WITHIN EIGHT HOURS OVERDOSE. BEYOND THIS POINT THIS DRUG IS NO LONGER EFFECTIVE. PATIENT LEFT OPTION IS LIVER TRANSPLANTATION. SO THE WAY WE DID THE EXPERIMENT IS THAT WE SYNTHESIZE HEPARAN OLIGOSACCHARIDES 18 SUGAR RESIDUE LONG, SYNTHESIZE APPROXIMATELY 400 MILLIGRAMS OF THIS COMPOUND THEN TEST IN MICE. AS YOU CAN SEE GIVING THE 18 MER WE CAN REDUCE LEVEL ALT MARKER FOR THESE LIVER DAMAGE. ALSO WE REDUCE TMF ALPHA CONCENTRATION IN PLASMA, INDICATED 18 MER HAS SUPPRESS THE INFLAMMATORY MOUSE SOMEWHAT. IN SURVIVAL SAW STUDIES WITH 18 MER TREATMENT IS IMPROVE THE SURVIVAL OF THE MICE. AFTER APP OVERDOSE. THEN WE SORT OUT MECHANISM OF ACTION OF THIS TUMOR. WENT TO THE LITERATURE TO BEGIN TO UNDERSTAND WHAT IS THE MECHANISM OF APAP OVERDOSE. INITIALLY IT'S CONVERTED TO A CHEMICAL REACTIVE SPECIES BY P 450. IT'S COATED MOLECULE, THIS IS A CHEMICAL STRUCTURE. THIS IS A CHEMICALLY REACTIVE COMPOUND AND IT'S CONJUGATED DAMAGE. THERE WAS THESE DAMAGE IS HAPPENING WITHIN FOR EXAMPLE LIKE WHAT I CIRCLED, THESE PARTICULAR CELLS, THE DEAD CELLS RELEASE NEW COLLIE EYE PROTEIN, KNOWN AS HPGB 1 PROTEIN. T THIS HMGB 1 UNDER HEALTHY CONDITION SUPPOSE TO BE ENCAPSULATED INSIDE THE NUCLEI. HOWEVER, UNDER THESE TOXIC CONDITION WHEN THE CELLS DIE THE HMG 1 BEGIN TO RELEASE. THESE RELEASE HMGB 1 TRIGGER THE NEUTROPHIL INFILTRATION TO THE SITE AND THEN TO START INFLAMMATORY SPOUT. SOMETIMES THE INFLAMMATORY SPOUT IS OVERREACTED AND AMPLIFY THE DAMAGE FROM THE SINGLE CELL SITE AND BEGIN TO AMPLIFY TO CAUSE LABTIVE -- COLLATERAL DAMAGE. THIS COLLATERAL DAMAGE LEAD TO THE LIVER INJURY. SO WHAT WE HYPOTHESIS IS THAT THERE'S PROBABLY THE HEPARAN SULFATE 18 MER STOPPED LATER STAGE OF THE SERO INFLAMMATION PROCESS TO PREVENT THESE LIVER AMPLIFICATION, THE INJURY AMPLIFICATION PARTS. HERE IS BECAUSE OF THE TIME LIMIT I'M USING HIGHLY SUMMARIZED SLIDE, ABOUT CONCLUSION FOR MECHANISM STUDIES. SO THERE'S A THREE LINES OF EVIDENCE TO SUGGEST 18 MER TARGETS TO HMGB 1 TO ACHIEVE HEPATOPROTECTION EFFECT. IF WE COMPARE, WE COMPARE THE PROTECTION EFFECT BETWEEN A TUMOR AND 18 MER HMGB 1 ANTIBODY. AND THEN AS YOU CAN SEE THAT HAS -- THEY BOTH 18 MER AND ANTIBODY DISPLAY A SIMILAR KIND OF PROTECTION BASED ON THE ALT LEVEL. AND IF WE USE A COMBINATION OF 18 MER AND ANTIBODY WE DON'T REALLY GAIN ANY ADDITIONAL PROTECTION SUGGESTING THESE TWO PROTEINS AND 18 MER TARGET TO SAME PROBE. SECOND LINE OF EVIDENCE TO SHOW THAT 18 MER PREVENT HMGB 1 MEDIATED NEUTROPHIL INTO THE INJURY SITE. WE SEE DRAMATIC DECREASE OF NEUTROPHIL INFILTRATION, IN THE PRESENCE OF 18 MER. THIRD LINE IS EVIDENCE OF GENETIC KNOCKOUT MICE EVIDENCE SO HERE USING A KNOCKOUT MICE, THERE'S NO HMGB 1 KNOCKOUT MICE AVAILABLE SO RAGE RECEPTOR FOR HMGB 1, THOSE NOT CALLED RAGE PRESUMABLY THE HMGB 1 PATHWAYS IS CONTINUED. SO AS YOU CAN SEE THAT ONCE IN THE RAGE KNOCKOUT MICE THE 18 MER LOSS THE EFFECT BECAUSE THE TARGET IS MISSING FROM THE MICE. THIS IS COMPARED TO THE CONTROL MICE WHICH YOU CAN SEE CLEARLY DECREASE IN HEPATOPROTECTION EFFECT. HEPATOPROTECTION EFFECT. SO THESE THREE LINES EVIDENCE SUGGEST THAT 18 MER TARGET TO HMGB 1 AND DISPLAY IS PROTECTING EFFECT. THE NEXT LINE IS WE BEGIN TO DO A LIMITED STRUCTURAL ACTIVITY MEASUREMENT AND USING THESE CO-LATED 18 MER WE SEE WHICH DIFFERENT SIZE OF THE SUGARS 6 MER 12 ME,R AND 18 MER. ONLY 8 MER BIND TO HMGB 1 AS DISPLAYED BY THE FEATURES HERE. WE INTRODUCE ALL THREE 6 MER 12 MER 18 MER ONLY 8 MER SUGGESTING COMPOUND USES CAPABILITY TO BIND TO HMGB 1 IT'S ALSO LOSES CAPABILITY TO PROTECT THE LIVER -- APAP INDUCE LIVER INJURY. THE NEXT EXPERIMENT IS THAT WHAT WE DID IS WE TRIED TO COMPARE 18 MER VERSUS EXISTING CURRENT THERAPEUTIC APPROACH SISTINE, IT'S CURRENTLY USED IN HOSPITAL FOR -- ACETYLSISTINE TO TREAT APAP OVERDOSE PATIENT. HOWEVER THE PROBABLE OF THESE ACETYLCYSTEINE HAS TO BE GIVEN IN EIGHT HOURS SO WE DID A MICE EXPERIMENT TO SEE WHAT IS PROTECTION EFFECT. WE COMPARE 18 MER AND CYSTEINE IF WE GIVE HALF HOUR THEY DISPLAY THE SAME HEPATOPROTECTION EFFECT. IF WE TREAT MICE IN DELAY FOR THREE HOURS THE ACETYLCYSTEINE LOSS THE PROTECTIVE EFFECT. THE 18 MER RETAINS. THE 6 HOURS SHOW SOME PARTIAL PROTECTION BUT IT IS ONLY COURSE IS NOT AS CLEAR AS THREE HOURS. WE DID SURVIVAL STUDIES SUGGEST THAT IS AFTER THREE HOURS DELAYED TREATMENT ACETYLCYSTEINE LOSS PROTECTIVE EFFECT BUT 18 MER MAINTAIN. BUT GO TO 6 HOUR, 18 MER AND 6 ACETYLSISTINE LOSS THE EFFECT. SO THIS SUGGESTS 18 MER CAN BE GIVEN LOW -- LATER STAGE WHEN THE INJURY HAPPEN AND WE THINK THIS WILL OPEN UP POTENTIALLY OPEN UP THE WIDEN THE THERAPEUTIC WINDOW FOR THESE APAP OVERDOSE INJURY PATIENTS. THEN THE NEXT EXAMPLE WHAT I'M TRYING TO TELL YOU IS WE ALSO USING A DIFFERENT OLIGOSACCHARIDE CHONDROITIN SULFATE TUMOR TO TREAT LIPOPOLYSACCHARIDE INDUCED SYSTEMIC INFLAMMATION. THE MAIN PURPOSE OF THIS, IT'S SYMPTOM FROM THE LIPOPOLYSACCHARIDE INDUCED ENDOTOXEMIA MICE HAS SIMILAR SYMPTOMS LIKE SEPSIS PATIENT WHICH HAS A WIDE IMPACT MANY THE PUBLIC HEALTH. THEN IN THIS PROCESS THE LPS ALSO ACTIVATE RELEASE OF HISTONE, ANOTHER DNA BINDING PROTEIN FROM NUCLEI THEN IS CONTRIBUTING FACTOR TO TRIGGER THE EXCESSIVE INFLAMMATION. SO HERE AS YOU CAN SEE, IF WE GIVE THE MICE LPS WE CAN CLEARLY SEE THERE'S INCREASE OF THE HISTOTONE RELEASED IN PLASMA, AND THEN THIS MIDDLE PANEL, IT SHOWS THAT IF WE CAN -- IT'S EXVIVO EXPERIMENT. WE TAKE THE PLASMA FROM LPS TREATED MICE. DEMONSTRATED THAT 18 ME,R, I'M SORRY, CHONDROITIN SULFATE CAN INTERACT WITH HISTONE. THIS WILL INDICATE THAT THERE'S A REAL INTERACTION. IN THE SURVIVAL EXPERIMENT ALSO DEMONSTRATED THE CHONDROITIN SULFATE 19 MER HAS CLEAR PROTECTION AGAINST MICE AFTER TREATED WITH THE LPS. HERE WHAT I'M GIVING THE EXAMPLE, IF SULFATED SUGARS OF GAG HAS WIDE RANGE OF BIOLOGICAL FUNCTION AND HERE WE ARE STARTED WITH ANTI-COAGULANT PROPERTY. NOW WE SEE HEPARAN SUP FATE HAS ANTI-INFLAMMATORY EFFECT. HEPARAN SULFATE AND CHONDROITIN SULFATE AND WHETHER OR NOT WE CAN USE DESIGN OF THESE OLIGOSACCHARIDE TO PROBE OTHER BIOLOGICAL FUNCTION OF THE HEPARAN AS POTENTIAL THERAPEUTICS, IT'S REMAIN TO BE SEEN BUT RIGHT NOW IT WILL BE HELPFUL BECAUSE TECHNOLOGY IS THERE, WE HAVE THESE OLIGOSACCHARIDE AND WE HAVE A METHOD TO BEGIN TO PROBE THESE PROPERTIES. THIS IS THE GROUP AT U INHC AND THEN IT'S EVERYBODY WORKING A DIFFERENT TYPE OF DIRECTION ON HEPARAN SULFATE AND WE ARE WORKING HARD ON THIS ISSUE. THIS IS THE -- I ALSO WANT TO THANK COLLABORATORS, FROM BCU. SO THE FUNDING IS WE GOT A GENERAL SUPPORT FROM NIH AND ALSO WE HAVE RECEIVED FELLOWSHIP THAT IS SUPPORT POST-DOCTORAL FELLOWSHIP. WHILE SHE'S COMPLETING THE LIVER PROJECT. I THINK I WILL STOP RIGHT HERE. >> THANK YOU SO MUCH, JIAN, APPRECIATE THE INTERESTING PRESENTATION. IN THE INTEREST OF TIME WE WOULD LIKE TO MOVE ON TO THE NEXT PRESENTATION. WE CAN HOPEFULLY USE SOME TIME LATER ON IF YOU HAVE SOME FEW MINUTES TO ADDRESS SOME QUESTIONS. SO THE NEXT PRESENTATION IS FROM DR. MIN DONG FROM BOSTON CHILDREN'S HOSPITAL. TITLE IS SULFATEED GLYCOSAMINOGLYCANS CONTRIBUTE TO C DIFFICIL TOXIN A BINDING AND ENTRY INTO CELLS. >> THANK YOU, CAN YOU HEAR ME OKAY? >> YES. >> THANK YOU TO THE ORGANIZERS FOR THIS WONDERFUL WORKSHOP AND IT WAS THIS OPPORTUNITY FOR ME TO INTRODUCE OUR WORK. WE DO NOT WORK ON THE -- WE WORK ON BACTERIAL TOXINS. TODAY I WILL TALK ABOUT RECENT FUNDING THAT SULFATED GAGS CONTRIBUTE TO THE BACTERIAL TOXIN C DIFFICIL TOXIN A BEHINDING ON ENTRY INTO CELLS. SO C DIFFICIL IS A MAJOR MEDICAL ISSUE NOW ADAYS IN THE HOSPITAL. ONE OF THE MAJOR OPPORTUNISTIC PATHOGENS IN HOSPITAL ACQUIRED INFECTIONS IN THE U.S. EVERY YEAR. THE PATHOLOGY FOR IT IS STARTED WHEN THE NORMAL GUT MICROBIOME IS DISRUPTED. FOR INSTANCE DURING ANTIBIOTIC TREATMENT. THAT CREATE A SPACE OF OPPORTUNITY FOR THOSE OPPORTUNISTIC PATHOGENS TO INFECT BECAUSE OF THE (INAUDIBLE) ENVIRONMENTAL PATHOGENS, THEY ARE SPORES EVERYWHERE IN THE ENVIRONMENT. THAT -- COLONIZE AN GROW IN THE COLON AND START TO PRODUCE TOXINS TO DISRUPT THE COMMON TISSUE THAT LEADS TO SEVERE DIARRHEA. AS WELL AS NEW RESPONSE. THE MAJOR TOXINS PRODUCED BY C DIFFICIL ARE THIS IS TCDA, ABOUT OF ABOUT 300 KD. THERE WERE SOME (INAUDIBLE) (OVERLAPPING SPEAKERS) RECEPTORS AND THEY ENTER CELLS THROUGH RECEPTOR MEDIATED ENDOCYTOSIS. THEN THE DROP OF PH WILL TRIGGER CONFIRMATIONAL ON THE TOXIN AND THEN THE TOXIN WILL PUNCTURE ACROSS THIS ENDOSOME MEMBRANE AND DELIVER PART OF IT WHICH IS THIS PART, IT'S A TRANSFERASE INTO THE CYTOSOL AND IN THE GLYCOSYLTRANSFERASE GRAB CO-FACTOR UDP GLUCOSE COVALENTLY LINK TO A KEY RESIDUE. SMALL GDPS TO INHIBIT GDPS ACTIVITY AND LEADS TO DISRUPTION OF CYTOSKELETON THEN YOU SEE THE CELLS AND THE CELLS WILL DIE, YOU CAN SEE HERE CELLS TREATED WITH INCREASE CONCEPTATION OF TCCB, YOU CAN SEE THE HELO CELLS, NORMALLY HAVE CELL SHAPE LIKE THIS, AND EVENTUALLY BECOME ROUND LIKE THIS. SO THIS IS A BASIC ASSAY WE USE TO MEASURE HOW SENSITIVE THE CELLS ARE TO THE TOXINS AND YOU CAN SEE ACROSS THE -- MY TALK THAT I HAVE ALWAYS -- WE ALWAYS USE THIS ASSAY TO TO MEASURE SENSITIVITY OF CELLS. THE PARTICULAR STEP WE ARE INTERESTED IN IS RECEPTOR BINDING, HOW THE TOXIN BINDS AND LAND ON CELL SURFACE. THE CLASSIC CONSIDERATION IS C TERMINUS OF THOSE TOXINS HAS REPETITIVE OLIGOPEPTIDE SEQUENCE TO THE CROP REGION. THIS REGION EXISTS IN BOTH TCDA AND TCDB AND HAS A STRUCTURE FEATURES WITH CARBOHYDRATE BINDING PROTEINS AND INDEED BIND WITH CARBON HYDRATES. THIS IS ONE OF THE EXAMPLE, IF YOU TAKE THIS PIECE AND CRYSTAL STRUCTURE IS SOLVED IN COMPLEX WITH TRISACCHARIDE MOTIF. SO THERE ARE INTERACTIONS WITH CELL SURFACE CARBOHYDRATE IN GENERAL. WHAT WE FIND AND OTHERS IN THE FIELD ALSO FIND IS THAT WHEN YOU TRUNCATE THIS CROP REGION, THE REST OF THE TOXINS, THE SHORTER VERSION IS STILL VERY POTENT TOXIN. WE DO ASSAY MEASURES CELL SENSITIVITY TO THE TOXINS YOU CAN SEE THIS IS CELLS, YOU CAN SEE A LITTLE REDUCTION BUT NOT MUCH. SUGGESTING THAT WITHOUT THIS CROP REGION THERE ARE ADDITIONAL REGIONS THAT RECOGNIZE ADDITIONAL RECEPTORS. THAT IS A START OF THE PROJECT OF POST DOC IN THE LAB, DECIDE TO FIND RECEPTORS, UNDERSTANDING HOW THE TOXIN ATTACH TO CELL SURFACE, USING GENOME WIDE CRISPER CAS 9 SCREEN. THE SCREEN ITSELF IS STRAIGHT FORWARD TO SET UP. TAKE CELLS, IN THIS CASE HELO CELLS, PUTTING CAS 9 THEN INFECT CELLS WITH GUARDED RNA LIBRARY USING LENTIVIRUS. THEN WE HAVE POPULATION CELLS LIKE GENES MUTATING. THEN SUBJECT TOXIN SELECTION THAT ACCRUE MOST CELLS, SOME CELLS BECOME RESISTANT BECAUSE SOME GENES CAN MUTATE. THE LEFT THEN COLLECT THOSE CELLS AND DO NEXT GENERATION SEQUENCING, FINE OUT WHICH GENE CAN MUTATE. THIS IS ONE FIND HERE, A FAIRLY CLEAN SCREEN, THE TOP IS A PROTEIN. ALDR. LOW DENSITY LIPOPROTEIN RECEPTOR, A CLASSIC CELL SURFACE CELL RECEPTOR. VERY WELL STUDIED RESTORE. -- RECEPTOR. ON THE TOP ARE BLUE ONES INVOLVED IN HEPARAN SULFATE BIOSYNTHESIS CELLS. THERE YOU SEE THESE GREEN ONES ARE THE ONES THAT INVOLVE LOCALIZED GENE INVOLVING GLYCOSYLATION ROCESSING IN GENERAL INCLUDING OF COURSE THE HEPARAN SULFATE BIOSYNTHESIS AS WELL. IF YOU COMPARE THE SCREEN RESULTS WITH OUR PREVIOUS SCREEN DOWN FOLATES SISTER TOXIN TCDB, THIS IS COMPONENT INVOLVING RECEPTOR, THIS IS COMPONENTS INVOLVED IN CELL BINDING AND RECEPTORS. THERE ARE 30 SEGREGATING. ONLY SEEM TO OVERLAP THOSE THREE GENES THAT INVOLVE THE INTRACELLULAR ACTION OF THE TOXINS WHICH IS A SHARED BETWEEN THOSE TWO TOXINS. THEY USE DIFFERENT RECEPTORS. SO DEMONSTRATING THE SPECIFICITY OF THE SCREEN. WE HAVE A PRETTY GOOD IDEA ABOUT THE BIOSYNTHESIS PROCESS OF HEPARAN SULFATE. LOOK AT ALL THE ENZYMES THAT INVOLVE IN BIOSYNTHESIS OF HEPARAN SULFATE WE LABEL ONE IN RED COLOR AND ONE LABEL IN PURPLE COLOR ARE ONES THAT CAUGHT IN OUR SCREEN. THEY ARE ALL RANKED HIGH. YOU CAN SEE PRETTY COMPREHENSIVE SCREEN WE GOT ALMOST EVERYTHING INVOLVED IN THIS PROCESS. TO VALIDATE THIS, WE LEARN FIRST DECIDED TO DO A LOSS OF FUNCTION APPROACH TO DO KNOCK OUT. SO YOU CAN CRISPER CAS 9 TO KNOCK OUT EXT 2 AND EXTL 3 THOSE ARE TWO GENES INVOLVED SPECIFICALLY FOR SYNTHESIZED HOPE BUT NOT OTHER GAGS. ALSO KNOCK OUT THIS GENE, SLC 35 B 2, REQUIRED FORESAIL PHAGO. IT IT -- FOR, SULFATION. IT'S REQUIRED FOR TRANSPORTING PRECURSORS FOR SULFATION PROCESS IN GAG GENE MEASURE SENSITIVITY OF TOXINS USING THE CELL ROUTING ASSAYS INCREASE TOXIN CONCENTRATION,CAL QUEUE LATING PERCENTAGE OF CELL ROUNDING. IN KNOCK OUT THOSE TWO GENES YOU SEE REDUCTION IN SENSITIVITY NOT COMPLETE RESISTANCE, THOSE CELLS ARE STILL SENSITIVE, THEY ARE OTHER COMPONENTS MEDIATING ENTRY. SENSITIVITY HAS CERTAIN DEGREE OF REDUCTION, ABOUT FOUR TO FIVE FOLD REDUCTION. IF YOU LOOK AT SISTER TOXIN, THIS IS TCDB CONTROL TOXIN NO REDUCTION OF CONTROL TOXIN SENSITIVITY. LOOK AT THE KNOCK OUT SLC 35B 2 YOU CAN SEE A LARGER DEGREE OF REDUCTION HERE. 10 FOLD REDUCTION IN SENSITIVITY. NO CHANGE ON THE SISTER TOXIN, TCDB SENSITIVITY, DEMONSTRATING DEFECTS HAPPEN RECEPTOR BINDING LABEL. THIS OF COURSE ARE ALL THE TRUNCATED TOXINS WITHOUT CROP REGION WITHOUT CARBOHYDRATE BINDING REGION. THIS IS EVERYTHING THERE. WE CAN SEE THREEFOLD REDUCTION IN SENSITIVITY. 35B 2. THIS IS LOSS OF FUNCTION APPROACH AND DID COMPETITION ASSAY USING TOOLS TO DEVELOP COMMUNITY, THIS IS ONE SMALL -- INITIALLY DEVELOPED BY IN JEFF'S LAB MANY YEARS AGO. SURFEN ESSENTIALLY WORKS BY BINDING TO HEPARAN SULFATE AND NEUTRALIZING THE ACTIVE CHARGE. YOU CAN SEE IT INDEED PROTECTED CELLS FROM CELL ROUNDING CONCENTRATION. DEPENDENTS MANNER YOU HAVE HIGH CONCENTRATION YOU CAN SUPPRESS THE CELL ROUNDING EFFECT OF TOXINS AN PREVENT TOXIN BINDING AND ENTERING TO CELLS. HAS NO EFFECT SISTER TOXIN OF TCDB TOXIN. NEXT DATA SCREEN DIFFERENT KIND OF HEPARAN SULFATE AND OTHER GAG INCLUDING HEPARAN AND DIFFERENT HEPARAN SULFATE. AND CHONDROITIN SULFATE, DERM ARK STIRKSN SULFATE AND NON-SULFATED GAGS. AS WELL AS SYNTHETIC HEPARAN SULFATE MIMICS THIS IS A COMMERCIAL PRODUCT FROM THIS COMPANY THAT DEVELOPED AS A HEPARAN SULFATE HOMOLOGUE. THAT REDUCE ITS ANTI-COGNITION ACTIVITY WHILE TRAIN TANNING MANY BENEFICIAL BINDINGS IN TERMS OF SUPPRESSING IMMUNE RESPONSE. SULFATED DEXTRIN MOLECULE DIFFERENT GROUPS BUT HAS VERY DIFFERENT BACKBONE. WHETHER WHAT YOU SEE THE -- ANYTHING THAT SULFATION GROUPS PROVIDE GOOD PROTECTION, NO MATTER WHAT BACKBONE YOU HAVE. IT IS MEDIATED BY SULFATION GROUPS ON HEPARAN SULFATE. REASON WE HAVE HEPARAN SULFATE IS DOMINANT FORM OF SULFATION GAG ON CELL WE ARE WORKING WITH, WHICH IS HELO CELLS. WE CAN DETECT DIRECT BINDING. USING IN VIVO ASSAY, THIS IS A BIOTIN HEPARAN USING THE BILAYER ASSAY WE CAN IMMOBILIZE HEPARAN. CONTROL NON-SULFATEED MOIETIES. WHAT ABOUT OTHERS WHICH IS PROTEIN LDLR, WE CAN GENERATE KNOCK OUT USING CRISPER CAS 9 KNOCK OUT THE GENE. YOU CAN SEE REDUCTION, NOT RESISTANCE BUT CERTAIN DEGREE OF REDUCTION ABOUT REDUCTION. HAS NO EFFECT ON SISTER TOXIN TCDB. WHICH SEE THREEFOLD DIFFERENCE, VERY SIMILAR WHEN WE KNOCK OUT HEPARAN SULFATE SULFATION PROCESS. WHEN WE TEST DIRECTING AND WE COULDN'T DETECT ANY DETECTIONS BETWEEN THESE PROTEIN TOXIN TCDA, THIS ONE OF THE ASSAY WE USE WITH -- WE USE SEVERAL ASSAYS FOR THIS. PURIFY LDLR EXTRA CELLULAR DOMAIN IMMOBILIZE ON PROBE, THIS IS CONTROL PROTEIN GRAPH, THIS IS ONE OF THE CLASSIC LIGANDS FOR LDLR. AS ROBUST BINDING TO IT. WHILE TOXIN HAS ESSENTIALLY NO BINDING TO LDLR. ON THE CELL SURFACE YOU CAN SEE WILD TYPE TOXINS FLUORESCENT LABELED TOXIN THAT BINDS WITH WILD TYPE CELLS. EQUALLY WELL TO THE LDLR KNOCK OUT CELLS. WHAT'S INTEREST INTERESTING IS THE BINDING IS SIGNIFICANTLY REDUCED TO THE HEPARIN SULFATE KNOCK OUT CELLS, KNOCK OUT CELLS INDICATING SULFATION IS ONE OF THE MAJOR FACTOR MEDIATING TOXIN BINDING ATTACHMENT TO CELL SURFACE. HOW DO THEY WORK TOGETHER? WE NEXT GENERATED DOUBLE KNOCK OUT. SO SLC 35B 2 KNOCK OUT WE FIRST KNOCK OUT LDLR ON THIS CELL. SENSITIVITY. WHAT YOU CAN SEE THAT DOUBLE KNOCK OUT GIVE YOU SOME DEGREE OF RESISTANCE TO TOXINS AS SINGLE KNOCK OUT OF THE SULFATION GROUPS. YOU DON'T SEE ADDITIVE EFFECT. WHAT THIS SUGGEST IS THOSE TWO THINGS. PROTEIN LDLR AND HEPARAN SULFATE WORK TOGETHER IN THE SAME PATHWAY. THEY ARE NOT REALLY REDUNDANT RECEPTORS, NOT SEPARATE RECEPTORS. THEY LIKELY FUNCTION TOGETHER IN THIS SAME PATHWAY. KNOCK OUT ONE WITH KNOCK OUT 2 YOU GET SIMILAR DEGREE OF RESISTANCE. THE NEXT STEP OR FOR US IS VALIDATE FIND INFORMATION VIVO IN A RELEVANT TISSUE WHICH IS A COLON, FIRST ASSAY IS INCUBATION , WE LIGATE COLON INJECT TOXIN DIRECTLY AND YOU CAN SEE A TOXIN BINDING TO THE COLON TISSUES IN RED COLOR HERE. YOU INJECT PROTEINS YOU CAN MAKE RECOMBINANT CROP DOMAIN AS COMPAQ TORR, IT REDUCES BINDING OF THE TOXINS SUGGESTING THAT CROP MEDIATING TOXIN ATTACHMENT IN P COLON TISSUE. NEUTRALIZING HEPARAN SULFATE BINDING AND WE CAN REDUCE BINDING THAT WAY. WHEN WE PUT THOSE TWO COMPETITORS TOGETHER, YOU GET BEST COMPETITION OF IT. SO SUGGESTING THAT THE BINDINGS MEDIATING BY BOTH COMPONENTS CARBOHYDRATE CROP INTERACTION AS WELL AS HEPARAN SULFATE GROUP MEDIATED ATTACHMENT. NEXT MEASURE TISSUE DAMAGE. THIS WAS HIGHLIGHTED BECAUSE REASON IS THAT WE CAN EASILY ACHIEVE INCUBATION, THIS IS 30 MINUTE INCUBATION AN PROTECTION OF BINDING BUT VERY DIFFICLT FOR US TO DO TISSUE DAMAGE ASSAY. WHICH REQUIRES TO INCUBATE TOXINS WHATEVER COMPETITORS FOR LONG PERIOD OF TIME, SIX TO EIGHT HOURS. WE FIND HEPARAN SURFAN AND DIFFERENT HEPARAN SULFATED GAGS WILL TRY CAN REDUCE TOXIN BINDING BUT BY THEMSELVES CAN ALSO INDUCE QUITE SEVERE BLEEDING IN THE COLON TISSUE, POSSIBLY BECAUSE OF THEIR INTRINSIC ANTI-COAGULATION ACTIVITY. SO EVENTUALLY WHAT WE FIND OUT IS GM 1111, SYNTHETIC COLON ACTIVITY REDUCE BINDING AND ALSO REDUCE DAMAGE TO THE COLON TISSUE. THIS IS DETAILED ANALYSIS OF THE COLON TISSUE. WHAT WE TRY THAT WORK FOR US. SO IN CONCLUSION WE DID IS GENOME WIDE SCREENING AND IDENTIFY BOTH SULFATE GAG AND LDLR AS CONTRIBUTORS FOR THIS TOXIN BINDING AN ENTRY TO CELLS. WHAT MATTERS IS THE SULFATE GROUP. THIS IS AN INTERESTING SYNTHETIC GAGS THAT CAN REDUCE TOXIN. TOXICITY LEVELS IN VIVO. THIS IS INFORMATION ABOUT THIS SYNTHETIC GAG. FINALLY I WOULD LIKE TO THANK PEOPLE WHO DID MY WORK. IN MY LAB. (INAUDIBLE) POST DOC IS NOW INVESTIGATOR INDEPENDENT INVESTIGATOR, JUST SET UP HIS LAB AT WEST LAKE UNIVERSITY IN CHINA. HE INSISTED -- ASSISTED WITH POST DOC AND (INAUDIBLE) IN THE LAB TOGETHER SENIOR SCIENTIST IN THE LAB WHO HELPED WITH ANIMAL EXPERIMENTS. ALSO THANKS TO MY COLLABORATORS. MORE RECENTLY MOVED TO WASHINGTON U, A BIOLOGIST WORKING ON HEPARAN SULFATE FOR AS CATCHMENT FACTOR FOR VIRUSES. WE COLLABORATED DEVELOPING MANY OF THE KNOCK OUT CELLS TOGETHER. AND (INAUDIBLE) PROVIDES US WITH TOXINS AND GM 1111 THANK YOU FOR YOUR ATTENTION. HAPPY THE TAKE QUESTIONS. >> THANK YOU VERY MUCH. APPRECIATE YOUR PRESENTATION. NOW WE ARE OPEN FOR QUESTIONS. WE CAN COVER BOTH MEN'S AND -- WE HAVE TWO MINUTES LEFT SO WE CAN PERHAPS COVER ONE QUESTION FROM -- ONE QUESTION. SORRY. WAS SOMEONE SAYING SOMETHING? >> JIAN WHAT KIND OF SCALE OF BAGS WILL MEAN IN VIVO EXPERIMENTS WILL ALLOW FOR TREATMENT TO NOT BE COST PROHIBITIVE? >> SO RIGHT NOW IT DEPENDS ON THE SUGARS. FOR EXAMPLE FOR US TO DO THIS BOTH CON JOINING SULFATE E 19 MER AND HEPARAN SULFATE, TO DO ANIMAL STUDIES WE NEED TO GET ABOUT PROBABLY 10 TO 100 MILLIGRAMS AMOUNT OF MATERIALS SHOULD BE ENOUGH. AND THEN ACTUALLY WE ARE DOING THIS SYNTHESIS IN 400-MILLIGRAM TO ONE GRAM SCALE SYNTHESIS. FOR SOME OF THESE COMPOUNDS WHAT I REFER TO LIKE A 12 MER OF HEPARAN OLIGOSACCHARIDE WE USE AS SYNTHETIC HEPARAN WE CAN SYNTHESIZE TO UP TO 100 GRAMS, WE'RE ABOUT TO START DOING GMP PRODUCTION FOR THE HUMAN CLINICAL TRIAL. SO IN MY VIEW ENZYMATIC SYNTHESIS OF HEPARAN OLIGOSACCHARIDE IS NO LONGER DIFFICULT. >> THANK YOU SO MUCH. SO I THINK WE CAN GO ON TO THE NEXT PRESENTATION FROM HIROSHI NAKATO. LESSONS FROM DROSOPHILA. >> THANK YOU VERY MUCH FOR THE INVITATION. MY NAME IS HIROSHI NAKATO, WE STUDY PROTEOGLYCANS USING DROSOPHILA MODEL. IF I CAN CONTROL THE SLIDE. CAN YOU MOVE FORWARD? >> SORRY, THERE'S -- THE SLIDES WILL COME UP IN ONE SECOND. >> I'M SHOWING YOU HERE DROSOPHILA A LEVEL ORGAN DEVELOPING INTO A -- DEVELOPMENT THREE MAJOR MORPHOGENERAL MOLECULES MAY A KEY ROLE, HEDGEHOG IS EXPRESSED IN THE POSTERIOR COMPARTMENT AND INDUCES EXPRESSION OF TARGET GENES IN THE ANTERIOR CELLS. ONE OF HEDGEHOG TARGET GENES IS ANOTHER MORPHOGEN CALLED DPP WHICH IS A DROSOPHILA HOMOLOGUE OF MORPHOGENETIC PROTEINS. ANOTHER MORPHOGEN IS EXPRESS AT THE DORSAL VENTRAL BORDER OF THE WINT DISC AN REGULATES PATTERNING ALONG THE DDP AXIS. ALL THESE MORPHOGEN MOLECULES ARE HEPARAN SULFATE DEPENDENT FACTORS AND THEY REQUIRE HSPG ON THE CELL SURFACE AS CO-REACCEPTER TO. CAN YOU MOVE SLIDE. NEXT SLIDE PLEASE. SO AS I SAID, BPP IS EXPRESS IN THE CENTRAL REGION OF THE WINT DISC AND ACTS AS A MORPHOGEN. WE HAVE PREVIOUSLY SHOWN THAT ONE OF TWO MOLECULES IN DROSOPHILA ACT AS CORECEPTOR FOR DPP AND REGULATED SCREENING FORMATION. -- REGULATED GRADIANT FORMATION SO IN ABSENCE OF DALLY DPP FAILS TO FORM NORMAL GRADIANT. THE SECOND MOLECULE CALLED THE DALLY LIKE DOESN'T SHOW MUCH ACTIVITY FOR DPP, INSTEAD DALLY IS A GREAT CORECEPTOR FOR HEDGEHOG BUT DALLY IS NOT. SO THERE'S SOME FUNCTIONAL SPECIFICITY IN THESE GLYCOCANS BUT WE DON'T KNOW THE UNDERLYING MECHANISMS YET. I SHOW HERE THE MOLECULAR MECHANISM FOR CORECEPTOR ACTIVITY, IT'S OUR MODEL FOR THE MECHANISM. RECEPTOR MOLECULE IS CRITICAL TO MEDIATE SIGNAL. BUT AT THE SAME TIME IT HAS NEGATIVE IMPACT ON SIGNALING BECAUSE THE RECEPTOR ELIMINATES THE LIGAND, RECEPTOR MEDIATED ENDOCYTOSIS FOLLOWED BY DEGRADATION. CORECEPTOR CAN CHANGE THE BALANCE. MOVE FORWARD. BALANCE OF THESE PLUS AN MINUS ACTIVITIES ON SIGNALING. FOR EXAMPLE, DALLY ENHANCES DPP SIGNALING, BY SLOWING DOWN THE PROCESS OF THIS ENDOCYTOSIS. STABILIZING LIGAND RECEPTOR COMPLEX ON THE CELL SURFACE. SO WE STILL DON'T KNOW HOW HSPG REGULATE ENDOCYTOSIS. THEY ARE LIKELY TO COOPERATE WITH OTHER FACTORS TO EXERT CORECEPTOR ACTIVITY. SO THERE MUST BE STILL A NUMBER OF MISSING PLAYERS IN THIS COMPLEX AND RECENT STUDY HAS SUGGESTED THAT SOME OF THESE MISSING PLAYERS MAYBE DIFFERENT TYPE OF PROTEOGLYCANS. NEXT PLEASE. IN ADDITION TO HS DROSOPHILA PRODUCES CHONDROITIN SULFATE CS. ALTHOUGH WELL ESTABLISHED MAJOR STRUCTURAL COMPONENTS OF ECM. SOME OF THEM ARE NOT TO FUNCTION IN CELL SIGNALING. GIVEN THE STRUCTURE SIMILAR SIMILARITIES BETWEEN HS AND CS, IT'S NOT SURPRISING CS HAS SUPPORTIVE COMPLIMENTARY FUNCTIONS THROUGH HS. UNFORTUNATELY HOWEVER, DROSOPHILA CSPG UNDERSTUDIED AND THEIR FUNCTIONS ARE NOT WELL CHARACTERIZED YET. THERE ARE ONLY TWO CSPGs HAVING AUGMENTED IN DROSOPHILA, WHILE 24 IDENTIFIED IN C ELEGANS. MOST C ELEGANS CSPGs DO NOT SHOW SEQUENCE HOMOLOGY TO BELIEVEIOUSLY IDENTIFIED CSPG EXCEPT COLLAGENATED FIBRIL LYNN AND KAPLAN. SO UNLIKE HSPG CSPG PROTEINS ARE NOT WELL CONSERVED BETWEEN SPECIES SO FINDING A NEW CSPG MOLECULE CANNOT RELY ON SEQUENCE HOMOLOGY TO KNOWN MOLECULES. NEXT PLEASE. SO WE COLLABORATE WITH DR. FREDERICK BORNE WHO ESTABLISHED A NOVEL GLYCO PROTEOMICS APPROACH TO IDENTIFY NEW PROTEOGLYCANS. AND FROM TISSUE LYSATES, PROTOGLYCANS CAN BE ENRICHED BY SAMPLES ARE TREAT WITH TRYPSIN HEPARINASE AND PASSED OVER COME AGAIN TO CS ATTACHED PEP TIDES. CS PEP TIDES ARE NOW TREATED WITH CHONDROITINNIST TO WHICH GENERATES RESIDUAL HEXA SACCHARIDE STRUCTURE ATTACHED TO THE PEPTIDE. THIS FRACTION IS ESTABLISHED BY LCSMS. AND CS ATTACHED PEP TIDES CAN BE IDENTIFIED. NEXT PLEASE. NOW THIS ANALYSIS IDENTIFIED A PROTEIN CALLED WINDPIPE AS A NOVEL DROSOPHILA CSVG. WHEN THE PIPE IS A SINGLE PASS TRANSMEMBRANE PROTEIN, WITH RICHARD REPEAT MOTIFS AND CS GENES. INTERESTINGLY, WHEN THE PIPE WAS PREVIOUSLY SHOWN TO NEGATIVELY REGULATE JACK SAT SIGNALING. WE WANT TO KNOW MORE ABOUT THIS MOLECULE AND WE NOTICED THAT OVEREGG EXPRESSION OF WINDPIPE IN THE DEVELOPING WINT USING GAL 4 U.S. SYSTEM, REDUCED WING SIZE ESPECIALLY THE AREA BETWEEN LONGITUDINAL WIND BANKS L 3 AND L 4. THIS PHENOTYPE IS CHARACTERISTIC OF REDUCED HEDGEHOG ACTIVITY. HEDGEHOG AS I SAID IS EXPRESSED IN THE POSTERIOR CELLS, FORMS A GRADIANT IN ANTERIOR CELLS AND INDUCES EXPRESSION OF TARGET GENES SUCH AS PATCHED DPP AND CURVETUS INTERACTUS. NEXT PLEASE. WHEN WE OVEREXPRESS EXPRESSED WINDPIPE SPECIFICALLY IN DORSAL COMPARTMENT. MARKED BY GFP EXPRESSION, IT REDUCED EXPRESSION DOMAINS FOR ALL HEDGEHOG TARGET GENES. ONLY IN THE DORSAL CELLS. THE QUANTIFICATION CONFER WITH IT SUGGESTING THAT WINDPIPE INHIBITS HEDGEHOG SIGNATURE MALLING. NEXT PLEASE. SO THIS IS A NEW CSPG, WE WANTED TO KNOW CS CHAINS IMPORTANT FOR WINDPIPE ACTIVITY. SO WE GENERATED A MUTANT FORM OF WINDPIPE, WINDPIPE DELTA GAG IN WHICH ALL THREE CS ATTACHMENTS SERINE RESIDUES WERE SUBSTITUTED TO ALANINE. SO THAT THE CORE PROTEIN CANNOT BE MODIFIED WITH CS. WE FOUND OVEREXPRESSION OF THIS MUTANT CONSTRUCT DID NOT AFFECT WING SIZE OR MORPHOLOGY. THE QUANTIFICATION IS SHOWN BELOW. WILD TYPE WIND PIPE REDUCE WING SIDE AND L 3 L 4 DOMAIN. WINDPIPE DELTA GAG FAILED TO DO SO. INDICATING THAT CS CHAINS ESSENTIALLY REQUIRE FOR WINDPIPE ACTIVITY. SO THEN GENERATED NULL MUTANT FOR WINDPIPE. BY CRISPER CAS 9 MUTAGENESIS. WE FOUND THE WINDPIPE NULL MUTANTS ARE VIABLE. BUT ADULT SHOWED VERY SPECIFIC PHENOTYPE IN SENSORY ORGANS. ON THE LEFT IMAGE, SHOW YOU TWO PAIRS OF MECHANIC KNOW SENSORY BRIS LES, SPECIFIC REGION OF THE ADULT AND WINDPIPE HOMOZYGOTE SHOW DUPLICATION OR SPECIFIC SET OF BRISTLES. THIS IS EXACTLY A PHENOTYPE KNOWN TO BE INDUCED BY HEDGEHOG OVEREXPRESSION. NEXT PLEASE. IN FACT, DELETING ONE COPY OF HEDGEHOG IN WINDPIPE HOMOZYGOTES ALMOST COMPLETELY SUPPRESS THIS MUTANT PHENOTYPE. THAT SUGGESTED THAT MUTANT SHOW THIS BRISTLE PHENOTYPE THROUGH OVERACTIVE HEDGEHOG SIGNALING. ALSO PERFORM MOSAIC ANALYSIS USING WINDPIPE MUTANT AND ALL THESE PANELS I'M SHOWING HERE IN THE SLIDE GFP NEGATIVE CELLS ARE WINDPIPE MUTANT. WE FOUND PATCHED EXPRESSION SHOWED MAGENTA IS HIGHER IN WINDPIPE NULL MUTANT CLONES. SO ALL OF OUR RESULTS CONSISTENTLY INDICATED THAT WINDPIPE IS A NOVEL NEGATIVE CORECEPTOR FOR HEDGEHOG SIGNALING. SO WE STILL DON'T KNOW HOW WINDPIPE DOWN REGULATE HEDGEHOG SIGNALING. BUT RECENT STUDIES SUGGEST THAT IT MAY AFFECT THE LEVEL OF SMOOTHENED OR SMO PROTEIN. SMO IS A KEY COMPONENT OF HEDGEHOG SIGNATURE MALL -- SIGNALING AND ITS TRANSLOCATION AN AICALLYMATION MANY CELL SURFACE TRIGGER THIS PATHWAY. THE TOP PANELS ARE A CONTROL WIND DISC. IF YOU COMPARE SMO STAINING AND THE DORSAL AND VENTRAL COMPARTMENTS, THE SMO LEVELS ARE COMPARABLE. IN ANOTHER WING THERE'S SHOWN ON THE BOTTOM WINDPIPE WAS KNOCKED DOWN BY RNAi ONLY IN THE DORSAL CELLS. WE FOUND WINDPIPE KNOCK DOWN INCREASES SMO LEVELS. SO IT'S POSSIBLE THAT WINDPIPE DISRUPTS SMO TRANSLOCATION OR DESTABILIZES SMO ON THE CELL SURFACE. BUT FURTHER STUDIES NEEDED TO DEFINE THE EXACT MECHANISM. NEXT PLEASE. IT'S WORTH NOTING THAT HEDGEHOG AND IMPAIRED SIGNALING, THE TWO PATHWAYS NEGATIVELY REGULATED BY WINDPIPE, ACTUALLY POSITIVELY REGULATED BY HSP GENES SUCH AS DALLY AND DALLY LIKE. THEIR MORPHOGEN SIGNALING AND ONCOGENIC PATHWAYS WHEN DISREGULATED AND DOSAGE CONTROL IS CRITICAL. A NUMBER OF STUDIES HAVE SHOWN, HS AND CS SHOW POSING EFFECTS ON NEW EXTENSION AND MULTIPLE VERTEBRATE MORAL. SO WE THINK THIS DUAL CSHSCS PROTEOGLYCAN CO-RECEPTOR SYSTEM MAY BE A JELL MECHANISM. TO FINE TUNE SIGNALING OUTPUT. THIS IS A SUMMARY SLIDE. I HAVE SHOWN YOU THE MODEL FOR HSPG CO-RECEPTOR ACTIVITY TO REGULATE THE STABILITY OF THE LIGAND RECEPTOR COMPLEX ON THE CELL SURFACE. BY CONTROLLING THE RATE OF RECEPTOR MEDIATED ENDOCYTOSIS. THIS ALLOWS PRECISE AND QUANTITATIVE CONTROL OF SIGNALING OUTPUT ESSENTIAL FOR MORPHOGEN SIGNALING AND PREVENT CANCER FORMATION. THERE IS FURTHER FINE TUNING OF SIGNALING DOSAGE BY CSPGs AND WE THINK THIS WILL PROTEOGLYCAN CORECEPTOR SYSTEM IS GENERAL MECHANISM. WITH THAT I WOULD LIKE TO ACKNOWLEDGE LAB MEMBERS, COLLABORATORS AND FUNDING AGENCY ACROSS NIGMS. HOTCH FOR YOUR ATTENTION. >> THANK YOU VERY MUCH FOR YOUR TALK. WE HAVE APPROXIMATELY THREE MINUTES LEFT AND WE CAN ENTERTAIN QUESTIONS FOR ALL THREE SPEAKERS AT THIS POINT. IF ANYBODY WOULD LIKE TO POSE SOME QUESTIONS. >> ANOTHER QUESTION THAT WAS RAISED FROM ONE OF THE EARLIER TALKS, WAS THAT (INAUDIBLE) INVOLVED IN INFLAMMATORY RESPONSE IN ADDITION TO NEUTROPHILS. >> WE DIDN'T TEST THAT, WE MAINLY FOCUS ON THE NEUTROPHIL IN OUR STUDY. OF THE -- IN THE STUDIES. >> CURIOUS IF I THINK THERE'S ONE GRAPH THAT SHOW TNF ALPHA WAS DECREASED, ALSO LOCK FOR IL 6 LEVELS BY ANY CHANCE? >> I DON'T THINK WE MEASURE IL 6 WE MEASURE TNF ALPHA. >> FINE. THANK YOU. QUESTION FOR NAKATO. ANY TO MODULATE DRUG IN MA MALARIAN SYSTEMS? >> I DON'T THINK THERE'S ANY SPECIFIC CORE PROTEIN KNOWN TO REGULATE SONIC HEDGEHOG BUT IN GENERAL, CS CAN BIND AND MODULATE HEDGEHOG ACTIVITIES. IT'S BEEN PROVEN IN STRUCTURAL STUDY. IT'S NOT VERY CLEAR THAT -- THE DIFFERENCE BETWEEN HS HEDGEHOG INTERACTION AND CS HEDGEHOG INTERACTIONS ARE DIFFERENT. THERE ARE MANY DISCUSSIONS DIFFERENCE BETWEEN LOW AN HIGH AFFINITY BINDING SITES AND HS AN CS OR FORM. HEDGEHOG IN TERMS OF MONOMER VERSUS OLIGOMER, MIGHT BE DIFFERENT. SO THE DIFFERENCE BETWEEN CS AND HS FUNCTION IN HEDGEHOG SIGNALING IS STILL ON THE WAY. -- STILL UNDERWAY. >> GREAT. THANK YOU. ONE MORE QUESTION FROM DR. WILSON. IS THERE A RODENT HOMOLOGUE WINDS PIPE ANY CHANCE? >> NOT REALLY, LIEU SEEN RICH REPIECE, IF YOU DO A HOMOLOGY STORAGE, IT SHOWS VERY, VERY WEAK HOMOLOGY TO MANY OF LUCINE RICH CHONDROITIN SULFATE PROTEOGLYCANS. THAT TOP OF THE LIST SHOWS MOLECULE, I CAN'T REMEMBER IN THE HUMAN HOMOLOGUE WHICH WHEN IT'S LOST SHOWS NIGHT BLINDNESS. I CAN'T REMEMBER THE NAME OF THE MOLECULE RIGHT NOW. BUT I WOULDN'T -- NOT SURE WE CAN CALL IT THE REAL HOMOLOGUE. BECAUSE OF THE LUCINE RICH REPEAT SHOWS WEAK SIMILARITIES. >> THANK YOU SO MUCH. SO I THINK WE ARE RIGHT ON TIME TO MOVE ON TO THE LAST TALK OF THE SESSION. THIS IS BY HANNES BUELOW. HEMATOPOIETIC CELL LINEAGE. >> GOOD MORNING, GOOD EVENING, GOOD AFTERNOON, WHEREVER YOU ARE. FIRST I WOULD LIKE TO THANK THE COMMON FUNDS AND PAMELA MORINO AS WELL AS NIH STAFF FOR ORGANIZING THE MEETING AND INVITE MEG AND GIVING ME THE OPPORTUNITY TO SPEAK. SECOND, I WOULD LIKE TO POINT OUT THAT I'M BUT THE MESSENGER HERE OF A LARGE COLLABORATIVE EFFORT THAT'S COMPRISED OF THE LABS OF -- COLLEGE OF MEDICINE AS WELL AS PEOPLE AT OTHER INSTITUTIONS. SO HEPARAN SULFATE HAS ALREADY BEEN INTRODUCED BY MOST SPEAKERS AHEAD OF ME SO I DON'T WANT TO BELABOR TOO MUCH STRUCTURAL CHARACTERISTICS. THE ONLY THING I WANT TO POINT OUT IN THIS LITTLE SCHEMATIC IS THAT WHAT REALLY IS IMPORTANT TO REALIZE IS THAT THESE CHAINS THESE GLYCAN CHAINS ARE NOT UNIFORMLY MODIFIED BUT THAT THEY SHOW MODIFICATION PATTERNS THAT COMPRISE DOMAINS OR MOTIFS OR EPITOPES, WHICHEVER WAY YOU WANT TO CALL IT AND THAT THOSE MOTIFS ARE THE BUSINESS END OF THE MOLECULE AND MANY CASES, AND ARE STRUCTURE MEDIATE PROTEIN PROTEIN INTERACTIONS AND THERE BY THE BIOLOGICAL FUNCTIONS OF THE GLYCANS. I WANT TO SUMMARIZE MANY THINGS SAID BEFORE IN ONE FORM OR ANOTHER FOR EXAMPLE, THE GLYCOSAMINOGLYCANS SUCH AS HEPARAN SULFATE ARE PRESENT ON ALL CELLS AND METAZOANS, NO MULTI-CELLULAR ORGANISM MAKES THROUGH GASTRULATION WITHOUT UNDERGOING MOLECULAR SIGNIFICANCE. THEY HAVE A MOLECULAR DIVERSITY SAID MANY TIMES BEFORE AND THEY ARE ALSO EXPRESSED WITH EQUALLY EXTRAORDINARY CELLULAR SPECIFICITY SUCH THAT MANY CELLS PROBABLY MOST CELLS HAVE LIGHTLY DIFFERENT MOLECULAR SIGNATURES OF HEPARAN SULFATE AND THEY ARE IMPORTANT FOR CELL CELL COMMUNICATIONS. BUT THE INALSIS AND CHARACTERIZATION OF THESE HEPARAN SULFATE REMAINS CHALLENGING AND BY AND LARGE IS ONLY PERFORMED BY SPECIALIZED LABS AND WE HAVE SEEN GREAT EXAMPLES, GREAT EXAMPLE EARLIER BY JOHN AMSTER WHO SHOWED US HOW WITH ADVANCE TECHNOLOGIES HE CAN DECONVOLUTE AND RECOGNIZE THESE STRUCTURE. BUT THEY MAY NOT BE READILY AVAILABLE TO COMMON LAB. DESPITE ALL THESE TECHNICAL ADVANCES, THE RESOLUTION OFTEN REMAINS SOMEWHAT LIMITED. THIS IS IN PART DUE TO THE NATURAL HETEROGENEITY OF HEPARAN SULFATE FROM NATURAL SOURCES BECAUSE MOLECULES ARE NOT GENETICALLY ENCODED THEREFORE HAVE CERTAIN BIOLOGICAL VARIABILITY. SO THE QUESTION WE ARE ASKING OURSELVES CAN WE LEVERAGE DIVERSITY OF HEPARAN SULFATE ON CELLS WITHOUT MUCH IF ANY STRUCTURAL UNDERSTANDING AND TO SOME EXTENT THIS WAS MOTIVATED BY THE FACT THAT I EVEN AFTER 15 YEARS AFTER STUMBLING TO THIS FIELD I REMAIN INTIMIDATED BY THE STRUCTURAL COMPLEXITY OF THESE MOLECULES. HERE WE WERE REMINDED OF REAGENTS THAT WERE ORIGINALLY ISOLATED BY IN THE ME NETHERLANDS IN THE 1990s, 36 SINGLE CHAIR VARIABLE FRAGMENT ANTIBODIES OR RECOGNIZE SPECIFIC HEPARAN SULFATE EPITOPES. WHAT I WOULD LIKE TO STRESS, THE SING CHAIN VARIABLE FRAGMENT ANTIBODIES WHEREVER TESTED, APPEAR TO RECOGNIZE DIFFERENT HEPARAN SULFATE EPITOPES OR DOMAINS THAT IS THEY RECOGNIZE COMBINATIONS OF MODIFICATIONS AND NOT INDIVIDUAL MODIFICATIONS. ALSO THAT NATURE OF THE EPITOPES HS DOMAINS THAT ARE RECOGNIZED BY THESE MOLECULES, REMAIN POORLY DEFINED. OUR IDEA WAS TO USE REAGENTS TO DEFINE CELLS PURELY BY ANALYZING WHICH OF THESE ANTIBODIES ARE BOUND BY PARTICULAR ANTIBODY. SO IN THIS THREE EXAMPLEIOUS SEE THREE CELLS THAT BIND DIFFERENT ANTIBODIES AND WE WILL DEFINE THIS AS THE GLYCO TYPE. OF A GIVEN CELL. SO WE RECREATED ANTIBODIES ENGINEERING THEM IN DIFFERENT WAYS FOR EXAMPLE WITH IMMUNOTAGS OR FC DOMAINS OR MAKE THEM COMPETENT FOR LABELING WHICH ALLOWS THE ADDITION OF FLUROPHORES OF ALL DIFFERENT KINDS. WE DECIDED TO USE -- I SHOW THAT IN A MINUTE, WITH EF NOW THIS TABLE JEFF CDNAs FOR ALL AGENTSES, INVEST VERSIONS FOR MOST AND SORTASE COMPETENT VERSUS MANY OF THOSE, SO WE DECIDED TO USE THEM IN FAX ANALYSES AND WE HAVE USED MOUSE CELL LINE CELLS MOUSE, ERYTHROLEUKEMIC CELLS. THESE ANTIBODIES RECOGNIZE ON MOST ANTIBODIES RECOGNIZE THESE CELLS AND THAT IF YOU DIGEST WITH HEPARINASE, YOU CAN SHIFT THE PEAK TO THE LEFT SHOWING THESE ANTIBODIES DO INDEED RECOGNIZE HEPARAN SULFATE. SO THEN WE APPLY THE GLYCOTYPE IDEA TO FOUR OR FIVE DIFFERENT CELL LINES HUMAN AN MOUSE LEUKEMIC CELL LINES CHRONIC AND ACUTE LEUKEMIC CELL LINES. AND WHAT YOU CAN SEE, THAT'S PROBABLY BEST ILLUSTRATED ON THE RIGHT, IN THIS HEAT MAP, INDEED EACH OF THESE CELLS IS CHARACTERIZED BY UNIQUE GLYCOTYPE. I SHOULD ADD THESE ONLY -- THESE EXPERIMENT ONLY USES 8 OF THESE 36 AVAILABLE ANTIBODIES. A CONTROL CELL LINE THESE MOUSE 32D MOUSE MYLOBLAST CELLS WERE KNOWN NOT TO EXPRESS HEPARAN SULFATE. NEXT WE APPLIED TO THE HEMATOPOI THE I CAN LINEAGE AND YOU HAVE SEEN IMAGES LIKE THIS IN ONE FORM OR ANOTHER. AND WHAT I WOULD LIKE TO POINT OUT HERE IS THAT THERE ARE TWO MAJOR ARMS, THAT DERIVE FROM THE STEM CELL POOL AND THAT'S CHARACTERIZED BY THE DESEN DENSE OF COMMON PROGENITOR CELL AND MYELOID PROGENITOR CELL AND MYLOPROGENITOR CELL SHOULD CONCERN US TODAY IN MY TALK. THAT IS SPLIT UP FURTHER IN THE GRANULOCYTE MONOCYTE PROGENITOR AND THE MEGA CAROCYTE ERYTHROPROGENITOR AND THIS IS WHAT I WILL BE MOSTLY TALKING ABOUT TODAY. HISTORICALLY THESE BLOOD CELL LINEAGE IS CHARACTERIZED BY BY BY THE CLUSTER OF DIFFERENTIATION OF CD MARKER SYSTEM SUCCESSFULLY USE OVER THE PAST DECADES TO CHARACTERIZE VARIOUS CELL TYPES. BUT IT SHOULD ALSO BE NOTED THAT RECENT SINGLE CELL TRANSCRIPTOMIC APPROACHES HAVE INDICATED THAT THE HEMATOPOIETIC LINEAGE CONTAIN MANY MORE CELL TYPES THAT CAN CURRENTLY BE RECOGNIZED BY THIS CD SYSTEM. SO WE STARTED TO ANALYZE THESE HEMATOPOIETIC CELLS AND WE DATED THEM THROUGH ESTABLISHED SCHEMES INTO THESE THREE TYPES OF PROGENITOR CELLS IN THE COMMON MYELOID PROGENITOR BRANCH. YOU CAN SO ETHEL HERE AS WELL, YOU SEE THE TWO BRANCHES HERE MEPs AND GEP MEGA CARO SITE AND GRANULOSITE BRANCH. WHAT YOU CAN SEE ONLY WITH THESE FOUR DIFFERENT ANTIBODIES MBB 49 REMAINS CONTROL IS THAT THEY SHOW SLIGHTLY DIFFERENT PATTERNS OF BINDING BUT THAT THE SIGNIFICANT AMOUNT OF BINDING APPEARS TO BE IN THE MEP. YOU CAN SEE THIS IN THESE THREE ANTIBODIES. AND SO WE DECIDED TO ANALYZE THOSE IN MORE DETAIL, MEP BRANCH AND THEIR ESTABLISHED GATING SCHEMES FOR THER REVENUEROID AND MEGA CARO SITE BRANCH. YOU CAN SEE MEGA KARYOCYTE BRANCH IS DEVOID OF BINDING FOR THE AT LEAST THESE FOUR ANTIBODIES. WHEREASER ITROID BRANCH SHOWS BINDING FOR THREE OF FOUR ANTIBODIES DECLINING AS THESE,ER ITROID CELLS BEGIN TO MATURE. FROM R 1 TO R 5, THIS IS ERYTHRO, THIS IS THE DIFFERENTIATION PATH OF THESE CELLS. WHEN WE TESTED THESE CELLS THEN IN FUNCTIONAL ASSAYS AND BASICALLY SORTED NOT THE PROGENITOR CELLS BUT ACTUALLY HEMATOPOIETIC STEM CELLS, SO MUCH MORE HETEROGENOUS POOL FOCUSING ON JUST ONE ANTIBODY HS 3A 8 ANTIBODY TO HIGH AND LOW EXPRESSING CELLS, WE FOUND THE LOW EXPRESSING CELLS CREATE RESULTED IN MANY MORE COLONIES AND COLONY FORMING ASSAYS THAN THE HIGH EXPRESSING CELLS. WHEN WE LOOK AT -- IN MORE DETAIL, AT THE TYPE OF COLONIES THAT WERE CREATED, WE FOUND THAT HIGH EXPRESSING CELLS HAD PROPENSITY TO CREATE COLONIES OF THE ERYTHROLINEAGE, THE LOW EXPRESSING CELLS APPEARED TO SHOW PROPENSITY TO MEGA KARYOCYTE TO THE MEGA KARYOCYTE TYPE OF CELLS. THIS IS MORE CLEARLY QUANTIFIED HERE WHERE YOU CAN SEE THAT THE LOW CELLS IF MEGA CAROCYTE COLONIES WHEREAS THE HIGH EXPRESSING CELLS ESSENTIALLY GIVE NONE. SO THIS ANTIBODY REALLY DIFFERENTIATES BETWEEN MEGA KARYOCYTE AND ERYTHROPROGENITOR, SO CREATES A WAY OF DIFFERENTIATING BETWEEN THESE CELLS THAT WASN'T POSSIBLE BEFORE. THIS IS JUST ONE EXAMPLE. WE THEN LOOKED SINGLE CELL TRANSCRIPTOMIC EXPERIMENTS AND THIS VERY NICELY REFLECTS WHAT I HAVE JUST TOLD YOU IN THESE FUNCTIONAL ASSAYS, TRAININGS SUBSCRIPTIONALLY WHERE YOU CAN SEE THIS THIS BRANCH THAT COMPRISES THE ERYTHROPROGENITORS THE EARLY MORE COMMITTED ONES, BOTH, IS ESSENTIALLY COMPLETELY MISSING IN THOSE CELLS THAT EXPRESS H 388 ANTIBODY VERY LOW. HS 3A 8 VERY LOW QUANTIFIED HERE ON THE RIGHT SIDE. SO OF COURSE, CONTRARY TO PRIOR ASSERTIONS, WE ARE INTERESTED IN THE STRUCTURES THAT ARE RECOGNIZED BY THESE ANTIBODIES. SO THE WAY WE HAVE BEEN TRYING TO APPROACH THIS, IS SLIGHTLY DIFFERENT THAN BY PURELY BIOANALYTICAL APPROACH. TO REMIND YOU DISACCHARIDE HERE HAS FIVE POSITIONS THAT CAN BE MODIFIED. WE DEVELOPED A CODING SYSTEM I SHOULD SAY THAT'S REALLY EXTENSION OF THE CODING SYSTEM CODING SYSTEM WITH FOUR LETTERS ORIGINALLY IDENTIFIED OR PROPOSED BY JEFF ESCO AND COLLEAGUES, THAT IN THESE FIVE DIGITS, DESCRIBES A DISACCHARIDE. WHAT THAT ALLOWS YOU TO DO IS TO WRITE A LINEAR SEQUENCE OF ANY PIECE OF HEPARAN SULFATE CHAIN. WHAT THAT INTERNAL ALLOWS YOU TO DO POTENTIALLY IS TO ALIGN OLIGOSEQUENCES, I'LL SHOW YOU IN A MINUTE WHY I THINK THIS COULD BE QUITE USEFUL. WHAT I DID FOR THE HS 3A 8 ANTIBODY, I DON'T HAVE TIME TO TELL YOU MORE PRECISELY HOW EXACTLY I DID IT, THERE WAS DATA IN 2002 USING INHIBITOR ELISA ASSAYS THAT PROVIDE IC 50 DATA FOR FIVE DIFFERENT OLIGOS. THAT INHIBITED BINDING OF THE HS 3A 8 ANTIBODY TO EPITOPE. WHAT I DID HERE IS ALIGN THOSE SEQUENCES AND WHAT YOU CAN SEE IS THAT THERE'S CERTAIN POSITIONS THAT ARE CONSERVED. INDICATE YOU KNOW THIS FROM AMINO ACID ALIGNMENTS, OTHERS SEEM TO BE MORE VARIABLE. AND TAKING INTO ACCOUNT THE IC 50 VALUES THAT WERE IN THIS PAPER I WAS ABLE TO COMPUTE A POSITION WEIGHT MATRIX THAT DESCRIBES THE HS 3 EPITOPE AT LEAST TO SOME EXTENT. OF COURSE THIS IS REMINISCENT OF WHAT Y'ALL KNOW IN TERMS OF TRANSCRIPTION FACTOR BINDING SITES IN DNA, IT LOOKS QUITE SIMILAR. WHAT IS REALLY SATISFYING, AND I DON'T HAVE TIME TO SHOW YOU ALL THE DETAILS HERE WAS THE FINDING THAT THE SIX SULFATION SEEMED REALLY IMPORTANT AND THE S -- THE NDST, SULFATION OF GLUCOSAMINE RESIDUE SEEM REALLY IMPORTANT. AND IF WE LOOK AT OUR SINGLE CELL TRANSCRIPTION TOE MIC DATA WE FIND -- TRANSCRIPT TOE MIC DATA. WE FIND ST 2 DEACETYLASE AS TRANSFERASE AS WELL AS TRANSFERASE, HS 3SD 1 IS HIGHLY REGULATED IN TERMINALLY DIFFERENTIATING ERYTHROLINEAGE, SHOWING THE TRANSCRIPTOMIC DATA NICELY CORRELATES WITH THIS EPITOPE. THE ADVANTAGE OF USING THE ANTIBODIES OF ISOLATING OR DESCRIBING THE CELLS IS THEY ARE ALIVE AND YOU CAN USE THEM AFTERWARDS AND DO EXPERIMENTS OR WHATEVER YOU WANT TO DO WHEREAS THE SINGLE CELL TRANSCRIPTOMIC APPROACH IS OBVIOUSLY DESTRUCTIVE APPROACH. SO WE ARE NOT TRYING TO DO THIS SYSTEMATICALLY USING APPROACH SYSTEMATICALLY AN THIS ACTUALLY PROBABLY JUST MORE OF A TEASER, COLLABORATING WITH DR. BOONS AND SOME EXTENT GLYCAN THERAPEUTICS AND THE HOPE IS THAT WE CAN USE THE ANTIBODIES TO CREATE THESE BINDING PROFILES AND THEN DEVELOP SOFTWARE IN COLLABORATION WITH -- THAT WILL DIRECTLY ANALYZE BINDING DATA AND CREATE POSITION WEIGHT MATRICES TO DESCRIBE -- MATRIXES TO DESCRIBE BINDING SITES OF ANTIBODIES THAT ARE APPLICABLE TO ANY PROTEIN YOU WOULD BE INTERESTED IN. AND THE INTERESTING OR THE ATTRACTIVE THING ABOUT THIS APPROACH I THINK IS THAT IT DOESN'T MATTER HOW MANY OLIGOS YOU HAVE, YOU WILL GET THE RESULT, IT WILL ONLY BE MORE PRECISE THE MORE ANTIBODIES THE MORE OLIGOS YOU WILL USE. IN YOUR ANALYSES. WITH THAT, I WOULD LIKE TO SUMMARIZE THAT YOU CAN LEVERAGE MOLECULAR DIVERSITY OF HEPARAN SULFATE OR GAGS FOR THAT MATTER TO CLASSIFY AND ISOLATE CELLS MANY THE HEMATOPOIETIC LINEAGE BUT PROBABLY WE HAVE DONE EMPERIMENTS WITH OTHER CELL TYPES AS WELL, OTHER CELLS AS WELL. WE THINK THE GLYCO TYPE PROVIDES A POWERFUL ORTHOGONAL TOOL TO ISOLATE CELLS IN ADDITION TO CD SYSTEM AND WITH THE DIVERSITY AND THE ADVENT OF MAYBE MORE IMMUNOREAGENTS WE EXPECT THAT WE WILL HAVE GREAT DISCRIMINATORY POWER. AND WHAT I FIND ATTRACTIVE ON SOME LEVEL IS THAT YOU CAN USE THESE REAGENTS WHILE BEING COMPLETELY AGNOSTIC TO THE HEPARAN SULFATE STRUCTURE. YOU DON'T EVEN STRICTLY NEED TO KNOW WHT THEY ARE. YOU CAN STILL LEVERAGE THE DIVERSITY FOR YOUR BIOLOGICAL APPLICATION. BUT OF COURSE WITH THE CHARACTERIZATION OF THE EPITOPES YOU WILL ALSO GAIN STRUCTURAL INFORMATION IN THE FUTURE. WITH THAT, I WOULD LIKE TO ACKNOWLEDGE THE GROUPS THAT HAVE PERFORMED THE WORK. HEMATOLOGIST, ONCOLOGIST AT EINSTEIN DID THE FACT SORTING. PROTEIN ENGINEERING. SPECIALIST AMONG OTHER THINGS AT EINSTEIN. THE BIOINFORMATICS PERSON AND OTHER PEOPLE THAT I HAVE MENTIONED. OF COURSE I WILL LIKE TO ACKNOWLEDGE THE COMMON FUND AND THE GRANT THAT SUPPORTING THIS WORK. WITH THAT I'LL BE HAPPY TO TAKE ANY QUESTIONS. >> THANK YOU SO MUCH DR. BULOW FOR YOUR PRESENTATION. ANY QUESTIONS RIGHT MOW FEEL FREE TO POST THEM. RIGHT NOW, FEEL FREE TO POST THEM. WE ARE RIGHT ON TIME. I CAN WAIT FOR A MINUTE. AND IF THERE ARE NONE WE CAN ADJOURN TO A LUNCH BREAK IF THERE ARE NONE. JUST WAIT FOR A MINUTE OR SO. GREAT. AT THIS POINT IT'S A GOOD OPPORTUNITY TO NOW ADJOURN FOR LUNCH. THANK YOU, EVERYBODY FROM THE FIRST SESSION FOR PRESENTING YOUR INTERESTING TALKS. FOR THE MANY TOOLS THAT YOU HAVE ALSO SHOWN US. WE ENCOURAGE ALL OF YOU TO TOUCH BASE WITH ANY SPEAKERS TO AVAIL YOURSELVES OF THESE TOOLS. AND TO USE THEM IN YOUR SYSTEMS AS WELL. SO ON THAT NOTE, I WOULD LIKE TO ADJOURN THIS FIRST SESSION AND THEN WE'LL COME BACK AFTER ONE HOUR AT 1:00 P.M. FOR THE NEXT -- FOR THE AFTERNOON SESSION. THANK YOU TO EVERYBODY. >> I WANT TO LET EVERYBODY OR AT LEAST LET ALL THE SPEAKERS KNOW I'LL BE GIVING A REMINDER INTERRUPTION ABOUT TWO MINUTES BEFORE THE WHEN YOUR TALK SHOULD END SO I WANT YOU TO BE AWARE OF THAT SO WE KEEP THING ON TIME. THINGS ARE GOING WELL SO FAR AND HOPE TO KEEP ON SCHEDULE UNTIL THE END OF THE AFTERNOON. IN STARTING I WOULD LIKE TO HAND THE PODIUM OVER TO UMESH DESAI FROM VIRGINIA COMMONWEALTH UNIVERSITY. ARE YOU READY? >> YES. THANKS, CARL AND I WOULD LIKE TO THANK PAM AND CARL AND ALL ORGANIZERS TO INVITE ME TO GIVE A TALK ON SOME OF THE TOOLS THAT WE HAVE DEVELOPED TO IDENTIFY GLYCOSAMINOGLYCANS SEQUENCES THAT COULD BE HELPFUL IN DISEASE AND IN HEALTH. THIS IS A GREAT WAY TO LEARN AND BRING TOGETHER EXPERTS MANY THE FIELD WORKING HARD FOR VERY LONG PERIOD OF TIME. MY TOOL HOPEFULLY WILL MAKE IT EASIER TO DISCOVER SUCH SEQUENCES. LET ME START BY PRESENTING A FIGURE. I HOPE EVERYBODY KNOWS THE FIRST DRAFT OF THE GLYCOSAMINOGLYCANS INTERACTOME HAS BEEN PUBLISHED. BY THE BLOUNT LAB IN HISTOCHEMISTRY AND PSYCHOCHEMISTRY. IF YOU LOOK AT THE NUMBER OF DISEASES AND GLYCOSAMINOGLYCANSES IT IS EYESTONESSING NOT COMMON ONES ARE OBVIOUSLY THERE, HEMOSTAYSIS, SIGNAL TRANSDUCTION, IMMUNE RESPONSE BUT ALSO QUITE A FEW THAT ARE BECOMING MORE AND MORE POPULATED IN TERMS OF GAG PROTEIN INTERACTIONS AND ESPECIALLY THOSE THAT SEEM UNIQUE AND MODEL, CROW MATTIC ORGANIZATION, NEURONAL SYSTEM, VESICAL MEDIATED TRANSPORT AND NUMBER OF OTHERS. THE GLYCOSAMINOGLYCANS INTERACTOME HAVE BEEN THE SUBJECT OF TWO MAJOR PUBLICATION, FIRST ONE CAME IN 2011. IN WHICH 435 PROTEINS WERE PROPOSED TO INTERACT WITH HEPARAN SULFATE. SO QUITE A BIT OF WORK THAT WENT INTO IDENTIFYING 435 PROTEINS. THE RECENT WORK WHICH ALSO TO LARGE EXTENT MAN Y'ALL CURATION OF PROTEINS THAT ARE YOU OF INVOLVED IN INTERACTK WITH O -- INTERACTING WITH THE GAGS THEY STUDIED THEY'D IDENTIFIED 827 PROTEINS THAT FORM 932 GAG PROTEIN COMPLEXES. WHICH IS ABSOLUTELY STUNNING. ONE WOULD THINK THAT WITH SO MANY INTERACTIONS THAT THERE SHOULD BE NUMBER OF DRUGS THAT WOULD HAVE BEEN REALIZED HOWEVER THE SITUATION IS VERY BLAKE. THERE ARE FEW DRUGS, GLYCOSAMINOGLYCANS DRUGS THAT COME INTO CLINIC. HEPARAN OBVIOUSLY EVERYBODY KNOWS. MOLECULAR WEIGHT HEPARAN AND THERE IS PARANEX CHONDROITIN SULFATE, WHILE RONIC ACID. THESE ARE FEW DRUGS HYALURONAN. THE ONLY DRUG WE COULD CALL AS HAVING BEEN DEVELOPED BY MEDICINAL CHEMISTS IS PARANEX. THE REST ARE BIOLOGIC AND CHEMO BIOLOGICS. I LIKE TO TALK TO MY STUDENTS AS AS OF NOW THE DRUG GAGOME RELATED THE DRUGS, SYSTEM ONE. WHILE RONIC ACID. WHICH I A SORRY STATE OF AFFAIR. WHY SO BLEAK? THERE ARE NUMBER OF CHALLENGES. WHICH HAVE PREVENTED DISCOVERY OF GAG SEQUENCES P. THAT REACH THE CLINIC. FIRST SYNTHESIS OR CHEMO ENZYMATIC SYNTHESIS, PREPARATION OF OLIGOSACK RIDES WE HEARD SEVERAL TALKS THE MORNING EMPHASIZING SYNTHESIS OF NOT DOZENS BUT HUNDREDS OF GAG SEQUENCES. HEXA SACCHARIDE OCTA SACCHARIDE AND DR. LIU PRESENTED 12 MER 16 MER AND 19 MER SEQUENCES WHICH IS PRETTY IMPRESSIVE. HOWEVER, SYNTHESIS IS VERY CHALLENGING AND IS RATE LIMITING. IN GENERAL IN ORDER TO DISCOVER DRUGS WE REALLY NEED A LIBRARY OF SOMEWHERE IN THE REGION OF A HUNDRED THOUSAND MOLECULES IN THIS CASE GAG SEQUENCES. TO REALLY DISCOVER SOMETHING THAT REACHES CLINIC. EVEN WITH ADVANCES MADE IN SEVERAL LABS OVER THE PAST DECADE OR SO, GENERATION OF 100,000 SEQUENCES IS A PIPE DREAM. THE OTHER PROBLEM, IT'S VERY DIFFICULT TO IDENTIFY DRUG LIKE SEQUENCES WE LIKE TO CALL NEEDLES FROM A HAY STACK, FEW TECHNOLOGY CAN DO THAT SO WHAT HAPPENS IS THAT WE ARE BASICALLY SCREENING MOLECULES SEQUENCES, ESSENTIALLY IN A BLIND FASHION AND TRYING TO IDENTIFY ONES THAT WORK AND THINGS OF THAT SORT. THE FINAL POINT WHICH IS EXTREMELY IMPORTANT FOR PHARMACEUTICAL INDUSTRY, THE REALLY LATCH ON TO GAG SEQUENCES IS INTELLECTUAL PROPERTY PROTECTION. IP PROTECTION. NATURAL GAGS MAY NOT BE REWARDING, WHY? BECAUSE WE MIGHT NOT BE ABLE TO CLAIM IP THEREFORE MODIFICATION OF NATURAL GAGS SUBTLE MODIFY MODIFICATION OF NATURAL GAGS MIGHT BE NEEDED TO GENERATE IP PROTECTION WHICH WILL HOPEFULLY ENABLE PHARMACEUTICAL INDUSTRY TO PICK UM GAG SEQUENCE. AS WE HEARD TODAY THERE WERE QUITE A FEW SEQUENCES THAT ARE VERY PROMISING AND HOPEFULLY DRUG GAGOME WILL INCREASE FROM ONE TO SEVERAL AS TIME FLIES BUT NOW IT'S STILL EXTREMELY CHALLENGING ENVIRONMENT. OUR SOLUTION TO THIS PROBLEM HAS BEEN WE HAVE COMPUTATIONAL TOOL WHICH WE CALL COMMON TORRIAL VIRTUAL LIBRARY SCREENING CVLS TECHNOLOGY. THE SPECIAL ASPECT OF OUR CVLS TECHNOLOGY IS THAT IT CAN IDENTIFY NEEDLES, IT CAN IDENTIFY NEEDLES IN A HAY STACK. THIS TOOL WAS DEVELOPED LONG TIME AGO WITH A FIRST PUBLISHED REPORT IN JOURNAL OF MEDICINAL CHEMISTRY IN 2006 BUT SINCE HEN WEAPON PROGRESSED QUITE A BIT AND SHOWN THAT THE SUCCESS RATE OF USING THIS STOOL IS PRETTY HIGH. WHILE THE UNIQUE ASPECTS ABOUT THIS TOOL THE CVLS TOOL IS WE CAN STUDY ALL POSSIBLE GAG SEQUENCES. IN SILICO. WE CAN ALL POSSIBLE GAG SEQUENCE. IN SILICO. AND THIS NUMBER COULD BE WAY MORE THAN 100,000 SEQUENCES SO WE COMPREHENSIVELY SCREENING ALL SEQUENCES FROM WHICH WE CAN IDENTIFY NEEDLESS IN A VERY SPECIFIC MANNER WHAT HAPPENINGS BECAUSE OF USE OF THIS TOOL IS IT CAN AID CHEMISTS, SO RATHER THIS THAN HUNDREDS OF SEQUENCES AND ASSESSING WHETHER PARTICULAR SEQUENCE WORKS COMPUTATIONALLY IF WE SCREEN HUNDRED THOUSAND SEQUENCES AND IDENTIFY TWO OR THREE SEQUENCES THEN WE CAN -- WE MAKE ONLY THOSE SEQUENCE, HOPEFULLY IT WILL SOLVE QUITE A LOT OF PROBLEMS IT CAN AID BIOCHEMISTS BECAUSE WE CAN ELUCIDATE MECHANISM OF ACTION AND FINALLY IT CAN AID MEDICINAL CHEMISTS BY DERIVING UNNATURAL GAGS IP WORTHY WHICH IS A SIGNIFICANT BOTTLENECK AS OF NOW. THIS TOOL IS ESSENTIALLY PRESENTED IN OUR WORK IN CURRENT OPINION AN STRUCTURAL BIOLOGY SOMETIME AGO. AND IS REPRESENTED IN THIS PARTICULAR MANNER. THE TOOL IS LITTLE MORE THAN SIMPLY DESIGNING GAG SEQUENCES. IT CAN ACTUALLY IDENTIFY COMPUTATIONALLY DOES PROTEIN BIND TO GAGS FOR THOSE INDIVIDUALS WHO DON'T KNOW THAT, COMPUTATIONALLY WE CAN IDENTIFY WITHOUT PERFORMING ANY EXPERIMENT IN A WET LAB, WE CAN IDENTIFY WHETHER THE PROTEIN CAN BIND GAGS. NUMBER TWO, WE CAN ALSO IDENTIFY WHERE DOES THE GAG BIND? AND THEN WE CAN ALSO IDENTIFY WHAT IS THE MOST OPTIMAL GAG SEQUENCE WHICH IS THE KEY ASPECT IDENTIFYING NEEDLES. FINALLY GOING FURTHER WITH IT ACTUALLY WORK IN VIVO BY FORMING A REALLY STABLE COMPLEX TOOL CAN ACTUALLY DO THAT FAIRLY EASILY. ALL COMPUTATIONALLY. LET ME JUST QUICKLY TELL YOU IT SOUNDS LIKE A VERY COMPLEX ALGORITHM SO I WANT TO DEMYSTIFY THIS, STEP ONE SIMPLY INVOLVES GENERATION OF LIBRARY OF SEQUENCES. WE ALL KNOW FOR EXAMPLE HEPARAN SULFATE THERE ARE QUITE A FUSSY QUEENSES. LIBRARY OF SEQUENCES WE CAN EASILY USE 48 UNIQUE STRUCTURE, 23 KNOWN FROM NATURE. YOU CAN GO WAY BEYOND AND USE 48 TETRA SACCHARIDES WOULD BE 2000 SEQUENCE, H SAW SACCHARIDE WOULD BE 100,000 AND OCTO CAN BE 5 MILLION SEQUENCES. ALL STUDIED WITH CVLS. THE SECOND STEP IS TARGET PROTEINS SO WE HAVE A STRUCTURE THAT'S THE NEED, YOU HAV TO HAVE A STRUCTURE WE TYPICALLY GENERATE ELECTROSTATIC POTENTIAL AND FROM THAT WE DIVIDE THE ENTIRE PROTEIN INTO DISCRETE BINDING SITES IN UNBIASED MANNER THEN STUDY EACH BINDING SITE WE CAN IDENTIFY WHERE A PARTICULAR SEQUENCE BIND. STEP THREE IS THE ACTUAL ALGORITHM, CVLS ALGORITHM, IT CONSISTS OF TWO MAJOR EXPERIMENT, ONE IN WHICH YOU ACTUALLY DO GOAL-BASED DOCKING FOR TEST TESTING AFFINITY OF DIFFERENT SEQUENCES AND THEN SIMILAR EXPERIMENT IN A MUCH MORE RIGOROUS MANNER WHERE YOU ASSESS CONSISTENCY OF BINDING. WHAT HAPPENS IS IF YOU ANALYZE THE RESULTS YOU GET SOMETHING LIKE THIS MAJORITY OF SEQUENCES ARE HERE BASICALLY BIND POORLY HIGH AFFINITY BIND TIGHTLY TO ESSENTIALLY GIVE YOU GOOD SCORE. FOLLOWED BY THAT, A SECOND FILTER, THESE SEQUENCES BIND CONSISTENTLY FOR EXAMPLE, ALL THE SULFATES ARE EXTREMELY IMPORTANT. THIS IS HOD YOU IDENTIFY THE NEEDLES. OVER THE YEARS WE PUBLISHED QUITE A BIT ON THESE AND HAVE SHOWN SEQUENCE ALGORITHM WORKS FOR MULTIPLE PROTEINS. TO GIVE SUMMARY HOW MANY DIFFERENT SELECTIVE SEQUENCES HAVE WE IDENTIFIED SO FAR. WE HAVE IDENTIFIED A HEXA SACCHARIDE THAT SELECTIVELY INHIBITS CANCER STEM CELL BUT NOT ADULT STEM CELLS. SELECTIVITY IS DIFFICULT TETRA SACCHARIDE DOES WORK, OKAY TOE SACCHARIDE DOESN'T WORK A HEXA SACCHARIDE OF SPECIFIC STRUCTURE WORKS. WE ALSO HAVE UNNATURAL HEXA SACCHARIDE THAT INHIBITING HEPARAN SULFATE FACTOR 2. NOT ANTI-THROMBIN. THIS IS ALSO A SIGNIFICANT SELECTIVE ACTIVATION. WE HAVE IDENTIFIED AN OKAY TOE SACCHARIDE THAT INHIBITS NEUTRA PHYLAE ALSOTASE TO INHIBIT INFLAMMATION IN CYSTIC FIBROSIS. THIS IS THE OKAY TA SACCHARIDE NOT THE HEXA SACCHARIDE OR ANYTHING LONGER. WHAT I'M GOING TO SHOW IN FIVE MINUTES IS DEVELOPING STORY OF INTEREST OBVIOUSLY A FEW GROUPS WE HEARD PROFESSOR BOONS PRESENTING THE SPIKE GLYCOPROTEIN. WE ALSO WORK ON THIS AND ITS INTERACTION WITH HEPARAN SULFATE SARS COV-2 SPIKE PROTEIN. THIS IS THE TRIMER OF WHICH THE RECEPTOR BINDING DOMAIN IS HERE, THERE ARE TWO ADDITIONAL HYPOTHESIZE BINDING SITES BS 2 AND 3 IN HERE. AND WHAT WE DID WAS TO FIRST SCREEN LIBRARY OF SOMEWHERE IN THE REGION OF 27,930 TOTAL SEQUENCES. FOR THE RECEPTOR BINDING DOMAIN, A SIMPLE FLOWCHART WHAT THE EXPERIMENT WAS. THE RESULTS WERE PRETTY STUNNING. THERE WERE QUITE A FEW SEQUENCES THAT ACTUALLY BOUND VERY WELL. WHEN YOU SEE HOW WELL THEY DOCK, IT TURNS OUT THESE RECOGNIZE SPIKE RESTORE BINDING DOMAIN VERY CONSISTENTLY. WHEN WE EXPANDED THE LIBRARY TO 93,312 HEXA SACCHARIDES, THERE WERE A FEW ADDITIONAL RESIDUES THAT BOUND REALLY WELL INDICATING THERE WAS SELECTIVITY IN RECOGNITION. COMPARED TO BINDING SITE 1, RECEPTOR BINDING DOMAIN, BINDING SITE 2 HOW FAR IT WAS ALL SCATTER ALL OVER THE PLAY, NO CONSISTENCY IN BINDING INDICATING THAT IT'S UNLIKELY TO RECOGNIZE BINDING SITE 2 AND SIMILAR RESULT WAS OBTAINED WITH BINDING SITE 3. THE PRIMARY SITE WAS RECEPTOR BINDING DOMAIN. WE ALSO STUDIED WHETHER SPIKE GLYCOPROTEIN AND ACE 2 COULD BE BROUGHT CLOSE TOGETHER BY HEPARAN SULFATE THROUGH A COMPLEX, TURNS OUTS ACE 2 ALSO BINDS HEPARAN SULFATE BUT THE BINDING SITE IS CLOSE TO SITE WHERE IT RECOGNIZES A RECEPTOR BINDING DOMAIN. THIS IS THE RECEPTOR BINDING DOMAIN, HEPARAN SULFATE, THIS IS THE HEPARAN SULFATE BOUND TO ACE 2, THERE'S LITTLE PROBABILITY OF FORMING ETERNAL COMPLEX BUT SIMULTANEOUSLY FORM INHIBITION AGAINST EACH OTHER. I CAN SUM SUMMARIZE THE OVERVIEW OF PREFERRED HS SEQUENCE WE HAVE IDENTIFIED SO FAR. RECEPTOR BINDING DOMAIN FAVORED HEPARAN SULFATE SITE OF BINDING. MINUTE MINIMUM CHAIN LINK IS HEXA SACCHARIDE. WE TYPICALLY USE 0.1% OF HEPARAN SULFATE SEQUENCE AND IF WE DO THAT, 92% OF THESE SEQUENCES CONTAIN AT LEAST ONE THREE SULFATE GLUCOSAMINE RESIDUE. WHICH IS STUNNING WITH PREFERRED GROUP BY SPIKE GLYCOPROTEIN. THE PREFERRED PLACEMENT OF THIS RESIDUE IS EITHER FIRST OR THIRD FROM THE NON-REDUCING END. AND IT APPEARS OVERALL THE SPIKE GLYCOPROTEIN FAVORS HIGHLY SULFATED SEQUENCES CONTAINING MORE THAN ONE SULFATE GLUCOSAMINE RESIDUES. THIS IS ONE OF THE CONCEPTS THAT CAN ENHANCE THE INTELLECTUAL PROPERTY VALUE AND RARE SEQUENCES MORE OF THEM ARE PART OF THE SAME SEQUENCE THAT WE CAN ACTUALLY DEVELOP AS DRUGS. IT TURNS OUT SULFATE RECOGNITION DO AND YOU HEARD PROFESSOR BOONS PRESENTING SOME OF THIS WORK. WE ALSO USE MICROARRAY OF 48 HEPARAN SULFATE SEQUENCE. IN WHICH WE FOUND ONLY 8 SEQUENCES CAME OUT REALLY HIGH. THE SMALLEST WAS CONTAINING A SULFATE GLUCOSAMINE, HEXA SACCHARIDE SIMILAR TO WHAT BOONS GROUP HAS DONE. IN GENERAL, LONGER SEQUENCES THAT CARRY HIGH SULFATION LED TO TURNS OUT >> I'LL ABOUT DONE. TWO OR THREE SLIDES. EXPERIMENTS PERFORMED IN COLLABORATORS LAB ACTUALLY SHOW THAT WHEN SULFATE HEPARAN SULFATE IS PRESENT IS USED ON INGESTION OR CELL FUSION OF INCREASES WITH DIFFERENT TYPES OF CELLS, THIS CAN BE PREVENTED BY USING 3O SULFATED ANTI-HS PEPTIDE. ALSO BE COMPETED WITH PYRINEX AND YOU CAN REDUCE THAT SIGNIFICANTLY. LIKEWISE WE HAVE CONVERTED THIS ENTIRE DISCOVERY INTO A DISCOVERY OF SMALL MOLECULE, SYNTHETIC MOLECULE CALLED SVGG A GAG -- TURNS OUT USING A SARS COV-2 INFECTION MODEL THE INHIBITION OF VIRUS ENTRY CAN BE EN-- SBGG IT HAS POTENCY OF 200 NANOMOLAR. OBVIOUSLY CAME FROM DISRY OF SULFATED SEQUENCES. THE MAJOR TAKE HOME MESSAGE HERE IS THAT HUNDREDS OF GAG PROTEINS SYSTEMS MAJOR OPPORTUNITIES TO DISCOVER THERAPEUTICS AND/OR CHEMICAL BIOLOGY TOOLS. OUR COMPUTATIONAL TOOLS CVLS HELP IDENTIFY GAG SEQUENCES RECOGNIZED BY TARGET PROTEINS. WE ARE RELEASING A WET TOOL CVLS FOR GAG SOON PART OF THE UO 1 PROJECT WE WERE AWARDED. THIS WILL ENABLE PEOPLE TO WORK ON THEIR PROTEINS AND GAG RECOGNITION. I WOULD LIKE TO THANK MY GROUP, MY ENTIRE GROUP. THE WORK PRESENTED TODAY IS FOR THREE INDIVIDUALS COMPUTATIONAL BIOLOGIST, BIOLOGY COMPUTATIONAL BIOLOGIST AND JOHN WHO IS DOING MICROARRAY ANALYSIS. WITH THAT, THANK YOU ALL FOR LISTENING. >> THANK YOU, DR. DESAI. RIGHT NOW NOT AWARE OF ANY QUESTIONS. WE ARE ACTUALLY RIGHT DOWN TO 1:20. SO YOU CAN STILL ASK QUESTIONS LATER ON AFTER ALL THESE TALKS. WE JUST AS SOON START WITH DR. MICHAEL SIMONS. DR. SIMONS, GO AHEAD. >> THANK YOU VERY MUCH. I WOULD FIRST LIKE TO THANK OUR THE ORGANIZERS OF THIS MEETING FOR GIVING ME CHANCE THE TALK TO YOU AND TALK TO YOU ABOUT OUR RECENT WORK. THIS HAS BEEN AN EXCITING MORNING AND HOPEFULLY I DON'T TELL YOU SOMETHING THAT YOU -- KEEP YOU INTERESTED AS WELL. SO I'M GOING TO TALK ABOUT OUR WORK HOW SMALL CHANGES IN SEQUENCE OVER THE COURSE PROTEOGLYCAN COURSE CAN AFFECT THE PATTERN OF THE CHAIN. THIS WORK CAME FROM HEPARANASE SYNDECAN, VASCULAR BIOLOGY AND THESE THINGS HAVE ALREADY BEEN MENTIONED SEVERAL TIMES TODAY PLAY A VERY IMPORTANT ROLE IN OUR GROWTH FACTOR. SO SYNDECAN FAMILY IS MADE UP OF SYNDECANS, 1 THROUGH 4, THOSE HAVE SIMILAR STRUCTURE. THESE ARE PLASMA MEMBRANE MOLECULES, CHAINS THAT HAVE CONSERVED INTERMEMBRANE, INTERMEMBRANE DOMAIN AND QUITE CONSERVED DOMAINS AS WELL. AS YOU WELL KNOW,S GAG CHAINS ARE IS ESSENTIALLY POLYMER SUGAR THAT CAN BE SECONDLY MODIFIED ADDITION OF NUMBER OF SULFATE GROUPS. THE TRANSMEMBRANE DOMAIN OF SYNDECANS IS (INAUDIBLE) AND THEY EXIST IN A DIMER, THEY DON'T EXIST AS ISOLATED MOLECULES. SYNDECANS ARE ALWAYS DIMERIZED. IF YOU (INAUDIBLE) PLASMID DOMAIN YOU CAN SEE THAT THESE LPCD BINDING PROTEINS SHOWN BY BFA MOTIF. BUT THEY ALSO HAVE WHAT'S CALLED THE VARIABLE DOMAIN THAT CAN PLAY A ROLE IN THE NUMBER OF SIGNALING EVENTS THAT HAPPENS IN CELLS. OUR WORK TRYING TO UNDERSTAND THE FUNCTION OF THIS MOLECULE SYNDECAN 2 LED TO DISCOVERY OF PROTEIN SULFATION. CODE. BUT BEFORE THAT LET ME JUST SAY IN CASE YOU HAVE NOT RECENTLY REFRESH MEMORY HOW CHAINS ARE MADE, IT'S A SEQUENCE, IT'S A THICK SEQUENCE OF EVENTS WHERE YOU HAVE ADDITION OF RESIDUES TO EXPANDING POLYMER CHAIN THEN COME THE -- THEN COME THE SULFATION ENZYMES, THAT ADD SULFATE RESIDUES IN DIFFERENT POSITIONS. IMPORTANCE OF THESE EVENTS IS THAT MAKE UP BINDING SITES TO VARIOUS GROWTH FACTORS. TWO SULFATED POSITIONS WHILE SULFATE POSITIONS, ET CETERA. SO WE MADE SIGNIFICANT TOOL KNOCK OUT MOUSE BECAUSE WE WERE INTERESTED IN HOW BLOOD VESSELS ARE FORMING IN THE SPINES. WHAT YOU ARE LOOKING AT IS A NEONATE RESIDENT LOOKING AT THIS, YOU CAN SEE IN THE KNOCKOUT MICE THE EXTENT FROM THE CENTER IS LESS THAN WHAT EWE SEE IN WILD TYPE MICE. NOT BIG DIFFERENCE, IT'S 25, 30% DIFFERENT BUT MEANINGFUL DIFFERENCE BECAUSE IT MEANS ENDOTHELIAL CELLS MAKE THIS -- PATTERN OF BLOOD VESSELS IS NOT AS LARGE AND NOT AS EXTENSIVE AS A WILE TYPE MICE WHICH MEANS YOU DON'T HAVE AS MANY ENTHELIAL CELLS THEY DON'T MOVE AS FAST. IF YOU LOOK AT THE PATTERN OF FRONT PATTERN OF HOW VESSELS FORMED YOU SEE WILD TYPE MICE LOTS OF -- NOT AS HIGH FROM THE KNOCK OUT MOUSE. THAT'S ALSO A SIGNIFICANT DIFFERENCE. BUT THAT TELLS YOU SOMETHING HAPPENED TO THE -- SOMETHING HAPPENED TO THIS -- SOMETHING HAPPENED TO THE ANGIOGENIC PROCESS HERE. KEY FACTOR REGULATING EXTENT OF ANGIOGENESIS IS GROWTH FACTOR AND HOW IT'S ACTUALLY VGFR 2 RECEPTOR. NORMALLY WHEN IT BINDS TO VGFR 2 YOU GUESS PHOSPHORYLATION EFFECT AND YOU SEE THIS INTENSE EVEN YOU WILL SEE THIS IN THE KNOCKOUT MICE NOT AS EXTENSIVE SO THE EXTENT OF ACTIVATION IS DOWN, WHILE -- NORMAL AND EVERYTHING IS EXPRESSED. NORMALLY. AN IDEA MAYBE SOMETHING IS HAPPENING THAT MAYBE SIGNIFICANT HOW INVOLVED IN VEGF SIGNALING. TO UNDERSTAND IT BETTER WE TOOK MOUSE CELLS AND IF YOU BACK INTO THEM YOU CAN SEE EXTEND OF VGSF ACTIVATION BECOMES KNOWN. IF YOU PUT SYNDECAN 2 BACK WITHOUT GAG CHAINS, THERE'S NO SULFATE CHAINS YOU CAN SEE THE EXTENT OF THE ACTIVATION IS THE SAME AS IF -- IF YOU PUT THE NAKED VIRUS. SO THERE'S A SIGNIFICANT DIFFERENCE IF YOU PUT SYNDECAN 2 BACK GAG CHAINS. INTERESTINGLY MUST HAVE IF YOU PUT THE SIMILAR -- YOU SEE NO RISK. THAT TELLS YOU SOMETHING SPECIAL ABOUT -- GAG CHAINS, NOT TRUE ABOUT SYNDECAN 4. SO WE TOOK ONCE AGAIN WE TESTED THE SYNDECANS AND ONLY SYNDECAN 2 RESCUES VEGF ACTIVATION. ONLY SYNDECAN 2 BINDS VEGF, DOING HERE THIS IS THE CO-AP. EACH SYNDECANS HAS (INAUDIBLE) BRING THEM DOWN AND DO WESTERN BLOT LOOKING TO SEE VEGF RECEPTOR COMPLEX AND YOU WILL FINE YOU HAVE SYNDECAN 2 IN THE SYSTEM. WE WENT A STEP FURTHER AND MADE CONSTRUCTS WHERE WE TOOK A SYNDECAN EXTRA CELLULAR DOMAIN PUT IT ON TO BE OF SYNDECAN 4 INTRACELLULAR OR OTHER WAY AROUND. OTHER WAY YOU HAVE THIS RESCUE IF YOU HAVE SYNDECAN 2 EXTRA CELLULAR DOMAIN, ATTACHED TO THE OWN CORE OF SYNDECAN 4 IT DOESN'T MATTER. YOU NEED SYNDECAN 2 EXTRA CELLULAR DOMAIN. SYNDECAN 4 EXTRA CELLULAR DOMAIN DOES NOT DO IT. IF YOU PUT -- GAG NO BINDING IF YOU USE DIFFERENT ENZYMES THAT DESTROY THAT WILL DESTROY SUGAR CHAINS RESIDUES HAVE 113 OR 1K 5 YOU INTERFERE WITH EGFR 2 BINDING TO SYNDECAN 2 AND EGFR 2 ACTIVATION. WHAT THAT TELLS US IS THAT SYNDECAN 2 FORMS VEGF AAV EGFR 2 COMPLEX AND FOR THAT TO HAPPEN YOU NEED SYNDECAN 2 EXTRA CELLULAR DOMAIN AND WHAT YOU REALLY NEED SYNDECAN 2 SULFATE CHAINS. THAT TOLD US MAYBE SOMETHING SPECIAL ABOUT THE CHAINS. SO BEFORE WE ISOLATE THE CHAIN, FROM SYNDECAN 2 AND WE USE MASS SPEC TO LOOK AT THE EXTEND OF SULFATION AT DIFFERENT SITES. WHAT YOU SEE HERE SHOWN HERE IN THE SYNDECAN 2 CELLS SHOWN IN THIS ARRAY, SYNDECAN 2 -- SIN CAN 4 CHIMERA EXTRA CELLULAR DOMAIN YOU HAVE A DRAMATIC INCREASE IN SULFATE, THAT'S WHERE EGFR OCCUPIES SO IN ALL SETS OF 6-0 SULFATION. BUT NOT IN ANY OTHER CHAINS. SO WE WENT A STEP FURTHER AND IF YOU SEE -- IF YOU USE LCMS MASS SPEC YOU CAN SEE INCREASE THESE SIZE BUT ALSO INCREASE IN 3-0 SITES. I WILL COME BACK WHY THIS IN 3-0 SITES AS WELL. THIS IS A FUNCTIONAL MEANINGFUL DIFFERENCE, THERE SHOULD BE FUNCTIONAL DIFFERENCE OF -- CHAIN BINDING ASSAY. AND THEY BIND TO SYNDECAN 2 CHAINS BUT NOT SYNDECAN 4 CHAINS. AS SHOWN. SO IT LOOKS LIKE THAT YOU NEED THE EXTRA CELLULAR DOMAIN OF SYNDECAN 2. WE WENT A STEP FURTHER AND WE DIVIDED EXTRA CELLULAR DOMAIN SYNDECAN 2 INTO TWO PARTS. WHAT WE CALL G1 IMMUNOASSAY (INAUDIBLE) 59101, 135, SORRY AND WE MADE SLOPES BECAUSE THIS PATTERN SYNDECAN 2 AND SAME SYNDECAN 4. AS YOU CAN SEE HERE WHEN WE EXPRESS THE 3-0 DOMAIN YOU HAVE VEGF BINDING AND VEGF OBSERVATION. SAME PATTERN FROM SYNDECAN 4. WE HAVE NOW GONE FURTHER AND LOCALIZED THIS SEQUENCE TO 10 IMMUNOASSAYS, THAT GIVES YOU THIS EFFECT. WHAT'S INTERESTING IS THAT YOU CAN PUT THE SEQUENCE WHETHER IT'S 59 AMINO ACID SEQUENCE WE HAVE NOT UPDATED FOR ACID YOU CAN PUT IT IN ANY PROTEOGLYCAN. ANY YOU PUT IT IN, WILL PUT IT IN SYNDECAN 1, IT WILL START MAKING 6-0 ENHANCE CHAINS AND REQUIRE THE ABILITY TO BIND VEGF AND PERFORM THE VEGF 2 COMPLEX. SO I THINK THIS IS THE FIRST -- THIS IS THE FIRST ILLUSTRATION THAT YOU CAN HAVE A SMALL PROTEIN SEQUENCE THEN DRIVE SULFATION PATTERN. HOW THAT HAPPENS WITH REGARD TO STUDY HAS TO DO WITH MOVEMENT OF THE PROTEOGLYCAN CHAINS IN THE GOLGI THAT'S AN EXCITING AREA BUT FOR ANOTHER TIME. THERE IS AN INTERESTING APPLICATION OF THIS. HEPARAN AS YOU KNOW IS A VERY IMPORTANT DRUG. THERE'S BEEN MULTIPLE EFFECTS MULTIPLE ATTEMPTS TO MAKE MEDICINAL HEPARAN USING MOLECULAR -- HEPARAN COMES FROM PIGOUTS. THE KEY FEATURE IS LARGE AMOUNT OF LOOPS. IN ORDER TO HAVE THIS SAL SULFATION YOU NEED TO H HAVE 6-0. WHAT YOU HAVE NOTICED THAT THESE -- THAT WHEN WE INCREASE EXPRESSION OF SYNDECAN 2 DOMAIN WE CAN INCREASE AMOUNT OF SULFATION. THAT GIVESIOUS AN IDEA THAT PERHAPS YOU CAN BIND BECAUSE 3-0 SITES IS WHAT'S INVOLVED IN BINDING INITIATION OF A (INAUDIBLE) IF YOU HAVE HEPARAN IT BINDS TO THE OO SITES AND FOR THAT REASON HEPARAN IS USED WITH BLOOD THIN PER. AS IT HAPPENS ESSENTIALLY ALL OF THIS HAPPENS AT COMES CHINA AND (INAUDIBLE) THERE IS SIMPLE SOURCE OF ALL HEPARAN THAT YOU HAVE. COMBINED WITH FACT THERE'S SWINE FEVER AFFECTING CHAINS IN PIGS AND WE HAVE A SERIOUS SHORTAGE OF THAT. THEREFORE IT BECOMES REALLY IMPORTANT TO SEE IF WE CAN FIND A WAY OF MAKING THIS DRUG THAT WOULD NOT DEPEND ON PIGS. NOW IS THE IDEA THAT LOOKING AT IS WE ARE USING IS FOR USING AL SERGLYCIN CHAINS EXPRESSED IN ENDOTHELIAL CELLS. AND IT'S EXPRESSED AT HIGH LEVELS. SO WE GENERATE COMPLEX WITH NUMBER OF PROTEIN SEQUENCES THAT WILL INCREASE 6 - 0 UNTIL SULFATION AND WE ARE HOPEFUL THIS WILL GIVE -- THAT SOMETHING THAT WILL AT LEAST RESEMBLE THE LARGE A. OF SULFATION IN HEPARAN AND MAYBE THIS WILL HAVE A PIE LOGICAL EFFECT SO WE ARE -- BIOLOGICAL EFFECT SO WE ARE PURSUING THE STRATEGY TO SEE IF WE CAN MAKE IN ESSENCE IN VITRO HEPARAN. TO SUMMARIZE END TERMINAL DOMAIN OF SYNDECAN 2 INCREASES 6 - 0 SULFATION OF REACTIONS. THE SAME SEQUENCE IMMEDIATE YATES 6-0 SULFATION OF EVENTS WHEN MOVED TO OTHER PROTEOGLYCANS. THE PRESENCE OF THE SEQUENCE ALSO ENHANCES ABILITY OF BOTH 3-0 SULFATION AND PERHAPS CAN BE USED TO MAKE HEPARAN LIKE DRUGS. WE ALSO HAVE THOSE WHO HAVE EVIDENCE ANOTHER SEQUENCE IN THE N TERMINAL SYNDECAN 4 DOMAIN INCREASES 2-0 SULFATION OF GAG CHAINS. SO THESE FINDINGS WOULD SAY THEY EXIST AMINO ACID SULFATION CODE AND MAYBE BY ACTUALLY MANIPULATING MNS SEQUENCES YOU CAN REALLYNA MANIPULATE COMPOSITION OF GAG CHAINS IN VIVO. AND IN CELLS. IT'S POTENT BECAUSE MAP SIGNALING AGAINST CELL TYPE DEPENDENT SIGNALING EVANS YOU PROBABLY USE NON-CELL BOUND GAG EFFECT SPECIFIC CELL SIGNALING EVANS BUT THE ABILITY TO MODIFY SULFATION PATTERN ON EXISTING CELL TYPE WOULD BE EXTREMELY VIABLE AND EXTREMELY BIG SCIENTIFIC CHALLENGE. THAT'S WHAT I AIM TO DO HERE. SO I WOULD LIKE TO THANK MEMBERS OF MY LAB WHO DID THIS WORK. WAS THE KEEPERS IN DOING THIS ASSISTED BY EMMA WHO HAS NOW TAKEN HER OWN ROLE STUDIES DISTINCTION IN GOLGI AFFECT HOW SULFATION HAVE BEEN CHANGED AND YOU HAVE -- WE HAVE WONDERFUL ASSISTANTS FROM -- GROUP AT UNIVERSITY OF GEORGIA AND THEY WERE ABLE TO PERFORM MASS SPEC SEQUENCE. SO I WOULD BE REMISS IF I DIDN'T MENTION EXTENSIVE SUPPORT FROM NATIONAL INSTITUTES OF HEALTH FOR THIS WORK IN THE LABORATORY. THANK YOU VERY MUCH. I WILL STOP HERE. >> THANK YOU VERY MUCH, DR. SIMONS. WE HAVE A COUPLE OF MINUTES. NOT AWARE OF ANY QUESTIONS THAT HAVE COME IN BUT I WOULD LIKE TO ASK YOU ONE QUESTION. PERHAPS YOUR END TERMINAL DOMAIN CAN BIND EITHER HEPARAN SULFATE OR THE SKULL FILL TRANSFERASES TO HELP ACCOUNT FOR WHY SEEING DRAMATIC AFFECT ON SULFATION? >> WHAT WE ARE SEEING THAT WHEN YOU MANIPULATE THAT PROTEIN SEQUENCE, THAT -- HAPPEN IN A DIFFERENT PART OF THE GOLGI. IT'S RATE OF MIGRATION THROUGH GOLGI IS DIFFER. SO IT'S POSSIBLE THE DIFFERENT PARTS OF GOLGI HAVE DIFFERENT STs AND SOME -- IT'S POSSIBLE THE RATE OF MOVEMENT ALLOWS A MORE PROLONG COMPACT, OSV BY INCREASING AMOUNT OF 6-0 SULFATION. >> OKAY. I DON'T SEE ANY OTHER QUESTIONS COMING IN RIGHT NOW. MAYBE WE CAN MOVE ON TO RALPH SANDERSON. THANK YOU VERY MUCH. VERY INTERESTING. DR. SANDERSON, I WOULD LIKE TO HAND IT OVER TO YOU NOW. >> THANK YOU, HANKS FOR THE OPPORTUNITY TO SPEAK TODAY. I'M GOING TO TALK A LOT TODAY ABOUT SYNDECAN 1 AND HOW HEPARANASE REGULATES SHEDDING OF SYNDECAN 1 AND EMPHASIZE HOW THE SHEDDING MECHANISM MAYBE IMPORTANT IN MULTIPLE DISEASE PATHOLOGIES. WE HAVE USED MODEL SYSTEM OF MULTIPLE MYELOMA TO STUDY THIS RELATIONSHIP FOR A NUMBER OF YEARS. MOST OF YOU PROBABLY KNOW THAT SYNDECAN 1 IS A TRANSMEMBRANE HEPARAN SULFATE PRO OWE GLYCAN, IT BINDS TO GROWTH FACTORS. AS WE HEARD MULTIPLE PRESENTERS TODAY THIS CAN BE IMPORTANT IN CO-FACTOR SIGNALING BE IT HEPARAN SULFATE CHAINS AND GROWTH FACTORS BOUND TO THEM. BUT INTERESTINGLY HEPARANNIST PLAYS A ROLE AS WELL IN BIOLOGY OF THIS MOLECULE, IT CAN CLEAVE HEPARAN SULFATE CHAIN, IT'S A GLYCOE RON DAYS, IT INHIBITS SULFATE FOR PROTEIN AND LEAVES SMALLER HEPARAN SULFATE CHAINS ATTACHED TO THE CORE. SO THE REMODELING IS VERY IMPORTANT, IT ALSO CAN SYNDECAN 1 CAN BE REMODELED BY PROTEASES TO CLIP THEM AND RELEASE EXTRA CELLULAR DOMAIN THE SHED PRO PROTEOBELIE CAN WHICH ALSO MAINTAINS BIOLOGICAL ACTIVITY. IN THE MULTIPLE MYELOMA SYSTEM WE STUDY, THIS IS PA FTT PET SCAN OF PATIENT, YOU CAN SEE LESIONS IN THE PATIENT, MOST LESIONS ARE IN BONE. IT'S A HIGHLY OSTEOLYTIC DISEASE AND CAUSES A NUMBER OF DIFFERENT PROBLEMS FOR PATIENTS DUE TO THIS OSTEOLOGYSIS. IF WE LOOK AT BONE MARROW BIOPSY AND STAIN FOR SYNDECAN 1 YOU CAN SEE RED STAINING IS HIGH LEVEL SYNDECAN EXPRESSION ON THE SURFACE OF THESE TUMOR CELLS. LOOK AT STAINING FOR HEPARANASE YOU CAN SEE INTRACELLULAR STAIN WITNESS BONE MARROW. BUT WE CAN MEASURE ACTIVE HEPARANASE IN FLUIDS FROM THESE PATIENTS INDICATING HEPARANASE IS ACTIVE ALSO IN THE EXTRA CELLULAR ENVIRONMENT AS WELL AS WITHIN THE CELLS. AND MYELOMA MICROENVIRONMENT. EARLY ON WE LEARN HIGH LEVELS OF SHED SYNDECAN COULD BE MEASURED IN MYELOMA PATIENT SERA. ANOTHER GROUP WAS ABLE TO SHOW THIS WAS ASSOCIATED WITH POOR PROGNOSIS THAT LED US TO ASK THE QUESTION WAS THE SHED SYNDECAN 1 IN FACT WHEN WE LOOK AT THIS IN MOUSE MODELS OF MYLOTHAT WE ARE ABLE TO SHOW SHED SYNDECAN PROMOTES METASTASIS AN GROWTH IN VIVO OF MYELOMA TUMORS. SO WE THEN TURNED FOCUS PREDOMINANTLY TO TRY TO UNDERSTAND WHAT WERE THE MECHANISMS SHED SYNDECAN 1 WAS EMPLOYS TO REGULATE DISEASE PROGRESSION. SO WE USED A MYELOMA CELL LINE, HUMAN MYELOMA CELL LINE CALLED CAG, THAT EXPRESSES RELATIVELY LOW LEVELS OF ENDOGENOUS HEPARAN ASE. WE ANSWER PECKED IT AND INCREASED HEPARANASE EXPRESSION TO A LEVEL SEEN QUITE OFTEN IN MYELOMA PATIENTS SO WE THINK THIS IS A PHYSIOLOGICALLY RELEVANT MODEL OF HEPARANASE EXPRESSION IN THESE TUMOR CELLS. WHEN WE UPREGULATED HEPARANASE IN THESE TUMOR CELLS WE SAW INCREASE IN E,K SICKNAL THAT DRIVES EXPRESSION AND SECRETION OF P 59. THAT WAS INTERESTING BECAUSE MMP 9 IS A SHED SYNDECAN 1 AND THAT DROVE ENHANCE SHEDDING OF THE ECHO DOMAIN. SO HEPARANASE WAS DRIVING SHEDDING THROUGH MMP T BUT ALSO LEARN WHEN HEPARANASE SHORTENS THE CHAINS HEPARAN SULFATE CHAINS ON THE SYNDECAN PROTEIN, THAT IT ALLOWS THE MMP 9 TO HAVE BETTER ACCESSIBILITY OF CORE PROTEIN AND DRIVE SHEDDING PROCESS ALSO. SO WHAT I HAVE TOLD YOU IS THERE'S TWO WAYS HEPARANASE DERIVES SHEDDING UPREGULATION OF NMP 9, OTHER SHORTENING OF CHAINS AN ENHANCED ACTIVITY OF MMP 9 AGAINST CORE PROTEIN. SO WE BEGIN TO STUDY SEVERAL MECHANISMS THEN OF HOW THIS SHEDDING OF THE PROTEOGLYCAN MIGHT BE REGULATING MYLO MYELOMA. ONE GROWTH FACTOR EXPRESSED IN THIS DISEASE IS HEPATOSITE GROWTH FACTOR. WHEN WE LOOKED AT THE EXPRESSION OF HGF BY CELLS WE LEARN WHEN WE UPREGULATED HEPARANASE WE DRAMATICALLY UPREGULATED EXPRESSION OF HGF. HGF BIND TO SHED SYNDECAN 1 AND WHEN IT DID THAT AND BONE MARROW MICROENVIRONMENT HAS ACCESS TO OSTEO BLAST, THAT HGF CAN BE PRESENTED BY THE SYNDECAN 1 AND ENHANCE CMET ACTIVITIES, CMET SIGNALING. CMET SIGNALING LEADS TO IL 11 EXPRESSION AND SECRETION. THE IL 11 BINDS TO RECEPTOR IN AUTOCRINE FASHION STIMULATING SIGNALING THAT RESULTS IN UPREGULATED EXPRESSION OF RANK LIGAND. RANK LIGAND IS A VERY IMPORTANT FACTOR IN THE DIFFERENTIATION OF OSTEOCLAST AND THERE BY SHEDDING MECHANISM LOOPS BACK THROUGH THIS PROCESS TO ENHANCE OSTEOPLAST GENESIS AND ACTIVITY AND PLACES IMPORTANT ROLE IN OSTEOLOGYSIS IN MYELOMA. SO WE LOOK AT ANOTHER MODEL BONE DESTRUCTION AND THIS IS THE SKID MODEL OF MYELOMA WHICH WE IMPLANT HUMAN BONES FRAGMENTS OF HUMAN BONES INTO SKID MICE WE CAN THEN INJECT BONES DIRECTLY WITH TUMOR CELLS. SO WE INJECTED THIS SERIES OF BONES WITH CELLS EXPRESSING LOW LEVEL OF HEPARANASE. THIS SERIES OF BONES INJECTED WITH CAD CELLS EXPRESSING HIGH LEVELS OF REPNASE. BY X-RAY YOU CAN SEE THE HIGH REPNASE EXPRESSING CELLS DRAMATICALLY DEGRADE THE BONE WHILE LOW HEPARANASE DIDN'T. AS A NICE FUNCTIONAL CORRELATE SHOW THAT HOW WE THINK THAT HEPARANASE IS DRIVING OSTEOLOGYSIS IN THIS DISEASE AN DOWNSTREAM OF THE SYNDECAN SHEDDING. WE LOOSE LOOKED AT ANGIOGENESIS IN A SIMILAR MODEL WE SAW THAT HEPARANASE INCREASE VEGF EXPRESSION, PRO DICKED BASED ON PREVIOUS STUDIES IN OTHER TYPES OF CANCER BUT WHEN WE LOOK AT THE CONDITION MEDIAN FROM THESE CELLS WE SAW VEGF WAS BINDING TO THE SHED SYNDECAN 1. IN A MODEL USING RAT AORTIC ORGAN CULTURE WE ADDED VEGF TO EACH ONE OF THESE CULTURES, WE COULD THEN GO BACK AN ADD CONDITION MEDIUM FROM EITHER CAG LOW HEPARANASE EXPRESSING CELLS OR HIGH HEPARANASE EXPRESSING CELLS. THE LOW HEPARANASE EXPRESSING CELLS WITH LOW LEVELS OF SHED SYNDECAN 1 THAT'S NOT EXTENSIVE ANGIOGENESIS. THE HIGH HEPARANASE EXPRESSING CELLS WITH HIGH AMOUNT OF SHED SYNDECAN 1 YOU SEE EXTENSIVE ANGIOGENESIS. INTERESTINGLY WE CAN TAKE LOW HEPARANASE CONDITION MEDIA, AND THE SHED SYNDECAN ONE AND THIS DRIVES THE ANGIOGENIC PROCESS. ORB WE CAN TAKE HIGH HEPARANASE HIGH SYNDECAN 1 MEDIUM AND WE CAN IP SYNDECAN 1 OUT, YOU CAN SEE THAT WE HAVE LOST THE ANGIOGENIC EFFECT AND THIS SIMPLY IS QUALIFICATION OF THE VESSEL FORMATION. SO CLEARLY REPNASE SHEDDING HEPARANASE INDUCE SHEDDING OF SYNDECAN 1 WAS HELPING DRIVE THE ANGIOGENIC PROCESS. INTERESTINGLY COMING FROM A TOTALLY DIFFERENT ANGLE, THIS IS COLLABORATION WITH ALAN (INAUDIBLE) WHAT HE DISCOVERED IN HIS LAB USING OUR MYELOMA MODEL AND SHED SYNDECAN 1, WAS IF THERE ARE -- WHEN SHED -- SYNDECAN 1 IS SHED THERE IS A CRYPTIC DOMAIN EXPOSED IN THE CORE PROTEIN. THAT THEN IS ABLE THE BIND TO VEGFR 2 AND BLA 4 FORMING A COMPLEX THAT ACTIVELY DRIVES RAC SIGNALING AND THIS RAC SIGNALING PROMOTE BOTH MYELOMA MIGRATION AN METASTASIS AS WELL AS ENDOTHELIAL TUBE FORMATION. VIA SINGLE MECHANISM ALAN EXPLAINED HOW HEPARANASE DRIVES MIGRATION METASTASIS AND ANGIOGENESIS. VERY IMPORTANT CONCEPT. ENDOTHELIAL TUBE FORMATION SHOW INHIBITED BY HEPARANASE INHIBITOR OR STATINS. SERIES OF PEPTIDES DEVELOPED MANY THE LAB THAT MIMIC THIS REGION OF THE CORE PROTEIN THAT INTERACTS WITH VEGFR 2 AND BLA 4 TO INHIBIT INTERACTION AND COMPLETELY DIMINISH THE ABILITY OF THESE ENDOTHELIAL TUBES TO FORM. ALSO INHIBITOR THAT BLOCKS SYNDECAN SHEDDING NONE OF THESE PROCESSES TAKE PLACE. IT'S A POWERFUL DEMONSTRATION HOW HEPARANASE INDUCE SHEDDING VIA CORE PROTEIN INDEPENDENT OF HEPARAN SULFATE CHAINS DRIVER THIS. SO IN SEPARATE STUDIES WE DISCOVER CELLS EXPRESSING HIGH LEVELS OF HEPARANASE SECRETED HIGH LEVELS OF EXSOMES COMPARED TO THE CELLS MAKING LOW LEVELS OF HEPARANASE, THAT QUESTION GENED LOW LEVELS OF EXSOMES WE CAN TAKE RECOMBINANT HEPARANASE TAKEN UP BY THESE CELLS AND UTILIZED. WE ADDED BACK TO THE LOW HEPARANASE EXPRESSING CELLS AND SHOW THAT THAT DRAMATICALLY ENHANCED EXSOME SECRETION. WE ALSO WITHER ARABLE TO SHOW TOTALLY DEPENDENT ON HEPARANASE ENZYME ACTIVITY AND SPECULATED IT WAS SHORTENING THE HEPARAN SULFATE CHAINS THIS WAS ENHANCING THE DRIVING OF THE EXSOME BIOGENESIS IN THESE CELLS. SO THAT TURNED OUT TO BE THE CASE AND MORE WORK BY DAVID THIS IS JUST A PANEL HERE FROM REVIEW COUPLE OF YEARS AGO THAT I WROTE BUT WHAT HE WAS ABLE TO SHOW IS EARLY IN COMPARTMENT WHEN EXSEOULS ARE BEGINNING TO BE FORMED YOU ACTUALLY GET HEPARAN SYNDECAN 1 INTERNALIZED AND HEPARANASE DEGRADE SULFATE CHAINS IN THE ENDOSOME CAUSING THE ALLOWING THE SYNDECAN CORE PROTEIN TO MORE CLOSELY ALIGN TO EACH OTHER. AS PROCESS MATURES YOU CAN SEE YOU GUT GET SHEDDING OF THE HE CAN TOE DOMAIN AGAIN WITHIN THE BUDDING VESICAL IN THE EXSOME FORMATION. ONCE THIS OCCURS IN THE EXTRA CELLULAR DOMAIN IS LOST, THEN YOU HAVE THE TRANSMEMBRANE AND CYTOPALACE MIC DOMAINS THAT INTERACT WITH THE COMPLEX WHICH ACTIVATE IT IS SKIRT COMPLEX WHICH IS THE MECHANISM THAT DRIVES THE BUDDING OF THE VESSELS. AND EVENTUALLY THE EXSOME BIOGENESIS. BY SHEDDING BY DEGRADING THE HEPARAN SULFATE RESULTING SHEDDING OF THE PROTEOGLYCAN YOU ARE DRIVING A BIOLOGICAL PROCESS THAT'S IMPORTANT PARTICULARLY IN CANCER. WITH EXSOME BIOGENESIS SO WE SPECULATED WITH ALL THESE POTENTIAL PATHWAYS THAT ARE BEING DRIVEN BY HEPARANASE INDUCE SYNDECAN SHEDDING, IF WE CAN INHIBIT HEPARANASE AN INTERFERE WITH MULTIPLE PAT WAYS THAT WE MIGHT IMPACT MYELOMA PROGRESSION. A NUMBER OF YEARS AGO WE STARTED A COLLABORATION WHICH WAS AT TIME HEADED BY (INAUDIBLE) ALONG WITH (INAUDIBLE) WE TESTED A MODIFIED HEPARAN WHICH WAS EVENTUALLY CALLED -- THIS MODIFIED HEPARAN IS POTENT INHIBITOR OF HEPARANASE ACTIVITY, VERY HIGH ANTI-COAGULANT ACTIVITY SO WE CAN USE IN HIGH DOSE IN ANIMAL MODELS AND WE SHOWED IN PRE-CLAIM STUDIES THIS WAS VERY GOOD INHIBITOR OF HEPARANASE ACTIVITY. GOOD BIOLOGICAL INHIBITION OF TUMOR GROWTH PARTICULARLY WHEN USED IN COMBINATION WITH OTHER DRUGS KNOWN ANTI-MYELOMA DRUGS SO THIS WAS FOLLOWED BY STUDY WITH BIOSCIENCES WE COLLABORATED IN AND PHASE 1 CLINICAL TRIALS, 19 PATIENTS. THEY SHOW THAT -- WAS INDEED SAFE IN PATIENTS WITH MINIMAL SIDE EFFECTS AND IMPORTANTLY, NO SIGNIFICANT COMPLICATIONS SO HOPEFULLY THIS WILL EVENTUALLY MOVE TO PHASE 2. WE KNOW THIS MECHANISM IS IMPORTANT IN CANCER, PROBABLY MULTIPLE TYPES OF CANCER BUT I WANTED TO TALK BRIEFLY GIVEN RELEVANT INTEREST MANY TALKS TODAY TOO ABOUT VIRAL PATHOGENESIS, I WANT TO TALK ABOUT HOW I THINK THIS MAYBE RELATIVE TO NUMBER OF DIFFERENT VIRAL INFECTION. THIS IS WORK BY DEEPAK AT THE UNIVERSITY OF ILLINOIS CHICAGO, LIKE MANY OTHERS WAS ABLE TO SHOW THE VIRUS BIND AVIDLY TO HEPARAN SULFATE, WORK IS PARTICULARLY ON HSV 1. THEY THAT THIS WAS IMPORTANT IN VIRAL ENTRY. BINDING TO HEPARAN SULFATE SYNDECANS IS IMPORTANT FOR VIRAL ENTRY. ONCE VIRUS IS GET INSIDE CELL THERE'S UPREGULATION OF THE NF KAPPA B PATHWAY LEADING TO HEPARANASE UPREGULATION. THIS HEPARANASE UPREGULATION HELPS TO DRIVE SECRETION EXPRESSION AND SAW CREASE OF NUMBER OF MOLECULES. INCLUDING MMP 3 AND 7, THESE ARE ALSO SHED ACES SYNDECAN 1. THIS BECOMES IMPORTANT IN VIRAL EGRESS BECAUSE AS THE VIRAL PARTICLES ARE MADE AND SHED BACK THEY BIND BACK TO HEPARAN SULFATE ON SELL CELL SURFACE AND HAVE TO BE RELEASED OUT INTO THE ENVIRONMENT. TO DO THAT RELEASE THEN, THE MMP 3 AND 7 HAVE CAUSED THE SHEDDING OF SYNDECAN 1 AND YOU CAN SEE THIS IS A SHED PARTICLE, THIS IS SHED PROTEOGLYCAN WITH BOUND VIRUS. AND ALSO THE HEPARANASE OUT IN THE MICROENVIRONMENT CAN DEGRADE HEPARAN SULFATE CHAINS SUBSEQUENTLY RELEASING VIRAL PARTICLES. SO THEY WERE ABLE TO SHOW THE MECHANISM IN CANCER THIS IDEA THAT HEPARANASE INDUCES EXPRESSION OF PROTEASE AT LEAST SHEDDING, ALSO CAN BE IMPORTANT IN VIRAL EGRESS. SO WE THINK WILL MAYBE VERY IMPORTANT ROLES FOR THIS MECHANISM OF HEPARANASE AND SHED SYNDECAN 1 IN MULTIPLE DISEASE PATHOLOGIES. SYNDECAN 1 SHEDING IS SHOWN TO ENHANCE VIRULENCE OF SOME BACTERIA, MUCH WORK HEAR DONE BY (INAUDIBLE) AND HIS LAB AT BOSTON CHILDREN'S AND WE THINK THAT THIS AGAIN, THE WHAT IS FOUND IN BACTERIAL INFECTION, WHAT'S FOUND IN VIRUS AND WHAT'S FOUND IN CANCER SUGGESTS THIS MIGHT BE A GENERAL MECHANISM THAT CONTRIBUTES TO PATHOGENESIS OF MANY DISEASES. PARTICULAR WILL I IMPORTANT IN TODAY'S DISCUSSION WITH ALL THE SARS COV-2 WORK, THIS MIGHT BE A MECHANISM THAT NEEDS TO BE EXPLORED. HEPARANASE INDUCE SHEDDING OF SYNDECAN INVOLVED IN SOME OF THE MECHANISMS OF COVID-19 PATHOGENESIS. MORE IMPORTANTLY WOULD HEPARANASE INHIBITORS IN BLOCK SOME OF THESE MECHANISMS, PERHAPS INTERFERE WITH PATHOGEN HOST INTERACTIONS LEADING TO DIMINISHED PATHOGENESIS IN THESE DISEASES. IN CONCLUSION I WILL SAY THERE WERE A NUMBER OF PEOPLE INVOLVED OVER THE YEARS IN THESE STUDIES. IT'S NOT ONLY FOLKS IN MY LAB BUT CLEARLY VERY IMPORTANT COLLABORATORS PARTICULARLY -- AND ISRAEL HAVE BEEN CRITICAL TO OUR SUCCESS WITH THIS WORK. AND CERTAINLY FUNDING FROM MULTIPLE AGENCIES AND THE NATIONAL CANCER INSTITUTE FUNDED DIFFERENT ASPECTS OF THIS WORK CONSISTENTLY SINCE EARLY 1990s. THANK YOU FOR YOUR ATTENTION. >> THANK YOU VERY MUCH DR. SANDERSON. VERY INTERESTING TALK. ONE A LITTLE CLOSE TO MY HEART SINCE I AM IN THE NATIONAL CANCER INSTITUTE. >> THANK YOU. >> WE HAVE GOT ONE QUESTION, WHAT IS KNOWN ABOUT THE ENDOGENOUS MECHANISMS FOR UPREGULATING HEPARANASE? >> SO THERE ARE NUMBER OF DIFFERENT THINGS WE KNOW CAN UPREGULATE REPNASE, TO SUMMARIZE THEM, WHAT HAS BECOME CLEAR AT LEAST TO ME OVER THE YEARS IS HEPARANASE IS A STRESS RESPONSE MOLECULE. WHEN YOU STRESS A CELL IN MANY DIFFERENT WAYS FOR EXAMPLE WE LEARNED THAT CHEMOTHERAPY WHEN YOU TREAT A CANCER CELL WITH A CHEMOTHERAPEUTIC DRUG CELLS BECOME STRESSED AND HEPARANASE IS UPREGULATED AND SECRETED RATHER RAPIDLY BEFORE THE CELL DIES. SO WE THINK IT'S A NUMBER OF THINGS THAT INVOLVED IN STRESS RESPONSE PROBABLY NOT ONLY IN CANCER BUT ALSO IN INFLAMMATORY DISEASE, MAYBE ALSO IN SOME OF THE VASCULAR INSULTS LIKE SEPSIS. ALSO IN VIRAL PATHOGENESIS, IT'S KNOWN FOR EXAMPLE THE VIRUS STORY THAT THERE'S CERTAIN MOLECULES RELEASED BY THE VIRUS THAT DRIVE HEPARANASE EXPRESSION SECRETION AS WELL. >> OKAY. I HAVE ANOTHER QUESTION. BESIDES MMP 9 ARE OTHER MMPs INVOLVED IN THE PROCESS AS WELL? >> YEAH. IF YOU TALK ABOUT THE PROCESS OF SYNDECAN 1 SHEDDING CERTAINLY. IN FACT IN THE VIRAL STORY THAT -- DEVELOPED, HE SEES MMP 3 AND MMP 7 AS IMPORTANT SHED ACES AND THE OTHER SHED ACES AS WELL SO IT'S DEFINITELY NOT JUST MMP 9 THAT CAN SHED SYNDECANS. MYELOMA CELLS WE THINK THAT IS THE MAJOR FACTOR OF SHEDDING BECAUSE WE CAN DO AN IN INHIBITION OF MMP 9 AND WE REMOVE THE SHEDDING PROCESS. OTHER CANCERS AND OTHER SYSTEMS, OTHER MMP METRICS MIGHT HAVE A PROTEONASES ARE INVOLVED DEFINITELY IN SHEDDING. >> THANK YOU. ALL RIGHT. WE ARE ABOUT ONE OR TWO MINUTES AHEAD OF SCHEDULE. WHICH HYALURONAN I THINK SINCE WE DON'T HAVE ANY OTHER QUESTIONS COMING IN, MAYBE WE'LL MOVE ON TO DR. PAUL NOBLE. FROM CEDAR SINAI. ALL YOURS. >> THANK YOU, SIR. APPRECIATE THE OPPORTUNITY TO PARTICIPATE IN THIS SYMPOSIUM. GOING TO CONTINUE ALONG THE DISEASE FRONT. I'M GOING TO TALK ABOUT THE ROLE OF HYALURONAN SYNTHASE IN PULMONARY FIBROSIS. I'M A PULMONOLOGIST AND PHYSICIAN INVESTIGATOR AND SPECIALIZE IN DISEASES THAT CAUSE SCARRING OF THE LUNGS. AND FIBROGENESIS IS A MAJOR SOURCE OF MORBIDITY AND MORTALITY WORLDWIDE. THERE ARE VERY FEW SUCCESSFUL ANTI-FIBROTIC APPROACHES. THERE'S TWO MEDICATIONS APPROVED TO SLOW THE PROGRESSION OF PULMONARY FIBROSIS IN LUNG DISEASE. BUT WORLD WILD IT'S A MAJOR PROBLEM. THE INCIDENCE OF SEVERE PULMONARY FIBROSIS, THIS IS SCARRING OF THE LUNGS THAT PROGRESSES IN PATIENTS TYPICALLY SUCK COME TO THE DISEASE IF UNABLE TO GET A LUNG TRANSPLANT, IT'S BEEN RISING IN INCIDENCE OVER THE LAST DECADE. IN FACT IDIOPATHIC PULMONARY FIBROSIS, THE MAJOR CAUSE OF DEATH IN FIBROSIS PATIENTS RIVALS MANY CANCERS YOU CAN SEE HERE IN TERMS OF AVERAGE -- FIVE YEAR MORTALITY IS VERY HIGH IN PATIENTS WHO HAVE PULMONARY FIBROSIS. THIS IS A CHEST CAT SCAN OF SOMEBODY WHO HAS IDIOPATHIC PULMONARY FIBROSIS, IT'S A DISEASE TA THAT CAUSES UNUSUAL PATTERN OF SCARRING, AFFECTS OVER A HUNDRED THOUSAND PATIENTS IN THE US. THERE'S TWO FDA MEDICATIONS BUT LUNG TRANSPLANT REMAIN IT IS ONLY CURE AT THIS POINT IN TIME. ONE OF THE THINGS WE SOMETIMES HAVE PATIENTS WHO GET EARLY CAT SCANS EARLY IN THE DISEASE PROCESS AND THIS IS WHAT THE LUNG LOOKS LIKE. YOU HAVE NORMAL LUNG, THEN YOU HAVE ESTABLISHED SCAR AND YOU HAVE WHAT WE REALLY BELIEVE IS THE HOLY GRAIL IN THE MIDDLE. IT'S ALMOST LIKE MESENCHYMAL TUMOR GROWING. I'M GOING TO TALK TO YOU ABOUT THIS OVERLYING ALVEOLAR EPITHELIAL CELLS, TYPE 2 ALVEOLAR EPITHELIAL CELLS, RESPONSIAL FOR MAKING SURFACTANT, THAT'S WHY WE CAN TAKE A TEAM BREATH. WHAT HAPPENS IN THIS DISEASE IS THESE CELLS PREMATURELY SENESCE AND THE MESENCHYME GETS ACTIVATED LIKE A SMALL TUMOR GROWING IN THE INTERSTITIUM OF THESE PATIENTS SUFFERING FROM THIS DISEASE. THIS IS WHAT THE LUNG LOOKS LIKE ADVANCE STAGE, THIS IS A PATIENT OF MINE WHO UNDERWENT A LUNG TRANSPLANT. YOU CAN SEE THE LUNG IS COMPLETELY REPLACED WITH SCAR TISSUE. MAKING IT IMPOSSIBLE FOR GAS EXCHANGE TO HAPPEN. SO WE BELIEVE THERE ARE TWO FUNDAMENTAL DEFECTS IN THIS DISEASE PROCESS. INTERESTINGLY BOTH OF THEM WE BELIEVE INVOLVE HYALURONAN. I MENTION THESE ALVEOLAR TYPE TWO CELLS, SURFACTANT MAKING CELLS. THEY PRE-MATURELY SENESCE IN PATIENTS WITH IDIOPATHIC PULL NO MARE FIBROSIS, THERE'S A GENETIC FORM WHICHTY ALMOST RACE ABNORMALITIES IN GENES ARE IDENTIFIED LEADING TO PREMATURE DEATH OF ALVEOLAR TYPE TWO CELLS. THERE'S ALSO A STRESS RESPONSE INVOLVED AND WHEN THESE CELLS ULTIMATELY SUCCUMB THE UNDERLYING MESENCHYME GETS ACTIVATED. I WILL FOCUS ON PROTECTIVE ROLE WE THINK HYALURONAN AND SYNTHASE PLAYS IN THE NORMAL LUNG AND HOW THIS IS LOST IN IPF, THAT'S GOING TO BE WHAT I WILL TALK ABOUT TODAY. WE ALSO HAVE A BODY OF WORK SHOWING THAT ACTIVATION OF HYALURONAN AND SYNTHASE TWO PROMOTES FIBROSIS IN THESE PATIENTS BUT I WON'T TALK ABOUT THAT IN THE INTEREST OF TIME. WE HAVE BEEN INTERESTED IN HYALURONAN FOR TWO DECADES, IT'S PRODUCED ALMOST EVERY ORGAN FOLLOWING INJURY. HYALURONAN IS PRODUCED, IT CAN PROMOTE INFLAMMATION, IT'S FUNDAMENTALLY IMPORTANT IN TUMOR GROWTH AND METASTASIS. SOME YEARS AGO NOW, WE BECAME INTERESTED IN ITS ROLE IN PULMONARY FIBROSIS. WHEN WE ENTER THE LUNG AND CAUSE INJURY INFLAMMATION AND FIBROSIS, THIS IS WITH GLIOMYSIN. HYALURONAN ACCUMULATES IN L A TREMENDOUS WAY IN THE NORMAL WILD TYPE MOUSE HYALURONAN ACCUMULATE AND IS CLEARED BUT WE PUBLISHED SOME YEARS AGO NOW THAT ADHESION MOLECULES CD4 4 WHICH IS A LIGAND FOR HYALURONAN IS IMPORTANT IN THE CLEARANCE OF THE FRAGMENTED HA FROM THE INFLAMED LUNG, IT'S ALSO INVOLVED IN SOME OF THE FIBROBLAST FUNCTION. SO WE HAVE BEEN INTERESTED IN THIS ROLE OF HYALURONAN FOR SOME TIME NOW. SOME YEARS AGO NOW, WE IDENTIFIED A FUNCTION FOR HYALURONAN ON THE ALVEOLAR LINING CELLS, THESE TYPE TWO CELLS. THESE TYPE TWO CELLS RESPONSIBLE FOR MAKING SURFACTANT ALSO EXPRESS INNATE IMMUNE RECEPTORS. TLR 2 AND 4. WHAT WE SHOWED SOME YEARS AGO IS THAT NORMALLY HYALURONAN WILL BIND TO THESE INNATE IMMUNE RECEPTORS AND THEY PREVENT ALVEOLAR EPITHELIAL CELL FROM INJURY. WHAT HAPPENS DURING TISSUE INJURY AND REPAIR IS THIS GETS FRAGMENTED. AND THESE BREAK DOWN PRODUCTS OF HYALURONAN INTERACT WITH INFLAMMATORY CELLS AND PROMOTE INFLAMMATION. WHAT I'M GOING TO TALK ABOUT TODAY IS SUBSEQUENT STUDIES TO BETTER UNDERSTAND THE PROTECTIVE ROLE OF CELL SURFACE HYALURONAN ON ALVEOLAR TYPE 2 CELLS IN PULMONARY FIBROSIS. THE QUESTIONS ARE WHY DO TYPE 2 CELLS HAVE HYALURONAN ON CELL SURFACE. TLR 4 IS A MOLECULE THAT'S RESPONSIBLE FOR RECOGNIZING BACTERIAL TOXINS LIKE LIPO POLYSACCHARIDE, INVOLVED IN HELPING TO FIGHT OFF INFECTION SUCH AS STAPHYLOCOCCUS. WHAT ARE SIGNALS GENERATED BY THIS INTERACTION? WHAT ARE THE CONSEQUENCES OF INTERRUPTING IT AND IS THIS RELEVANT TO HUMAN DISEASE? ONE OF THE THINGS WE HAVE DEVELOPED OVER LAST FEW YEARS WITH COLLEAGUES, COLLEAGUE OF MINE BARRY STRIPP A COLLEAGUE OF MINE AT DUKE AND HE'S BEEN MOVED OUT HERE TO LOS ANGELES WITH US, HE'S BEEN INSTRUMENTAL IN DEVELOPING THE ABILITY TO PURIFY PRIMARY ALVEOLAR TYPE 2 CELLS FROM NOT ONLY THE MOUSE LUNG BUT ALSO HUMAN LUNG. THIS IS A SLIDE SHOWING JUICING ALVEOLAR TYPE 2 CELL LABEL WITH GFP USING THE SELECTION PROCESS WE CAN PURIFY TYPE 2 CELLS THEN STUDY BIOLOGICAL FUNCTION. WHEN WE INJURE THE LUNG WHAT HAPPENS IS THE TYPE 2 CELLS ARE KILLED AND THEN THEY START TO REGENERATE. THIS REGENERATIVE PROCESS IS ESSENTIAL FOR REMITTING FIBROSIS. WHEN THIS RENEWAL PROCESS IS IMPAIRED, THE FIBROSIS PROGRESSES. SO AS I MENTION ALVEOLAR TYPE TWO CELLS EXPRESS TLR 4. VERY LITTLE EXPRESSION OF THIS INNATE IMMUNE RECEPTOR ON FIBROBLASTS BUT VERY POTENT OF COURSE ON MACROPHAGES BUT UNEXPECTEDLY WE FOUND EXPRESSION ON TYPE 2 CELLS WE TOOK MICE DEFICIENT IN TLR 4. THIS WAS GLOBAL KNOCK OUT. WHEN WE INJURED THEY WERE SUSCEPTIBLE TO FIBROSIS. ACCUMULATED MORE COLLAGEN FOLLOWING INJURY. INTERESTINGLY, I SHOWED YOU THAT PHYSIOLOGIC CURVE FOR INJURY AND REGENERATION OF TYPE 2 CELLS IN WILD TYPE YOU SEE BY TWO WEEKS THERE'S A SUBSTANTIAL REPOPULATION OF THE INJURE TYPE 2 CELLS. WE KNOW ALVEOLAR TYPE TWO CELLS ARE PROGENITOR IN THE DECEMBER TALL SPACE. THESE PROGENITOR CELLS CAN REPAIR, IT'S ESSENTIAL FOR RECOVERY FROM FIBROTIC INJURY FOR THEM TO REPAIR. IN ABSENCE OF TLR 4 THIS REGENERATIVE PROCESS WAS DELAYED. AND IMPAIRED SUGGESTING TO US THERE MIGHT BE A ROLE FOR HYALURONAN AND TLR 4 IN ALVEOLAR PROGENITOR CELL RENEWAL. DURING MY TIME AT DUKE, WE COLLABORATED WITH BRIGE TEXASE HOGAN. SHE IS RETIRED BUT ONE OF THE LEGENDS OF DEVELOPMENTAL BIOLOGY IN THE LUNG. SO WHAT KRISTINA BARSCOSCIS A PULMONOLOGIST WORKING IN -- JOURNAL OF CLINICAL INVESTIGATION SHOWED YOU COULD GROW LUNG IN A DISH. BY ISOLATING PURIFIED TYPE 2 CELLS AND PURIFIED MESENCHYMAL FETAL CELLS. USING NORMAL MOUSE CELLS YOU CAN FORM THIS LITTLE ALVEOLUS. WHAT WE FOUND USING IN THIS COLONY FORMING ASSAY, WHICH IS A SURROGATE FOR PROGENITOR CELL RENEWAL, IN THE ABSENCE OF TLR 4 ALVEOLAR TYPE 2 CELLS PURIFIED IN THIS MANNER WERE DEFICIENT IN IN THEIR ABILITY TO REGENERATE WE COULD AUGMENT THE NORMAL RENEWAL CAPACITY. OF TYPE 2 CELLS WITH EXOGENOUS HYALURONAN. THIS WAS NOT EFFECTIVE IN ABSENCE OF TLR 4 SUGGESTING IT WAS REQUIRED FOR THIS PROCESS. TO, EMPLOYER THIS WE SELECTIVELY DELETED HYALURONAN SYNTHASE 2, MA I YOUR SYNTHASE FOR HYALURONAN PRODUCTION IN THE LUNG. WE DELETED THESE MANY THE ALVEOLAR TYPE TWO CELLS. WHAT WE FOUND WAS THAT MICE WHO DEFICIENT IN CELL SURFACE HYALURONAN DEMON DEMONSTRATED BY FLOW PSYCHOLOGY INDUSTRY, THEY PRODUCE LESS HYALURONAN INTO THE ALVEOLAR FLUID. WHEN WE INJURED MICE THEY DEVELOPED SEVERE PROGRESSIVE FIBROSIS. MICE THAT DON'T HAVE HYALURONAN ON CELL SURFACE TYPE 2 CELLS MORE SUSCEPTIBLE TO FIBROTIC INJURY. WHEN WE LOOK AT RENEWAL CAPACITY OF THESE TYPE 2 CELLS IN OUR COLONY ASSAYS THE MICE THAT DIDN'T HAVE CELL SURFACE HA WERE DEFICIENT. INTERESTINGLY WE WERE ABLE TO SHOW THAT EXOGENOUS HYALURONAN REVERSE THIS PROCESS. SO WE ARE INTERESTED IN HUMAN FIBROSIS. WHICH DEVELOP ABILITY LARGE PART BECAUSE OF DR. BARRY STRIP'S WORK TO PURIFY TYPE 2 CELLS FROM RUNG TRANSPLANTS FOR IDIOPATHIC PULL NO MARE FIBROSIS. THERE WAS TREMENDOUS LOSS OF PROGENITOR CELLS, IN THE LUNGS OF PATIENTS SUCCUMBING TO IDIOPATHIC PULL NONARY FIBROSIS. AND WHAT WAS FASCINATING, WE LOOKED TO SEE WHAT THE EXPRESSION OF HYALURONAN WAS ON NORMAL TYPE 2 CELLS AND TYPE 2 CELLS FROM IPF PATIENTS. WE FOUND A DRAMATIC REDUCTION IN CELL SERVICE HYALURONAN. IN PATIENTS WITH IPF. SIMILAR IN OUR CONDITIONAL KNOCK OUT. OF HAS 2 ALVEOLAR TYPE 2 CELLS. THERE WAS AN IPF A TREMENDOUS REDUCTION MANY THE CELL SURFACE HA. INTERESTINGLY HAS TWO MRNA EXPRESSION WAS ALSO REDUCED. USING COLONY FORMING AWE SAYS WE SHOED PATIENTS WITH IPF HAVE REDUCED PROGENITOR CELL FUNCTION IN ADVANCED STAGES OF THE DISEASE. SO WE WANTED TO SEE WHAT THE ROLE OF HYALURONAN WAS IN THIS PROCESS. SO WHAT WE FOUND WAS IN THE NORMAL LUNG 80% TYPE TWO CELLS EXPRESS HYALURONAN ON THE CELL SURFACE. BUT THERE IS A SUBSET THAT ARE LOW EXPRESSING. THIS RATIO IS REVERSED IN DISEASE. EVEN FROM NORMAL LUNG TYPE TWO CELLS LOW EXPRESSING ON CELL SURFACE IMPAIRED PROGENITOR CELLS REGENERATIVE CAPACITY. WE FOUND WE COULD PARTIALLY RESTORE THIS IN PATIENTS WHO HAVE IDIOPATHIC PULL NO MARE FIBROSIS. IN IF INTEREST OF TIME I WON'T GO INTO ADDITIONAL MECHANISM IN THIS STUDY, HYALURONAN BINDS WITH TLR 4 ON CELL SURFACE OF TYPE 2 CELLS AND THEY PRODUCE NUTRIENTS AND CYTOKINES THAT PROMOTE PROGENITOR CELL FUNCTION. THE MAIN ONE WAS IL 6 WE DEMONSTRATED IN OUR MOUSE MODEL WHERE EXPRESS IS REDUCED REDUCED THE IN TYPE 2 CELLS AND PATIENTS THEY WERE UNABLE TO PRODUCEICSL 6, WHEN WE RESTORE IL 6 OR ELIMINATED WITH ANTIBODIES WE CAN MODULATE FATE OF PULMONARY FIBROSIS. THIS SUGGESTS WE HAVE IDENTIFIED A NOVEL MECHANISM WHICH THE NORMAL TYPE 2 CELLS REPAIRS ITSELF AFTER INJURY. AND THAT THIS MECHANISM IS DEFICIENT IN PATIENTS WITH PULMONARY FIBROSIS. CURRENT EFFORTS ARE TO BETTER UNDERSTAND THIS PATHWAY TO IDENTIFY MECHANISM WHICH IS WE COULD RESTORE CELL SURFACE HYALURONAN EXPRESSION. SO I WOULD LIKE TO THANK MY LONG TIME COLLABORATORS CAROL YANG AND THE FOLKS THAT ARE RESPONSIBLE FOR THIS WORK AND WE HAVE HAD THE GOOD FORTUNE OF FUNDD BY NIH FOR THIS AS WELL. THOUGH WE HAVE FIRES NOW IN LOS ANGELES WE ARE LOOKING FORWARD TO RESTORING BLUE SKIES AND 72 DEGREES. THANK YOU VERY MUCH. >> THANK YOU VERY MUCH, DR. NOBLE. SO YOU ARE RIGHT ON TIME. WE HAVE A FEW MINUTES TO WAIT AND SEE IF QUESTIONS COME IN. FOR ANYBODY ELSE OUT THERE YOU CAN TAKE A QUICK FIVE MINUTE BREAK, WE HAVE UNTIL 2:20. TO START THE NEXT TALK. IF YOU NEED TO RUN OUT THIS IS YOUR ONE CHANCE THIS AFTERNOON WHERE YOU CAN DO IT. DO STAINING OF HA DISTINGUISHED BETWEEN HA OF DIFFERENT MOLECULAR WEIGHT. THE SECOND QUESTION, DO THEY EXPECT CHANGES OF PROTECTIVE EFFECTS WITH HA SUPPLEMENT OF DIFFERENT MOLECULAR WEIGHT HA? >> GREAT QUESTION. UNFORTUNATELY TO MY KNOWLEDGE NONE OF THE STAINING TECHNIQUES DIFFERIATE HA MOLECULAR WEIGHT. YOU CAN OF COURSE WE AND OTHERS HAVE SHOWN THAT IN INFLAMMATION THERE IS AN INCREASE PREPONDERANCE OF LOWER MOLECULAR WEIGH HA SPECIES THAT WE THINK MAY CONTRIBUTE TO CHRONIC INFLAMMATION. SO IT'S INTERESTING THAT THERE IS A THERAPEUTIC APPLICATION OF HIGH MOLECULAR WEIGHT, HYALURONAN INTO THE JOIN SPACE FOR OSTEOARTHRITIS. IT'S BEEN SHOWN TO HAVE SOME BENEFIT IN THAT DISEASE PROCESS. IT'S INTERESTING WE DO THINK THAT THE HIGH MOLECULAR WEIGHT IS PROTECTIVE. IT'S BETTER BINDING TO CELL SURFACE TLR 4. SO WE THINK THAT PROTECTIVE MECHANISM IS PROBABLY LOST IN INFLAMMATION AND WE THINK THIS CONCEPT MIGHT ALSO BE APPLICABLE TO OTHER CELL TYPES SUCH AS IN OSTEOPOROSIS. >> ANOTHER QUESTION THAT CAME IN. WHAT CAUSES HA DEGRADATION IN IPF? GREAT QUESTION. THERE'S INCREASED EXPRESSION OF HYALURONANASEs, BUT LOW PH. THERE'S SOME CASES HYALURONAN, MOSTLY IT'S OXIDATIVE STRESS, COMBINATION OF NUTRITION AND OXIDATION BOTH WHICH HAVE BEEN SHOWN TO OCCUR IN IPF, THERE'S A TREMENDOUS INCREASE IN THE OX DATIVE STRESS BURDEN. THAT'S PROBABLY THE MAJOR MECHANISM. YEARS AGO WE SHOW THAT SOD COULD INHIBIT THE BREAK DOWN OF HA IN ANIMAL MODELS OF PULMONARY FIBROSIS AN AN AMELIORATE THE DISEASE PROCESS. SO WE THINK ANTIOXIDANT APPROACH IS BENEFICIAL. THAT'S A LITTLE BIT OF DATA THAT IN ACETYLSISTINE IS A MODEST BENEFIT BUT WE DON'T THINK THAT'S THE RIGHT ANTI-OXTANT. -- ANTIOXIDANT. >> THANK YOU VERY MUCH, DR. NOBLE. LOOKS LIKE THAT MIGHT BE IT FOR THE QUESTIONS. LESS THAN A MINUTE LEFT SO WE CAN PROBABLY MOVE TO OUR NEXT SPEAKER DR. XIAOFANG WANK. >> I APPRECIATE THE OPPORTUNITY FOR GIVING ME THIS OPPORTUNITY TO PRESENT OUR RECENT WORK. I'M NOT A GLYCO BIOLOGIST. MY RESOURCE IS INVOLVED IN -- (INAUDIBLE) (OVERLAPPING SPEAKERS) HAPPY TO SHARE MY FUNDING WITH ENVIRONMENT ABOUT THE ROLE OF GLYCANS IN GLYCANS. IN THE PAST 20 YEARS ABOUT -- WE HAVE ACCUMULATED PRETTY MUCH KNOWLEDGE ABOUT THE GROWTH FACTORS AND TRANSCRIPTION FACTOR S IN -- BOTH ORGANS ARE VERY EARLY STAGE INTERACTION BETWEEN THE EPITHELIUM AND THE MESENCHYME. IT'S VERY INTERESTING TO LOOK AT THE EXTRA CELLULAR INPUTS INTO THIS PROCESS WHICH I SHOW YOU TODAY. SO FIRST SECTION I WOULD LIKE TO INTRODUCE THE ROLE OF GAG CHAINS IN DATA REMISSION OF THE MONOP PHYODONT PHENOTYPE IN MICE BY RESTRICTING FGFR 2B SIGNALING. FAM 20B IS A PHYLOS KINASE WHICH PHOSPHORYLATES THE FILOSE WHICH IS IN THE REGION GAG CHAIN. PHOSPHORYLATION OF XYLOSE IS ESSENTIAL FOR LINK FORMATION AN SUBSEQUENT EXTENSION OF THE GAG CHAIN. WITHOUT FARM 20B WE CANNOT PHOSPHORYLATE XYLOSE AND WE HAVE MOST GAG CHAINNAL -- THAT'S BASICALLY THE CONSEQUENCE OF F FAM 20B KNOCK OUT. SO BEFORE I GO INTO THE TOPIC HERE ARE SOME BACKGROUND KNOWLEDGE ABOUT TOOTH REPLACEMENT IN CASE MOST OF YOU MAY NOT BE USED TO HAVE SOME GENERAL IDEA WHAT I'M TALKING ABOUT. SO TOOTH REPLACEMENT IS ACTUALLY -- DURING EVOLUTION TO KEEP THEIR DENTATION, RABDO AND FISH, THEY REPLACE THROUGHOUT THEIR LIFE SO THEY HAVE MULTIPLE DENNATIONS. BUT HIGHER VERTEBRATES SUCH AS MAMMALS HAVE VERY LIMITED TOOTH REPLACEMENT CAPACITY. MOST OF ONLY REPLACE ONCE IN LIFETIME SO THEY -- AT THAT TIME THE DENTAL LAMINAR WILL ORIGINATE FROM THE ORIGINAL DENTAL LAMINAR AND FORM NEW REPLACEMENT IN THE TOOTH PRIMARY TOOTH. SO THAT'S WHAT HAPPENED IN THE TOOTH REPLACEMENT. BUT FOR RODENTS THEY DO NOT REPLACE TEETH. AND THEY HAVE REDUCED NUMBER OF TOOTH NUMBERS. HERE THEY DON'T HAVE THE PRE-MOLARS SO THEY RENEW THEIR BY HUGE UPPER AND LOWER MANDIBLES. SO RODENTS ESPECIALLY MOUSE ARE CALLED MONOPHYODONT ANIMALS. WHEN WE KNOCK OUT -- IN ORDER WHEN WE DISRUPT THE GAG CHAIN FORMATION IN THE DENTAL EPITHELIUM, WE FIND THAT WE REVIVE REPLACEMENT TOOTH FORMATION IN MICE. YOU CAN SEE TWO REPLACEMENT TOOTH FROM MEDIUM AND SIDE OF THE LOWER AND UPPER INSIDER -- INSIZERS. THIS REPLACEMENT IS SPECIFICALLY ASSOCIATED WITH THE DENTAL EPIHELIUM GLYCAN BUT NOT DENTAL -- IMMUNOGLYCAN. IN LOWER MICE WE CAN SEE THIS IS THE TRANSSECTION OF THE INSIZER, DIFFERENT TAKES DURING THE DEVELOPMENT. WHEN WE KNOCK OUT 120B FROM THE EPITHELIUM, STARTING FROM -- STAGE, WE CAN SEE THERE'S A NEW ECTOPIC DENTAL EPITHELIUM FROM THE MESO SIDE OF THE PRIMARY ORGAN AND WITH TIME THOSE ALONG YOU CAN SEE SUCCESSION OF DENTAL LAMINAR FORM AND TO THE END YOU HAVE NEW ORGAN AND FOUND REPLACEMENT PIECE. BUT WHEN WE KNOCK OUT FROM DENTAL MESENCHYME THE TOOTH NUMBER DID NOT CHANGE THOUGH THERE'S MANY OTHER CRANIOFACIAL DEFECT BUS THE TRUE NUMBER IS COMPLETELY NORMAL. NOW WE'LL TRY TOPHI GUSH OUT WHAT'S GOING ON HERE. THE TRYING TO FIGURE OUT WHAT'S GOING ON HERE. FIRST LOOKING TO WHAT'S THE APOPTOSIS IN THE KNOCK OUT PIECE. HERE THE RIGHT INDICATE THE -- YOU CAN SEE MUCH MORE PROLIFERATION IN KNOCK OUT. IN BOTH DENTAL EPITHELIUM WHICH IS PLOTTED BY THE -- ALSO IN THE DENTAL MESENCHYME WHICH IS SURROUNDING THE DENTAL EPITHELIUM. SO FROM THIS WE CAN TELL OCCURS WAY EARLY AT CERTAIN POINT WHICH MEAN THE CELL CHANGE HAS OCCURRED BEFORE THIS STAGE, VERY INITIAL STAGE OF -- ARE THE ARE THEY DATA MINED. NOW WE TRY TO FIGURE OUT THE EXACT TIMING FOR THIS CHANGE. SO WE TO THIS END WE USE SYSTEM TO KNOCK OUT TIME COURSE MANNER. WHAT WE FOUND HERE IS THAT THE REPLACEMENT PIECE CAN BE ONLY WHEN WE KNOCK OUT 120B EARLY STAGE BETWEEN TEN POUND FILE AND 12-POUND FILE. KNOCK OUT AFTER THIS STAGE OR LATER THAN THIS TIME WINDOW WILL NOT CAUSE REPLACEMENT TOOTH FORMATION. SO THAT MEANS SULFATE CONSISTENT WITH OUR PROLIFERATION AND SURFACE HAS BEEN -- VERY INITIAL STAGE OF THE TOOTH DEVELOPMENT. SOX 2 EXPRESSION CELLS I BELIEVE DENTAL EPITHELIAL PROGENITOR OR STEM CELLS WITH CAPACITY FOR REPLACEMENT TOOTH FORMATION. IN THIS CASE SERIAL AFTER SOX 2 HOMEOSTATIC WE HAVE THE -- THAT GOES ALONG SOX 2 EXPRESSION FADE AWAY FROM THE WILD TYPE ORGAN BUT IN THE (INAUDIBLE) B KNOCKOUT MICE, THE SOX 2 EXPRESSION RETURNS HERE AND HE CAN TOMMICLY -- ECTOPIC EVEN AT THE LATER STAGE. SO THAT MEANS SOX 2 HOMEOSTATUS HAS PROBLEM WITH KNOCKOUT MICE. THEN WE DO A TC CLEARANCE AND LINEAGE FACING SPECIFICALLY FOR SOX 2 MARKER, YOU CAN SEE THE SOX 2 CELL ECTOPIC HOMEOSTATUS IN THE KNOCKOUT MICE COMPARED WITH THE NORMAL MICE. SO THAT MEANS THE GAGS CONFINED HOMEOSTATUS OF THE SOX 2 POSITIVE CELLS IN KNOCKOUT MICE. THE LAST QUESTION WE TRY TO ASK IS IF THE GAG CHAIN SPECIFICALLY REQUIRE BY SOX 2 EXPRESSING CELLS TO THIS ON THE WAY SOX 2 CRE ARE TO KNOCK OUT SPECIFICALLY FROM SOX 2 EXPRESSING STEM CELLS. SO THIS IS THE -- OF THE SOX 2 EFFICIENCY. THIS IS THE KNOCK OUT EFFECTS IN THE SOX 2 CREER COMPARE WITH THE STEMMING OF SOX 2 STEMMING SO FULLY EFFICIENCY THE GOOD ENOUGH TO DO DEDEPRIVE EXPRESSION OF B GENE FROM EPITHELIUM. SO THE RESULT OF THIS KNOCK OUT MOUSE TURN OUT DEPRIVING THE SOX 2 FROM SOX 2 EXPRESSION, EXPRESSION CELLS, DOES NOT CAUSE TOOTH NUMBER CHANGE. THAT MEANS THE GAG RESTRICTS SOX 2 HOMEOSTASIS IN NORMAL MANNER. SO IN OTHER WORDS GAG FROM OTHER CELLS RESTRICT SOX 2 HOMEOSTATUS. THEN WE TRY TO FIGURE OUT SIGNAL PHENOTYPE WE SYSTEMATICALLY SCREEN SIGNALING PATHWAYS AT INITIAL STAGE, WHAT WE FOUND HERE IS ROBUST UPREGULATION OF 9 KNOCKOUT MICE AND THE MOST IMPORTANT EPI EPIHELIUM AND MESENCHYME, THIS CONVERGE TO KNOCK OUT MOUSE HAVE ECTOPI AND HYPERACTIVITY FGF SIGNALING. CONSISTENT WITH THESE RESULTS WE FOUND FGF DOWNSTREAM SIGNALING WHICH IS SONIC HEDGEHOG ALSO UPREGULATED. YOU CAN SEE THE EXPANDING TERRITORY OF SONIC HEDGEHOG ON BOTH LOWER AND UPPER MANDIBLE IN THE LOCATION OF THE FUTURE FORMATION SITES. CONSISTENT WITH THIS, DOWNSTREAM MARKERS DO SONIC HEDGEHOG WHICH IS GLE 1 AND PATCH 1 YOU CAN SEE BOLDER RESPONSIVE RESPONSIVE TERRITORY IN THE KNOCKOUT MICE COMPARED TO THE WILD TYPE, SIMILAR WITH GLIAL 1 RESPONSIVE TERRITORY WHICH IS INDICATED BY THE GLE 1 MICE. BECAUSE THERE ARE MANY FGF AND FGF RECEPTORS EXPRESSED IN THE EARLY STAGE TOOTH DEVELOPMENT, SO THAT'S THE -- SOMETHING WE NEED TO CONSIDER WHICH IS THE CANDIDATE FOR THIS OR ACTIVATION. WE DID MANY SCREEN AND EXCLUDED SOME FGF CANDIDATES BASED ON EXPRESSION TIMING UNDER LOCATION. SO WE TARGETED FGF 2 BECAUSE AT THAT TIME VERY INITIAL STAGE OF TOOTH DEVELOPMENT, FGF IS THE DOMINANT FGF GROWTH FACTOR IN THE DENTAL MESENCHYME. AND INTERESTING PART IS FGF TURN RECEPTOR FGF OUGHT TO BE EXCLUSIVELY EXPRESSED IN THE DENTAL AXILLA AT THAT STAGE. SO THIS COMPLIMENTARY EXPRESSION PATTERN REQUIRES THE VEGF FROM THE DENTAL MESENCHYME TO THE DENTAL EPITHELIUM TO PERFORM AS FUNCTION. SO IN THIS CASE WE ARE TRYING TO PASS GENETICALLY IF WE CAN RESCUE THIS PHENOTYPE BY INHIBITING FGF R 2B SIGNALING. IN THIS WAY WE USE THE SYSTEM TO OVEREXPRESS DOMINANT NEGATIVE FGF INHIBITOR WHICH IS FGF R 2B EDGE EDGE AND WE SUCCESSFULLY RESCUED THE PHENOTYPE FULLY RESCUED OR PARTIALLY RESCUED AS SHOWN HERE. THE RESCUE TENDS TO BE HOW SIMILAR TREND AS WE INDUCE REPLACEMENT TOOTH FORMATION, THAT WILL BE THE EARLIER THE INHIBITION WILL HAVE BETTER RESCUE EFFECTS IN SIMILAR TIME WINDOW AS WE SHOWED IN THE INDUCTION KNOCK OUT EXPERIMENTS. SO AGAIN, THAT'S CONFIDENCE THAT PHENOTYPE WAS CAUSED BY FGFR 2B HYPERACTIVATION OF SIGNALING. NOW WE TRY TO FIGURE OUT WHAT IS GOING ON IN GAG PROFILING. DURING THIS KNOCK-OUT. SO WE COLLABORATED WITH -- TO DO GAG SPECIFICALLY IN THE GUT EPITHELIUM, AS YOU CAN SEE THE KNOCKOUT MICE HAS DRAMATICALLY REDUCED THE AMOUNT OF HS AND CS BUT THE TOTAL COMPOSITION DID NOT CHANGE THERE'S SOME SUBTLE CHANGE IF YOU PREFER FURTHER OLIGOSACK RIDES, THERE'S SOME DIFFERENCES HERE BUT OVERALL -- OLIGOSACCHARIDES BUT OVERALL THE CS DID NOT SHOW MUCH DIFFERENCE. TRYING TO FIGURE OUT THE DIFFERENCES SHOWN IN THE OLIGOSACCHARIDES WILL CHANGE THE FGF REACTIVITY IN CELL CULTURE. WE USE RESOURCE THAT IS SPECIFICALLY ENGINEERED FOR EXPRESSING FGFR 2B RECEPTOR AND LIABILITY OF THE CELL CULTURE WILL REFLECT FGF REACTIVITY SO IN THIS CELL SYSTEM, WE USE THIS GAG TO TEST IF ANY OF THEM HAVE SYNERGISTIC OR INHIBITOR EFFECTS COMPARED TO HEPARAN WHICH IS USED AS POSITIVE CONTROL. AS YOU CAN SEE POSITIVE CONTROL HAVE DRAMATICALLY SYNERGISTIC HAVE GRADUALLY SUGGESTED THE FACT IN CELL CULTURE BUT NONE OF THIS PASSAGE GAG SHOW EITHER SYNERGISTIC EFFECT OR INHIBITOR EFFECTS. NOW WE TRY TO BUILD A HYDRO GEL CELL CHURL SYSTEM TO MIMIC THE ACTUAL CELLULAR MATRIX ENVIRONMENT TO SEE IF GAGS ARE DEFINED OF FGF DURING THIS PROCESS. SO HERE WE HAVE THE RESULT OF DIFFERENT CONCENTRATIONS OF GAG CHAINS WHEN AIDED THE GEL CULTURE SYSTEM AND LOWER CONCENTRATIONS WE DO SHOW DIFFERENTIAL CONFINING EFFECTS ON FGF 2. THIS EFFECTS PRETTY MUCH CONSISTENT WITH THE PREVIOUS SPR RESULTS BY DR. LEE'S LAB THAT THERE'S A PREFERENCE TREND BETWEEN DIFFERENT ATTEMPTS OF GAG CHAINS. SO OVERALL BECAUSE -- ONCE SELECTIVELY THE ACTIVITY -- DISTURB GAG FORMATION FOR BOTH GAGS. MOST LIKELY THIS IS A GENE EFFECT OF GAG CHAIN DYSFUNCTION. >> GOT ABOUT A MINUTE LEFT. >> SO AT LATER STAGE BASICALLY WE FOUND HYPERACTIVATION OF AS SHOWN BY BETA COMMUNICATOR AND DOWN REGULATION OF WNT INHIBITOR NOW AGAIN WE CAN RESCUE THIS PHENOTYPE BY OVEREXPRESSION OF DQQ 1. THAT MEANS THE UPREGULATION OF WNT SIGNALING IS ESSENTIAL BUT NOT ORIGINAL REPLACEMENT TOOTH REPLACEMENT IN MICE. FGF IN DENTAL MESENCHYME LEAD TO AXILLA TO PERFORM FUNCTION AND DENTAL REPLACEMENT TOOTH FORMATION. DURING THIS PROCESS GAG CHAIN PLAYS INHIBITOR ROLE FOR THIS PROCESS. AT LATER STAGE THE WNT SIGNALING IS ESSENTIAL REPLACEMENT FOR TOOTH FORMATION. AT CELLULAR ROLE, GAG CHAIN FOR FGF 2 DIFFUSION AT THIS TIME YOU HAVE LIMITED FGF SIGNALING TO BIAS BETWEEN RENEWAL AND THE DIFFERENTIATION OF THE SOX 2 STEM CELLS. WHEN YOU HAVE NO -- YOU HAVE MUCH MORE FGF SIGNAL THAT BREAK THE VIOLENCE AND YOU HAVE MORE ECTOPIC RENEWAL OF HOMEOSTASIS OF THE STEM CELL, YOU GET REPLACEMENT IN THE -- FOUND IN MICE. THE SECOND PART BRIEFLY, THIS IS THE -- STILL ON THE DEVELOPING PROJECT, WE I'LL BRIEFLY INTRODUCE WHAT WE FOUND IN THE KNOCKOUT MICE THAT -- (INAUDIBLE) IN THE LIMB BUD MESENCHYME. SO THIS -- >> WE PROBABLY WON'T HAVE TIME FOR THAT WE ARE STARTING TO RUN OVER NOW. SO YOU'LL HAVE TO WRAP UP. >> OKAY. LASTLY I WOULD LIKE TO THANK TO MY LAB MEMBERS AND MY COLLABORATORS AND ALSO FROM THE NIH. THANK YOU FOR ATTENTION. >> THANK YOU, DR. WANG. NOW WE ARE A MINUTE BEHIND. WE HAD A COUPLE OF QUESTIONS COME IN FROM PREVIOUS SPEAKERS. WE WILL HOLE THOSE OVER TO THE END. DR. LUO, YOU ARE WELCOME TO GET STARTED. DR. LUO YOU ARE STILL MUTED. >> SORRY. YEAH. I WOULD LIKE TO THANK THE COMMITTEE TO GIVE ME THIS OPPORTUNITY TO SHARE WITH YOU OUR RECENT IMMUNE STUDY ON CSPG AND PTP SIGNAL RECEPTOR PATHWAY IN NEUROREPAIR. I THINK I DON'T HAVE THE CONTROL. SO WE -- OUR LAB IS INTERESTED IN NEUROREPAIR MECHANISMS AND STROKE IS VERY GOOD MODEL TO STUDY THIS. FIRST OF ALL STROKE IS A VERY SIGNIFICANT MEDICAL PROBLEM FOR OUR SOCIETY. SEEN MT. UNITED STATES EACH YEAR THERE'S ABOUT 800,000 PEOPLE WHO EXPERIENCE A STROKE. AND ABOUT 807 ARE NEW STROKE CASES. IT'S ALSO THE LEADING CAUSE OF ADULT DISABILITY IN THE UNITED STATES. THE ONLY FDA APPROVED TREATMENT DRUG TREATMENT FOR STROKE IS RECOMBINANT TISSUE ACTIVITY TPA A THROMBO LYTIC AGENT THAT CAN REMOVE THE CLOT. HOWEVER, DUE TO SEVERE SEDATIVE AFFECT AND RAPID DEATH OF NEURON WHICH I WILL GET INTO, THIS TREATMENT IS ONLY RECOMMENDED WITHIN SIX HOURS OF STROKE ONSET. AS A RESULT, ONLY ABOUT 5% OF THE STROKE PATIENTS CAN GET THIS TREATMENT. MAJORITY OF THEM, THERE'S NOTHING WE CAN DO FOR THEM. SO THERE'S NO FDA APPROVED DRUGS THAT ARE AVAILABLE TO ENHANCE THE FUNCTIONAL RECOVERY AND REPAIR DURING THIS SUB ACUTE OR CHRONIC STAGE OF STROKE. THE LACK OF TREATMENT IS NOT DUE TO LACK OF EFFORT, IT'S BECAUSE RESCUING NEURON TURN OUT TO BE VERY CHALLENGING. IN THE CASE OF NEURONAL INJURY, NEURONAL DEATH INITIATE QUICKLY AND IT'S IRREVERSIBLE. SO SHOWN HERE, IS JUST SOME DATA SHOWING YOU HOW QUICKLY WE ARE LOSING THE NEURON AN SYNAPSES AND THE MYELINATED FIBERS, EVERY MINUTE WHEN PATIENT IS UNDERGOING STROKE. SO AS A RESULT, THOUGH THERE'S HAVE BEEN MORE THAN A HUNDRED DRUGS THAT HAS BEEN TESTED IN CLINICAL THEY ALL FAIL TO TRANSLATE TO CLINICAL TREATMENT WITH EXCEPTION OF TPA. THAT'S BECAUSE THE RAPID IRREVERSIBLE DEATH OF NEURON. HOWEVER, I WANT TO POINT OUT THAT NEURONAL REPAIR IS STILL ACTIVE DAYS EVEN WEEKS AFTER ONSET OF DISEASE. THEREFORE, THERE'S A WINDOW SECOND OPPORTUNITY FOR US, THERE MIGHT BE SOME HOPE. SO THERE ARE TWO MAJOR MECHANISMS FOR NEURONAL REPAIR. THE FIRST ONE IS AXONAL SPROUTING, SHOWN HERE NEURON IN CORTEX THAT PROJECT TO THE CONTRALATERAL SIDE CORTEX OR IPSA-LATERAL STRIATUM. IN THE CASE OF STROKE LAST OXYGEN AND GLUCOSE BY ISCHEMIA COULD LEAD TO DEATH OF NEURON. CAUSING DENERVATION OF AFFERENT DIVERSE TO OTHER AREAS. HOWEVER, WHILE IT WAS TIME THE BRAIN DOES HAVE SOME CAPACITY TO REGENERATE IN THE SENSE OF EXONAL ROUTING AND SYNAPSE GENESIS. SO THESE SPROUTED AXONS AND NEWLY FORM SNAPSIS CAN REGENERATE THE THE DINNER VETVATED AREA TO CERTAIN EXFEINT. THIS IS TO SHOW YOU THERE'S KEY MOLECULES IDENTIFIED TO CONTROL POSITIVELY OR NEGATIVELY THIS PROCESS. SHOWN HERE IS CSPGs, AS INHIBITORY FACTOR FOR AXONAL GROWTH. BASICALLY IT'S SHOWING THE CSPDs COMBINED TO A VARIETY OF RECEPTORS. SUCH AS PDP SIGMA, RECEPTOR 1 OR 3 AND THROUGH BINDING AND DOWNSTREAM SIGNALING PATHWAY THERE BY INHIBITING FOR EXAMPLE AKT OR STIMULATING ROLE A PATHWAY AS ENDSPG SERVES AS INHIBITOR QUEUE FOR AXONAL GROWTH. THIS INHIBITORY ROW IS IMPORTANT DURING DEVELOPMENT OR OTHER HOMEOSTASIS PATHOLOGICAL CONDITION TO KEEP THE STABILITY OF NEURONAL NETWORK. HOWEVER, IN THE CASE OF NEURONAL INJURY, WHEN THE BRAIN IS TRYING TO REGENERATE OR REPAIR ITSELF,S THESE INHIBITOR QUEUES CAN BECOME AN OBSTACLE. OUR LAB ARE INTERESTED IN NEUROGENESIS AN AXONAL SPROUTING AND THIS PROJECT IS A COLLABORATIVE EFFORT THAT WAS -- WHILE I WAS FACULTY MEMBER AT CASE WESTERN RESERVE UNIVERSITY. THIS WORK HAS REALLY BEEN PIONEERED BY DR. JERRY SILVER AND HIS GROUP. THEY HAVE ELEGANTLY DESIGNED A PEPTIDE KNEW MATTIC PEPTIDE CALLED INTRACELLULAR SIGNAL PEPTIDE. THIS IS SHORT ABBREVIATED AS SP. THIS PEPTIDE MIMICS THE -- PTP SIGMA RECEPTOR THAT JUST RESIDED IN INTRACELLULAR PART OF THE RECEPTOR. THIS DOMAIN HAS PREVIOUSLY BEEN SHOWN TO BE CRITICAL AND HAVE INHIBITOR ROLE TO THE CSPD PDP SIGNALING AND COUPLE OTHER RECEPTORS AS WELL. SO BY USING THIS PEPTIDE DR. JERRY SILVER'S GROUP HAS SHOWN FOR EXAMPLE IF YOU CAN SEE HERE BY COATING OR HIGH CONCENTRATION RING OF CSPGs, THE AXONS OF NEURON FAIL TO CROSS THIS BOUNDARY. WHEN THEY ADD ISP TO THE MEDIA NOW AXONS IGNORE HIGH CONCENTRATION AND CAN FREELY CROSS. SO USING THIS NOVEL PEPTIDE JERRY'S GROUP AND SEVERAL INDEPENDENT GROUPS HAVE SHOWN THAT THIS PATHWAY IS INDEED VERY CRITICAL FOR THE AXONAL SPROUTING PROCESS IN MULTIPLE DISEASES. SO FIRST WE ARE INTERESTED IN WHETHER THIS PROCESS IS ALSO INVOLVED IN STROKE RECOVERY. SO FIRST WHAT WE CHECKED IN MOUSE MODEL OF STROKE WE FOUND THAT BOTH SEVEN DAYS AND POST STROKE 14 DAYS WE SEE ABUNDANT ACCUMULATION OF THIS SPGs NEAR THE GLIAL SCAR SURROUNDING THE INFARCT CORE. THIS IS MOUSE DATA, WHAT ABOUT IN HUMAN? SO IT HAS BEEN PREVIOUSLY RECORDED ALREADY THAT IN HUMAN STROKE POSTMORTEM TISSUE WE SEE ENRICHMENT OF CSPGs THERE AS WELL. THESE MOLECULES ARE RIGHT PLACE RIGHT TIME. ARE THEY INVOLVED IN THIS. BUT I WANT TO REMIND YOU EARLIER I TOLD YOU THERE WERE TWO MAJOR MECHANISM FOR NEUROREPAIR, ONE AXONAL SPROUTING, THE OTHER WE HAVEN'T TALK ABOUT WHICH IS NEUROGENESIS. SO NOW WE KNOW THROUGH PIONEERING WORK OF JOSEPH AND MANY OTHERS THAT IN THE ADULT RING WE HAVE CAPACITY TO GENERATE NEURON. ONE VENTRICULAR ZONE, AND THE OTHER ONE IS IN HIPPOCAMPUS WHERE CONSISTENTLY ADDING MANURANCE IN THE MAMMAL BRAIN. THESE ARE INDICATED IN MA'AM MAILIAN FORMATION AND OTHER CONDITIONS SO THESE CELLS DO RESPOND TO INJURY SHOWN HERE BY (INAUDIBLE) PREVIOUS WORK, THEY CAN PROLIFERATE AND MIGRATE INTO THE INJURED AREA TRYING TO REPAIR. OUR WORK HAVE SHOWN MODULATING THIS PROCESS WE WERE ABLE TO ENHANCE ENDOGENOUS NEUROGENESIS IN STROKE ANIMALS BY UPREGULATING PATHWAY THESE ANIMALS SHOW NOT ONLY ENHANCE NEUROGENESIS BUT ALSO BEHAVIOR RECOVERY. IMPROVEMENT AS WELL. SO FIRST WE WANTS TO SEE AXONAL SPROUTING WELL STUDIED FOR THIS PATHWAY, HOWEVER IN ADULT NEURON GENESIS, IT'S HAVE NOT BEEN EXTENSIVELY LOOKED AT YET. HOWEVER SOME EVIDENCE SUGGESTING, MIGHT BE IMPORTANT, FOR ONE THE CSPGs ARE ENRICHED IN THE NICHE WHERE THESE CELLS RESIDE. YOU CAN SEE MY -- BY STAINING DOUBLE CORD MATURE NEURON, SURROUNDED BY THIS CSPG. WE ALSO CULTURED THE CELLS, YOU CAN MAKE CULTURES FROM ADULT BRAIN AND THEY'LL SELF-RENEW GROWING THROUGH NEUROSPHERES AND YOU CAN DIFFERENTIATE THEM. YOU CAN SEE HERE THESE CELLS IN CULTURE AS NEUROSPHERE MAKE THEIR OWN CSPG AS WELL. TO STUDY THE ROLE OF THIS PATHWAY IN THESE CELLS WE USE A COMBINED METHOD, ONE PHARMACO LOGICALLY, IN COLLABORATION WITH DR. JERRY SILVER, WE ARE USING PEPTIDE TO INHIBIT THIS PATHWAY. WE ALSO GENERATED SPECIFIC CONDITIONAL KNOCKOUT MICE WHICH HAS BEEN REPORTED PREVIOUSLY. THIS ALLOWS US TO GENERATE NEUROSTEM CELLS SPECIFIC INDUCIBLE KNOCK OUT PPD MICE. YOU CAN SEE HERE BY TOMATO REPORTER GENE WE CAN SHOW WE GET SPECIFIC LABELING STEM CELL BOTH SBZ AND SGZ AND PROJECT INTO THE CS 3 REGION THROUGH THE FIBERS. IN THE CULTURE PRIMARY CELLS WE GET HIGH EFFICIENT RECOMBINATION. USING THESE CELLS, WE WANT TO SEE IF THEY REGULATE NEUROOUTGROWTH. WHEN WE DIFFERENTIATE THIS ADULT STEM CELLS INTO NEURONS COMPARED TO CONTROL PEP SIDE, AND THE SCRAMBLED AS P PEPTIDE WHICH HAS THE SAME AMINO ACID COMPOSITION BUT DIFFERENT SEQUENCE. SIGNIFICANTLY IT HAS NEURORIOT OUTGROWTH IN DIFFERENTIATED NEURON. CONFIRMING THAT, WE ALSO LOOK AT IN THE CONDITIONAL KNOCK OUT CELLS AS YOU CAN SEE HERE AGAIN, SIMILARLY THAT THE CONDITION KNOCK OUT IN NEURAL STEM CELLS WHEN YOU DIFFERENTIATE THEM THEY GROW MUCH LONGER NEURONS. SO WHAT ABOUT MIGRATION? BECAUSE THESE CELLS ARE HIGHLY MOBILE. THEY NEED TO GO TO THE INJURY SITE WHERE THEY CAN DO THEIR JOB AND REPAIRING THE BRAIN. SO INTERESTINGLY YOU CAN SEE HERE WE REPORT NEUROSPHERE ON CULTURE DISH, THE CELLS AUTOMATICALLY START MIGRATING OUT. THIS IS 24 HOURS AFTER THE DEPOSIT. YOU CAN SEE HERE THERE'S NICE CONCENTRATION DOSE RESPONSE IN INHIBITION OF CSPG WHICH WE USE, INHIBITING MIGRATION OF THESE CELLS. INTERESTINGLY, IN THE PRESENCE OF ASP WE INHIBITED, THE CELLS IGNORE THE INHIBITORY QUEUES AGAIN. AND THEY MIGRATE JUST THE SAME AS WITHOUT CSPGs. SO HERE IS QUANTIFICATION OF THE MIGRATION. AGAIN, TO ACCOUNT VALIDATED IN GENETIC MANIPULATION, WE USE A CONDITIONAL KNOCK OUT CELLS AND SIMILAR SIZE OF NEUROSPHERES ARE DEPOSITED AND YOU CAN SEE FIRST THING NOTICE IS CONDITIONAL KNOCK OUT CELLS MIGRATING MUCH FURTHER. COMPARED TO WILD TYPE. AND IN THE INHIBIT THIS MIGRATION WILD TYPE BUT KNOCK OUT CELLS STILL MIGRATE MORE. AND YOU -- WE DO NOTICE STILL INHIBIT CONDITIONAL KNOCKOUT MICE IN CELLS MIGRATION, THAT COULD BE DUE TO EITHER NOT 100% DELETION OF THE GENES OR OTHER RECEPTORS WILL ARE ALSO IMPORTANT IN MEDIATING THIS INHIBITION. THEN WE USE INHIBITORS TO VARIETY OF SIGNALING PATHWAYS. TO SEE WHICH ARE RESPONSIBLE FOR THIS PHENOTYPE. YOU CAN SEE HERE ABCB CON JOINING MICE WAS ABLE TO HAVE THE SAME PHENOTYPE, HOWEVER, WHEN WE ADDED THE ERK PATHWAY INHIBITOR, OR MMP 2 INHIBITOR WE REVERSED THIS PHENOTYPE SUGGESTING BY ACTIVITY ERK AND PRODUCING MMP 2. WHAT ABOUT IN VIVO P WE KNOW EVENTUALLY CELLS DO RESPOND TO THIS PATHWAY, IT'S CRITICAL. WE TRANSLATE TO BENEFIT IN STROKE MODEL. WE I WANT TO POINT OUT TWO IMPORTANT THINGS. ISP TREATMENT WAS NOT GIVEN UNTIL 24 HOURS AFTER STROKE. SO THIS IS IN REFERENCE TO THE CURRENT TREATMENT WINDOW OF SIX HOURS. SECOND FOR EVERY STROKE MICE WE GAVE THEM MI IMAGING SCAN. THIS ALLOWS US TO GROUP THEM INTO TWO EQUAL GROUPS MIMICKING A GOOD CLINICAL TRIAL. NOW WE MEASURE BEHAVIOR TEST WEEKLY TO TRACK THEIR IMPROVEMENT. WHEN WE OBSERVE A DRAMATIC INCREASE OF NEUROGENESIS. RED STAINING NEWLY BORN NEURON, WE SEE MUCH MORE NEURONS BORN MIGRATE INTO THE LESION STROKE. STRIATUM. AND ALSO WE SEE ENHANCED AXONAL SPROUTING SHOWN HERE. BY INJECTIONING TRACER TO THE CONTRALATERAL SIDE OF THE CORTEX. THESE WILL BE TAKEN UP BY NEURONS AND YOU CAN TRACE EXACTLY WHERE THEY SEND THEIR PROJECTIONS. WE SEE SIGNIFICANT IMPROVE ENHANCED AX OBJECTIONAL PROUDING TOWARDS CONTRALATERAL SIDE THROUGH THE CORPUS CLOSE SUM AS WELL AS CORTICAL SPINAL CORD TRACT ALL THE WAY DOWN WHICH THE SPINAL CORD. THE MECHANISM BOTH MAJOR MECHANISMS ARE ENHANCED. DOES THIS TRANSLATE TO BEHAVIOR IMPROVEMENT? YOU CAN SEE HERE WE MEASURE THOSE LOCAL MOTION FUNCTION AND SENSORY MOTOR FUNCTION AS WELL AS COGNITIVE FUNCTION. I WANT TO POINT TO ONE OF THEM. TO GIVE YOU ATTENTION HERE. ANIMALS ARE THE SAME BEFORE STROKE. AND THREE DAYS LATER THERE IS THE SAME BECAUSE WE GROUP THEM ACCORDING TO SAME LESION. THEY HAVE ONLY GOT TWO DAYS OF SP TREATMENT. HOWEVER WITH TIME YOU CAN SEE THAT THEY REALLY SEPARATE WITH RECOVERY. INDICATING INDEED WORKING ON IMPROVING RECOVERY. >> TRY TO WRAP UP IN TWO MINUTES. LOOKS LIKE -- THANK YOU. WITH THAT I WANT TO REMIND EVERYBODY BESIDES NEUROPROTECTION MECHANISM, WE HAVE THE NEURAL REPAIR MECHANISM WE CAN TAP INTO AS WELL. WITH THAT I WOULD LIKE TO THANK JERRY SILVER. IT'S BEEN SUCH A FUN PROJECT. SO EXCITING WORKING WITH JERRY. I'M NEW TO THE GAG FIELD SO THIS IS A BRAND NEW FIELD FOR ME, BUT IT HAS BEEN TREMENDOUS DISCOVERY AND VERY MUCH EXCITING. DR. (INAUDIBLE) WAS A POST DOC FELLOW IN MY LAB WHO DID MAJORITY OF THE WORK, HAS -- WHO HAS STARTED HIS INDEPENDENT GROUP NOW AS A PI. THE LAB, THIS IS THE LAB AT CASE AND RECENTLY MOVED TO CINCINNATI, THESE ARE NEW LAB MEMBERS AND MY COLLABORATORS AT CINCINNATI CHILDREN, AND THE STROKE CLINICAL TEAM. WITH THAT I'LL TAKE ANY QUESTIONS. THANK YOU. >> THANK YOU, DR. LUO. ONE QUESTION. COULD YOU PLEASE REMIND US OF THE MECHANISM OF ISP INHIBITORY FUNCTION AND HOW DOES IT ON PTP SIGNAL? -- SIGMA? >> YES. IT'S BELIEVED THAT THE CSPG GENES CLUSTER THE PDP SIGMA TOGETHER AND WHEN CLUSTERED TOGETHER THE DOMAIN BINDING TO EACH OTHER INHIBITING THE DOWNSTREAM SIGNAL. I THINK THAT'S THE REASON WHY HEPARAN -- HSPG HAVE OPPOSITE BECAUSE THEY STRETCH OUT HAVE RECEPTOR SEPARATED OUT. THAT'S HOW ISP WORKS. >> ANYTHING -- >> THANK YOU VERY MUCH. SO WE ARE BACK ON TIME AGAIN. WE HAVE A COUPLE OF QUESTIONS IN THE QUEUE IF WE CAN HOLD OFF UNTIL THE END BECAUSE THEY WERE FOR SOME OF THE PREVIOUS SPEAKERS. I'LL ASK DR. RISBUD TO GO AHEAD. >> THANK YOU. CAN YOU HEAR ME? I WANT TO THANK PAMELA AND ALSO AMANDA AND ALL THE NIH STAFF FOR GIVING ME THE OPPORTUNITY TO SPEAK WITH YOU TODAY. SO MY TOPIC IS SLIGHTLY ON A DIFFERENT ISSUE. SO WHAT I'M GO TO DO IS -- OKAY. SO FIRST I WOULD LIKE TO THANK PEOPLE WHO HAVE DONE THE WORK. BECAUSE USUALLY I DON'T WANT TO PUT THAT AS A LAST SLIDE BECAUSE THESE ARE THE FOLKS WHO HAVE ACTUALLY DONE WORK. I HAVE TWO TALENTED WHO HAS DONE MOST OF THE WORK I'M SHOWING TODAY AND ALSO PREVIOUS LAB MEMBERS. I ALSO WANT TO THANK MY RECENT COLLABORATOR WHO IS AT CHILDREN'S HOSPITAL PHILADELPHIA AND ANOTHER EXCELLENT COLLABORATOR WITH BIOINFORMATICS WASHINGTON UNIVERSITY AND SOME OTHER COLLABORATORS AS WELL. SO JUST TO GIVE YOU A BRIEF BACKGROUND, IF YOU LOOK AT THE GLOBAL BURDEN OF DISEASES, BACK PAIN IS -- IF YOU LOOK AT THE -- DISABILITY, LOW BACK PAIN AND NECK PAIN, THESE ARE SOME OF THE TOP DISORDERS. LOW BACK PAIN IS ONE. IF YOU LOOK AT THE GLOBAL STATISTICS NOW, NECK PAIN RANKS FOUR. SO THESE TWO DISORDERS ARE REALLY A MAJOR CONTRIBUTORS TO THE GLOBAL DISABILITY. ONE OF THE STRONGEST CONTRIBUTORS TO THE LOW BACK PAIN IS ENTERVERTEBRAL DISC LESION. THIS IS A SORT OF AN UNCOMMON TISSUE NOT PEOPLE THINK ABOUT TOO MUCH BUT WE ALL THINK AT SOME POINT BECAUSE OF LOW BACK PAIN OF COURSE. THAT THIS DISC HAS INTERESTING EMBRYO GENESIS. THE TISSUE MY LAB FOCUSES ON THE NUCLEUS WHICH IS RIGHT AT THE CENTER OF THE INTERVERTEBRAL DISC IS DEVELOPED FROM EMBRYONIC PROTOCOL WHEREAS THE FIBROSIS, CONCENTRATE TISSUE AROUND NUCLEUS AND THE -- IS DERIVED FROM THE SCLERODORM. WHY INTERVERTEBRAL DISC IS IMPORTANT? WHEN WE ARE STANDING WALKING BENDING ANY MOTION FIRST OF ALL, V CAN STAND UP RIGHT BECAUSE WE HAVE A SPINAL COLUMN. AND BASICALLY SPINAL COLUMN GETS A PUSH BECAUSE WE HAVE INTERVERTEBRAL DISC WHICH ARE NOTHING BUT CARDIAL JOINT WHICH ALLOW YOU A THREE DIMENSIONAL MOTION. SO WHEN SANDING MOST LOWER OUT OF BODY EXPERIENCE 4% COMPRESSSIVE LOAD WHEN WETANT OR SIT PROCESS THROUGH THIS ENTERVERTEBRAL DISC. AND ONE OF THE MAJOR CONSTITUENTS OF -- ONE OF THE IMPORTANT THE COMPONENT WHICH GIVES FUNCTION IS THE HIGH GLYCOSAMINOGLYCANS CONTENT OF THE NUCLEUS PURPOSIS. IT IS BASICALLY I WOULD IN A LAYMAN'S TERM, A BACK FILL OF PROTEOGLYCANS. THE MOST PREDOMINANT IN THE DISC IS A LOT OF OTHER DIFFERENT INTERESTING PROPERTY OWE GLYCANS FORMED IN THIS TISSUE. AND OBVIOUSLY BECAUSE I DON'T IMMEDIATE TO EXPLAIN HIGH LEVELS OF KERATIN SULFATE CHANGE, BASICALLY THIS ALLOWS THIS TISSUE, CLOSE CONTAINER SO IT ALLOWS THIS TISSUE PULL IN IN WATER AND GIVE TISSUE HYDRATION. AS -- BECAUSE OF AGING BECAUSE OF TRAUMA, ONE OF THE FEATURES OF THIS IS THERE IS UPREGULATION OF MATRIX DEGRADING ENZYME, YOU DECREASE SYNTHESIS OF THESE MOLECULES, SO BASICALLY YOU COULD IMPLAIN TERMS DESCRIBE THIS AS DISEASE OF THE MATRIX. BECAUSE YOU ARE USING THE MATRIX, YOU ARE LOSING THE BIOMECHANICAL FUNCTION OF THE TISSUE. SO WE HAVE BEEN OVER INTERESTED IN UNDERSTANDING DIFFERENT MECHANISMS WHICH ARE ESSENTIAL TO THIS DEGENERATION AND THE MATRIX KATABOLISM WHICH IS SEEN AND WE HAVE DONE WORK AGAIN, I DON'T NEED TO EXPLAIN TO AUDIENCE ABOUT SYNDECANS BUT THESE ARE INTERESTING MOLECULES WHICH WE HAVE BEEN STUDYING A WHILE NOW AND ONE OF THE MOLECULES ET CETERA SPECIAL -- ESPECIALLY IS SYNDECAN 4 WE HEARD A GREAT TALK BY DR. SIMONS TODAY. WE HAVE SEEN THAT SYNDECAN 4 WAS REALLY ONE OF THE MOLECULES WHICH ARE INVOLVED IN DRIVING SOME OF THE INFLAMMATORY SIGNALING WHICH WAS HAPPENING IN THE DISC. WE SAW SYNDECAN 4 WAS IMPORTANT IN TERMS OF PROMOTING DEGRADATION, WE ALSO SAW SYNDECAN 4 CONTROL LARGER TRANSCRIPTIONAL RESPONSE IN TERMS OF INFLAMMATORY CYTOKINE SIGNALING. WITHIN INTERESTING OBSERVATION WHICH FORM THE BASIS OF THIS RECENT INQUIRY WE ALSO SAW THAT SYNDECAN 4 CONTROL EXPRESSION OF SOX 9 WHICH IS CONSIDERED A TRANSCRIPTION FACTOR. SO WE STARTED GETTING INTERESTED INTO AGAIN SYNDECAN 4 SEEM TO REALLY CONTROL BROADER TRANSCRIPTIONAL RESPONSE INTO THIS TISSUE AND ONE OF THE MOLECULES WAS SOX 9 SO WE STARTED TO LOOK INTO WHAT SOX 9 DOES. SOX 9 IS AN IMPORTANT TRANSCRIPTION FACTOR IN CONTRA GENESIS. THIS IS THE DOMAIN STRUCTURE OF SOX 9. HIGH MOBILITY GROUP TRANSCRIPTION FACTOR, IT FUNCTIONS AS A DIMER, IT HAS A TRANSACTIVATION DOMAIN. AND IT USUALLY BINDS TO ENHANCE ERS THROUGH PAIRS OF INVERTED RECOGNITION SITES AND LOT OF TIMES SOX 9 BINDS IN COLLABORATION WITH OTHER SOX TRANSCRIPTION FACTORS DESCRIBED A A SOX 3 SOX 5 AND 6 AND ALSO SOX 9 IS ALSO SHOWN OTHER TRAININGS SUBSCRIPTIONAL INHIBITORS. SOX 9 PLAYS A MAJOR ROLE IN GENESIS, SO IT'S VERY IMPORTANT OF UNDIFFERENTIATED PROGENITOR CELLS, IT IS ALSO VERY IMPORTANT FOR DIFFERENTIATION OF CONTRA SITES HOWEVER SOX 9 GETS TURN OFF WHEN SAY FOR EXAMPLE OSTEO PROGENITORS DIFFERENTIATE INTO OSTEO BLAST BUT PLAYS VERY IMPORTANT ROLE IN CHONDROCYTE DIFFERENTIATION. THERE HAVE BEEN SOME REPORTS ABOUT SOX 9 CONTRIBUTING TO THE PROTOCOL DEVELOPMENT. AS SAID EARLIER THAT'S THE TISSUE WHICH THE NUCLEUS IS DERIVED. EARLY DELETION IN SOX 9 NULL MUTANTS PEOPLE HAVE REPORTED THAT THE NOTOCHORD UNDERGOES DEGENERATION, IT DOESN'T DEVELOP SO TO ORIENT YOU TO SECTIONS IF YOU CAN SEE THIS IS THE SPINAL COLUMN, THIS IS WHERE THE PRIMARY CENTER OF OSSIFICATION IS AND WHERE THE VERTEBRA ARE FORMING. THIS TISSUE HERE, THIS IS BASICALLY THE NUCLEUS TISSUE WHICH WE STUDY. YOU CAN SEE IN THE MUTANT BASICALLY YOU ARE SPINAL COLUMN DEVELOPMENT IS REALLY SEVERELY AFFECTED, NUCLEUS DOESN'T FORM PROPERLY. -- GROUP ALSO LOOKED INTO ROLE OF SOX 9 INTO OVERALL SKELETAL TISSUE AND CARTILAGE. WHAT THEY HAVE SEEN WAS AGAIN, VERY BRIEF MENTION IN THIS PAPER THAT THEY HAVE SEEN ALSO THERE WERE SOME DISC PHENOTYPE, HOWEVER, THIS PHENOTYPE WAS NOT REALLY DEFINED BUT SEEMED FROM SOME OF THE PICTURES THEY PRESENTED THAT THERE WAS ALSO SOME LESION HAPPENING IN THE DISC. SO WE WANTED TO LOOK INTO THIS WHAT IS THE ROLE OF SOX 9 INTO ADULT INTERVERTEBRAL DISC. SO WE IMPLIED CONDITIONAL STRATEGY TO DO SOX 9 A AGGREGAT CRE IS HIGHLY E PRESSED IN CARTILAGE AND INTERVERTEBRAL DIETSK IT TARGETS TARGET FIBROSIS AND CARTILAGE COMPONENTS OF THE THE INTERVERTEBRAL DISC. THESE ARE TISSUES. THESE MICE -- SO WE HAVE SOX 9 PHLOX CONTROL ANIMALS AND AGGEGAN KRE ER ANIMALS SO THESE ARE INDUCIBLE CRE. AT THREE MONTHS THE MOUSE WAS GIVEN -- WAS GIVEN TO THESE ANIMALS AND WE HAVE DONE ANALYSIS UP TO ONE YEAR. AGAIN, THIS SOX 9 ALLELE HAS SIZE FLANKING EXON 2 AND 3, HALF OF YOUR TRANSACTIVATION DOMAIN DNA BINDING DOMAIN WOULD BE DELETED SO IT RESULTS IN -- WE STARTED TO LOOK AT THESE ANIMALS WHEN FOUR MONTHS OLD SO THIS WAS ONE MONTH POST DELETION. WE LOOK AT ONE WEEK POST DELETION, THERE IS NO PHENOTYPE, ONE MONTH POST DELETION, THE PHENOTYPE WAS MILE. BUT AS THESE ANIMALS AGE, TWO MONTHS POST DELETION, WHEN HAHN ANIMALS WERE FIVE MONTHS OLE WE CAN SEE DEGENERATION TO NUCLEUS PURPOSIS HERE THIS TISSUE IN THE CENTER. WE ALSO STARTED TO SEE CHANGES IN THE FIBROSIS, THE TISSUE HERE. AND THERE WERE ALSO CHANGES IN THE END PLATE CARTILAGE ON EITHER SIDE OF THE NUCLEUS PURPOSIS. THESE CHANGES WERE AGAIN QUANTIFIED HERE SO YOU CAN SEE EARLY TIME POINT NO CHANGES BUT LATER TIME POINT YOU CAN CLEARLY SEE MORE DISC SHOW DEGENERATIVE PHENOTYPE. AGAIN WHEN WE WORK ON THE SPINE, WE HAVE SEEN MANY PEOPLE HAVE SEEN BEFORE YOU GET EVERY DISC PROCESS DIFFERENT PHENOTYPE OUTCOME AS OPPOSED TO FOR EXAMPLE IF YOU ARE WORKING ON CARTILAGE OF LUNG BONES YOU HAVE BASICALLY TWO CARTILAGE TO LOOK AT AND THERE IS NO DIFFERENCE IN PHENOTYPE. HOWEVER DEPENDING ON WHERE YOUR DISC IS IN THE SPINE, WHETHER IT IS AN UPPER LUMBAR LEVEL OR WHETHER LOWER LUMBER LEVEL PHENOTYPIC PRESENTATION IS SLIGHTLY DIFFERENT. IN A SENSE, WE GET TO ANALYZE MULTIPLE DISC IN EACH ANIMAL. AND YOU CAN SEE THAT WHEN YOU LOOK AT THE AVERAGE AGE OF DEGENERATION AND YOU ALSO HAVE A PHENOTYPICALLY CHARACTERIZED DIFFERENT NUMBER, THESE 3, 4 NUMBER, 45, AND YOU CAN SEE AT FOUR MONTHS THESE CHANGES ARE VERY MILD YOU START TO SEE SOME CHANGE HERE AT THE N 6 S 1 LOW LOWEST LUMBER LEVEL IS L 6S 1 IN HUMANS, IT IS BASICALLY FIS 1. ALSO THE DISC THAT SHOWS THE MOST LEARNIATION IN HUMANS. YOU CAN SEE THAT AT HIVE MONTHS SEE HIGHER GRID OF DEGENERATION. WHEREAS UM PER LUMBAR LEVELS DON'T SEE THE CHANGE YET SO IT'S A PROGRESSIVE CHANGE THAT AFFECTS LOWER LUMBAR LEVELS RATHER THAN UP PER LUMBAR LEVELS. WHEN WE LOOK AT THE MICE ONE YEAR OLD WELCOME SEE THE DEGENERATION PROGRESSED. THE NUCLEUS PURPOSIS WAS DEVOID OF PROTEOGLYCAN OR GAG STAINING, PROCESS WAS EXTREMELY DISRUPTED AND THE GROWTH PLACE HERE ON EITHER SIDE OF THE DISC, THESE ARE GROWTH PLACE BASICALLY GONE. YOU CAN CLEARLY SEE THAT THE DEGENERATION VERY PROMINENT AND REGARDLESS OF THE LEVEL NUMBER LEVEL DEGENERATION WAS HIGHLY DETERMINED. WE ALSO STARTING TO LOOK AT THE SENSE THERE WERE CHANGES IN THE GROWTH PLACE WE ALSO STARTED TO LOOK INTO WHAT HAPPENS TO THE BONES. WE COULD CLEARLY SEE THAT SOME OF THE BONE PARAMETERS WERE AFFECTED, VERTEBRAL BONE PARAMETERS AND WELL AS CORTICAL BONE PARAMETERS. TO SEE WHAT WAS HAPPENING TO THE CELLS, WE PERFORMED ASSAY AND WE CAN CLEARLY SEE THAT AS HAS BEEN REPORTED IN CARTILAGE WHERE SOX 9 SHOWN TO BE IMPORTANT SURVIVAL FACTOR WE SAW SOMETHING SIMILAR HAPPENING INTO THE DISC. NUCLEUS PURPOSIS AS WELL AS ANNULUS FIBROSIS WE SEE INCREASE IN CELL DEATH AS SEEN BY POSITIVITY AND QUANTIFIED HERE. TO SEE IF THERE WAS COMPENSATORY CELL PROLIFERATION HAPPENING WE DID AN EDU CHASE ASSAY. BASICALLY INJECTED ANIMALS WITH EDU SIX HOURS BEFORE SACRIFICE, THEN LOOK FOR EDU SIGNAL AND DIDN'T SEE COMPENSATORY INCREASE IN CELL PROLIFERATION. TO SEE IF THE CELLS FROM OTHER COMPARTMENTS BECAUSE THERE HAVE BEEN SOME REPORTS WHICH SHOWED THAT CELLS FROM OTHER COMPARTMENTS CAN CONTRIBUTE TO THE DISC COMPARTMENTS WHEN UNDERGOING DEGENERATION. SO WE USE LINEAGE TRACING APPROACH WHERE WE USE AN R 26 REPORTER. AND IDEA IS THAT YOU WOULD LABEL EVERY CELL RED WHICH EXPRESSES YEAR 3. THIS IS HOW COMPARTMENT LOOKS O SO THE IS STAINING IN CONTROL ANIMALS, AND THEN WE BASICALLY FOLLOWED WHAT HAPPENS TO THE CELLS IN THE COMPARTMENT WHEN SOX 9 IS DELETED. WE DID THE CHASING AFTER ONE MONTH, TWO MONTHS, SEVEN MONTHS AND AFTER 12 MONTHS. SO WE COULD CLEARLY SEE THAT THERE WAS DECREASE IN NUMBER OF CELLS IN THE COMPARTMENT AS YOU CAN SEE THERE WAS DECREASE IN NUMBER OF POSITIVE CELLS BUT WE DID NOT SEE ANY SIGNIFICANT NUMBER OF CELLS NOT POSITIVE WITHIN COMPARTMENT, IT CLEARLY SHOWED US THAT THESE CELLS FROM OTHER COMPARTMENTS DID NOT INTEGRATE INTO THE DISC. WE USED AN IMAGING FBIR TO LOOK INTO WHAT WAS HAPPENING TO THE DISC. SO WE DID A SPECTRAL CLUSTERING ANALYSIS BASICALLY LOOKED INTO PROTEOGLYCANS REALING TO COLLAGENS AND WE LOOK INTO TOTAL PROTEIN AND WE COULD CLEARLY SEE THERE WAS DECREASE IN PROTEOGLYCAN OVERALL TO THE COMPARTMENT. AGAIN AS I WAS INITIALLY MENTIONING, AGGREGATE IS A MAJOR PROTEOGLYCAN INTO THE DISC. WE LOOK AT THE LEVELS AND SEE THAT THERE WAS DRAMATIC DECREASE IN PROTEIN LEVELS WE CONFIRM BY STAINING -- LEVELS AND WHOA WE ALSO LOOKED INTO WAS INCREASE AGGREGATE, AND WE USED SOME ANTIBODY SPECIFIC G1 EPITOPE FOLLOWED BY CLEAVAGE 4 AND 5 SO WE CAN CLEARLY SEE THERE WAS INCREASE IN AGGEREGATE TURN OVER. >> CAN YOU WRAP UP IN TWO MINUTES. >> YES. THERE WAS DECREASE IN COLLAGEN CONTENT. SO SOX 9 SEE OVERALL DECREASE IN MATRIX COMPONENTS AND PROTEOGLYCANS AFFECTED. WHICH LOOKED INIF A SPECIFIC PEPTIDE CALLED CHP PEP SIDE WHICH MEASURES HOW MUCH DENATURED COLLAGEN INTO TISSUE AND WE CAN CLEARLY SEE THAT COLLAGEN NOT REDUCED BUT WHAT WAS COLLAGEN WAS THERE STARTED TO DEMATRIX SO THE MATRIX WAS GETTING TURNED OVER. NOW ONE OTHER INTERESTING THING WE WANT BED TO SEE WAS,WHAT WERE THE BROADER TRANSCRIPT TRANSCRIPTOMIC CHANGES? WE COLLECTED THESE TISSUES FIVE DAYS POST INJECTION. SO ENRICH FOR SOX 9 TARGETS AND THEN WE USE A VERY INTEREST INTERESTING RECENTLY DEVELOPED TOOL CALLED -- THIS TOOL HAS BEEN DEVELOPED AT WASHINGTON UNIVERSITY. THESE ARE BIOINFORMATICS FOLKS WHO HAVE COME FROM PFIZER. AND WE ARE COLLABORATING WITH ONE OF THE INFORMATITION WHO IS A FACULTY MEMBER THERE SO THIS IS A NEW TOOL THEY CAME UP WITH, A VERY POWERFUL TOOL, BASICALLY AN AI BASED TOOL AND WHAT IT DOES IS THAT IT BASICALLY TAKES DIFFERENTIALLY EXPRESSED GENE LIST AND USING THIS SIGNAL DETECTION NATURAL LANGUAGE PROCESSING ALGORITHM, IT BASICALLY LEAVES ALL PUBMED AND THEN GENERATES INTELLECTUALLY BIOLOGICAL CONCEPTS TO GIVE YOU BIOLOGICAL INSIGHTS. SO WHAT IT DOES IS THAT DEVELOPS THIS DIFFERENT TEAMS AND DEVELOPS DIFFERENT CONCEPTS AND YOU CAN PUT THIS INTO SOME SORT OF A THREE DIMENSIONAL MAP AND WE CAN CLEARLY SEE WHEN WE LOOK AT DIFFERENTIALLY EXPRESS GENES IN THE TISSUE AFTER SOX 9 DELETION LOT OF ECM GENES WE ALSO SAW LOT OF GAG SO THESE ARE BASICALLY TEAMS, CLUSTERS OF THE TEAMS AND THEN YOU HAVE CONCEPTS. IF YOU LOOK AT THOSE CONCEPTS BASICALLY IT SHOWS ME WHAT ARE THE CONCEPTS WHICH ARE CONTRIBUTING TO THIS TEAM AND THEN WHAT ARE THE GENES WHICH ARE CONTRIBUTING TO EACH OF THE CONCEPT WITNESS THAT TEAM. SO WE SAW THAT AGAIN MATRIX GAG BIOSYNTHESIS ARE MAJOR CONCEPTS WHICH ARE AFFECTED. AS WELL AS CYTOSKELETAL LOT OF INNATE IMMUNE GENES WERE AFFECTED. SO IN SHORT LAST SLIDE IN SHORT WHAT WE ARE SEEING NOW, IS THAT SOX 9 IS A MAJOR REGULATORY OF NUCLEUS CORPOSIS AND FIBROSIS FUNCTION. ONE OF THE GROUPS OF GENES ARE THE MAJOR TEAMS AFFECTED INCLUDES NOT JUST ECM BUT PRIMARILY GAG BIOSYNTHESIS AND WE HOPE TO UNDERSTAND COMPLEXITY OF WHAT CHANGES ARE HAPPENING IN THESE TISSUES. SO LOT OF GREAT TALKS TODAY AND REALLY GAVE US A LOT TO THINK ABOUT. SO WITH THAT,-MY LAST SLIDE. THANK YOU. >> THANK YOU VERY MUCH, DR. RISBUD. WE STILL HAVE HAVE A LITTLE BIT OF TIME, IF YOU HAVE QUESTIONS FOR HIM, MEANTIME, WHILE WE ARE WAITING FOR QUESTIONS FOR DR. RISBUD, WE DO HAVE A QUESTION FOR DR. LUO THAT CAME IN. THAT IS, CHONDROITIN SULFATE PROTEOGLYCANS ARE ESSENTIAL COMPONENTS OF THE NEURAL STEM CELL NICHE. WHAT ARE YOUR THOUGHTS ON THEIR ROLES IN NEUROSTEM CELL SELF-RENEWAL AND HOMEOSTASIS? IT'S A GREAT QUESTION.QUESTION, SO THIS -- WE DIDN'T KNOW BEFORE MY LAB HAS DONE SOME EXPERIMENT ON THIS. WE ARE RELEGATING IT IN THE CONDITIONAL KNOCK KNOCKOUT MICE AS WELL. I THINK THE MAIN ROLE OF CSPGs ARE TO KEEP THEM STABILIZED IN THE NICHE. I DIDN'T HAVE TIME TO SHOW IT SO I DIDN'T PUT IT UP THERE WHEN WE USE THE CRE REPORTER MICE WHEN WE SAW WAS THAT TOTAL TOMATO POSITIVE CELLS DIDN'T INCREASE MIGRATING. IT DID NOT INCREASE BUT THE DOUBLE POSITIVE -- WAS INCREASED MIGRATING TOWARDS INJURY. SO MY THOUGHTS IS THAT THE -- ON THE CONTRALATERAL SIDE YOU CAN SEE DOUBLE CHONDROITIN IS SAME COMPARED TO CONTROL SP TREATMENT. SO I THINK IT MIGHT NOT BE PLAYING AS PERMANENT ROLE IN SELF-RENEWING RATHER THAN MIGRATION OF THESE CELLS. THAT'S IT. >> THANK YOU. AS OF NOW, WE DON'T HAVE ANY OTHER QUESTIONS IF JERRY SILVER IS LISTENING, SEEMS DR. NOBLE HAS LEFT THE ROOM BUT I'LL CONTACT YOU SEPARATELY. OR ACTUALLY YOU COULD -- I CAN SEND YOU -- IF I HAD YOUR EMAIL ADDRESS, I CAN GIVE YOU PAUL NOBLES' EMAIL ADDRESS. SO ARE THERE ANY OTHER QUESTIONS FOR ANY OF OUR SPEAKERS FROM THIS AFTERNOON? JUST WAIT A MINUTE OR TWO TO SEE IF ANY COME IN. OKAY. HERE IS A QUESTION FOR DR. SANDERSON. CAN THE INTERACTION OF THE SYNDECAN PROTEIN BACKBONE IN CELL SURFACE RECEPTORS AFTER SHEDDING THE INHIBITED BY SMALL PEP TIDES DERIVED FROM THE BINDING DOMAIN OF SYNDECAN? DR. SANDERSON, ARE YOU STILL THERE AS MUCH >> YEAH I'M HERE. I WAS TRYING TO INTERPRET THE QUESTION HERE. >> DO YOU WANT ME TO TO TO REPEAT IT AGAIN? >> I'M LOOKING AT IT. I SEE IT HERE. THE INTERACTION OF THE I GUESS THE SYNDECAN CORE PROTEIN -- OKAY. YEAH. THAT'S ACTUALLY I THINK WHAT YOU ARE ASKING IS ACTUALLY WHAT ALAN HAS DONE. HE'S MADE PEPTIDES THAT BLOCK -- THAT BASICALLY COMPETE AGAINST THE CORE PROTEIN, THE BLOCK THE BINDING TO THE VEGFR 2 AND ALPHA 4 BETA 1 INTEGRIN. SO I THINK THE QUESTION IS DIRECTED AT PRECISELY WHAT HE'S DONE AND THE PEPTIDES HE CALLS STATIN ARE EFFECTIVE IN BLOCKING THAT SIGNALING MECHANISM AND ALSO I THINK HE'S USED SOME OF THESE PEP TIDES IN VIVO NOW LOOKING AT THEIR ABILITY TO BLOCK TUMOR GROWTH. >> STILL WAITING FOR ANY MORE QUESTIONS TO COME IN. IF YOU ARE WRITING YOUR QUESTION OUT GO AHEAD BUT I'LL JUST USE THIS OPPORTUNITY TO THANK ALL THE SPEAKERS FOR GIVING -- BEING ON TIME AND GIVING SUCH STIMULATING TALKS. I REALLY ENJOYED THIS, I THINK MOST OF THE OTHER ATTENDEES HERE ALSO APPRECIATED HEARING SOME GREAT SCIENCE HERE AND APPRECIATING THE CRITICAL ROLE THAT GLYCOSAMINOGLYCANS PLAY IN MANY ASPECTS OF BIOLOGY. RIGHT NOW I'M NOT SEEING ANY MORE QUESTIONS COMING IN. MAYBE I'LL HAND IT OVER TO PAM TO GET THE ROUND TABLE DISCUSSION STARTED. PAM, ARE YOU THERE AS MUCH >> PAM, ARE YOU THERE? >> I'M HERE. I WANT TO THANK -- AND MERCY AND SALVATORE FOR LEADING THESE SESSIONS AND ALL THE SPEAKERS FOR SOME WONDERFUL TALKS TODAY. AS YOU KNOW THE COMMON FUND GLYCOSCIENCE PROGRAM IS ONE OF THE MANY COMMON FUND PROGRAMS, THIS ONE IS DEDICATED TOWARDS MAKING TOOLS, TECHNOLOGIES, LIBRARIES, AVAILABLE TO THE BROADER SCIENTIFIC COMMUNITY TO MOVE GLYCOSCIENCE FORWARD. WHEN WE ARE LOOKING FOR TARGETS FOR THESE TOOLS AND TECHNOLOGIES, GAGS WAS ONE OF THE AREAS THAT WE REALLY WANTED TO FOCUS ON. AS YOU HEARD TODAY, GAGS ARE EXTREMELY IMPORTANT MOLECULES IN JUST ABOUT EVERY BIOLOGICAL PROCESS AND NUMEROUS DISEASES. AND THEY SUPPORT FOR INVESTIGATORS TO STUDY THESE MOLECULES TO SPREAD OUT ACROSS MULTIPLE NIH INSTITUTES WHICH MAKES THESE MOLECULES REALLY FRONT AND CENTER FOR THE COMMON FUND. IN TERMS OF WHAT WE ARE TRYING TO DO IS TO PUT TOGETHER LIBRARIES DEFINE SULFATED LIBRARIES, LONGER LIBRARIES, MORE ROBUST LIBRARIES TO MAKE THESE THINGS AVAILABLE IN BULK SO THAT PEOPLE CAN JUST BUY THEM. OR WORK WITH OUR INVESTIGATORS TO GET THEM TAYLOR MADE. WE ARE INTERESTED IN BEING ABLE TO SYNTHESIZE AS WELL AS SEQUENCE THESE MOLECULES AND YOU HEARD TALKS TODAY FROM COMMON FUND INVESTIGATORS WHO CAN HAND TOOLS AND TECHNOLOGIES TO RAPIDLY SEQUENCE MOLECULES NOW. AND HIGH THROUGH PUT ASSAYS FOR THESE MOLECULES. SO TODAY YOU HEARD FROM FIVE DIFFERENT COMMON FUND INVESTIGATORS, IT'S INCUMBENT ON THEM TO ACTUALLY REACH OUT TO THE SCIENTIFIC COMMUNITY AND TO WORK WITH YOU TO BETA TEST THEIR TOOLS. SO ALL OF THEM ARE EAGER TO MEET YOU AND IF YOU HAVE TIME AND INCLINATION TO BRING SOME OF THESE TOOLS INTO YOUR LABORATORIES, PLEASE CONTACT OUR COMMON FUND INVESTIGATORS DIRECTLY. AND ASK TO WORK WITH THEM. THAT TAKES US TO THE QUESTIONS THAT THE ROUND TABLE HERE IS TO DISCUSS. COMMON FUND HAS SET UP CERTAIN THINGS AND IT'S BEEN ABLE TO DO BUT CERTAINLY GIVEN BREADTH OF SCIENCE THAT GAGS ARE INVOLVED WITH, WE HAVE HEARD MANY APPROACH TO STUDY THESE MOLECULE S AND IF YOU HAVE IDEA OR NEEDS ABOUT OTHER TOOLS THAT WILL BE HANDY TO HAVE OR THAT YOU WOULD LIKE TO MAKE AND YOU NEED FUNDING FOR WE WOULD LIKE TO HEAR ABOUT IT. NIGMS PUT SPECIAL INITIATIVES ON THE BOOKS. FOR RO1s AND R21s SPECIFICALLY FOR TOOLS AND TECHNOLOGY TO BE ABLE TO ADDRESS MAKING ADDITIONAL TOOLS. AND CERTAINLY THIS FIELD IS WELCOME TO TAKE ADVANTAGE OF THOSE PROGRAM ANNOUNCEMENTS. THE ROUND TABLE IS YOUR TIME TO DISCUSS SOME OF THE WORK THAT'S E TAKING PLACE. WE HEARD SOME 'ALLY INTERESTING TALKS TODAY FROM THREE GROUPS, DR. BOONS, DR. DESAI AND DR. SANDERSON, ABOUT HOW GAGS ARE INVOLVED IN VIRAL INFECTION. AND CERTAINLY THERE MAYBE A COMMON MECHANISM AS WAS POINTED OUT. IF YOU HAVE THE FLU, YOU COME IN AND YOU BIND TO SIALIC ACID TIPS OF NUISANCE. AND THE GET THE VIRUS OUT WE USE NEUROMINIDASE. IT WAS SEEN WITH COVID YOUR BINDING TO HEPARAN, SEVERAL PAPERS INCLUDING ONE FROM JEFF ESCO THAT SUPPORTS THE IDEA THAT HEPARAN IS INVOLVED IN THE BINDING MANY BUT HEPARANASE MAYBE INVOLVED IN RELEASE OF THE VIRUS SO THIS MAYBE A COMMON MECHANISM, COMMON PATHWAY. ANYBODY HERE WANT TO TALK ABOUT OR CAN SOMEBODY PROPOSE A GENERAL MECHANISM? I THINK ALL LEE INVESTIGATORS HINTED AT THE FACT THAT THE VIRUS HAS TO BIND AND THAT THERE ARE DIFFERENT BOTH RECEPTOR IN THE VIRUS -- AND THE VIRUS ITSELF MAYBE INVOLVED IN BINDING AND ORIENTING THE MOLECULE FOR INFECTION. BUT I DIDN'T HEAR ANYBODY TAKE IT TO THE POINT WHERE WE ARE ACTUALLY PROPOSING A WORKING MODEL. HAS WORK PROGRESSED WHERE WE HAVE A WORKING MODEL FOR VIRAL INFECTION? WE HEARD SEVERAL PRESENTATIONS ABOUT SULFATION PATTERNS AND HOW SULFATION PATTERNS MAYBE IMPORTANT IN BOTH NEURAL GROWTH AND ADHESION. IN SEVERAL DISEASES LIKE CANCER FOR METASTASIS. ARE WE MISSING TOOLS TO BE ABLE TO STUDY THIS? SO I WANT THE OPEN THE TABLE TO YOU AND TACKLE WHAT YOU LIKE. THIS IS YOUR TIME. PLEASE GO AHEAD. >> DO YOU WANT TO SAY MORE ABOUT THE MINING, POTENTIAL BINDING OF SASHES COV-2 TO HEPARIN? >> YEAH. I THINK FROM MANY LABS, THERE IS NO COMPELLING EVIDENCE THAT SARS COV-2 EMPLOYEES HEPARAN SULFATE HAVE INITIAL ATTACHMENTS AND THAT HEPARAN HEPARAN SULFATE INHIBITS THIS INTERACTION. PRIOR PRIOR TO COV 2 THERE WERE A LOT OF DATA SUPPORTING THAT MANY VIRUSES HEPARAN SULFATE SOME FOR INITIAL ATTACHMENT SOME AS RECEPTOR FOR CELL AND SUPPORTING THE POINT THAT YOU WERE MAKING. MANY VIRUSES NEED TO BATTLE THE GLYCO LIPIDS BEFORE THEY CAN REACH THE CELL MEMBRANE FOR ATTACHMENT AND GLYCANS MEANS THE VIRUS NEEDS TO TEAL WITH THAT AND CONVERSELY WHEN -- IT NEEDS TO GO THROUGH THAT GLYCO HELIX AGAIN SO BIOLOGY AND GLYCOBIOLOGY ARE INTIMATELY THIS CONSORTIUM HELPS UNRAVEL HOW THIS HAPPENS AT MOLECULAR LEVEL. AND WILL PROVIDE OPPORTUNITIES FOR THERAPEUTIC INTERVENTION. >> TODAY WE HEARD THREE SPEAKERS STARTING OUT I'M NOT A GLYCO BIOLOGIST, I BACKED INTO THE FIELD, THERE'S FEW CARD CARRYING GLYCOBUY JOISTS WHO DID THIS FROM UNDERGRADUATE TO GRADUATE TO POST DOC. THERE'S ONLY A HANDFUL. OF PROGRAMS, TRAINING PROGRAMS AND GLYCOSCIENCE RIGHT NOW. THOSE HAVE ONLY COME ON THE BOOKS RECENTLY. FROM SO LITERALLY EVERYBODY IS BACKED INTO THIS FIELD. >> I THINK SO TOO. >> IT'S REALLY, VERY IMPRESSIVE TO SEE THE PLIO TROPIC PATHWAYS VIRUSES ARE USING. I HAVE TO WONDER IF YOU ARE CUTTING A VEGETABLE YOU HOLD WITH THE LEFT HAND AND CUT WITH THE RIGHT. SO DOES THE HEPARAN SULFATE HOLD THE VIRUS IN PLACE SO THAT THE SPIKE CAN ACTUALLY DO ITS THING? IT CAN BE RELEASED AN PENETRATE CELL? DOES IT HELP ORIENT (INAUDIBLE)? >> I THINK IT HELPS. (OVERLAPPING SPEAKERS) (OVERLAPPING SPEAKERS) >> EXACTLY. >> AND ALL THESE PROTEOGLYCANS ARE JUST A WAY FOR THE SPIKE PROTEIN TO GET A HANDLE ON THE OUTER MEMBRANE OF THE CELL. THEN CAN START TO SEARCH AROUND LOOK FOR THAT ACE 2 RECEPTOR. THERE YOU GO. >> BOTH THE CELL AND RECEPTOR SEEM TO HAVE BINDING SITES ON THE VIRUS. I WAS JUST WONDERING IF THEY HAVE TO ORIENT EACH OTHER. >> DENSITY OF PROTEOGLYCANS IS HIGHER THAN THAT OF ACE 2 RECEPTOR. SO BE VERY SPIKE PRO PROTEIN TO GET HANDLE ON THE THE PROTOYES GLYCANS AN IT HAS ROOM TO SURVEY THE CELL SURFACE UNTIL IT FINDS ACE 2 RECEPTOR. >> THAT'S WHAT I WAS WONDERING IF IT WAS A TETHERING AFFECT. >> ONE OF THE INTERESTING QUESTIONS IS WHETHER THERE'S ANY INTERACTION BETWEEN THE RECEPTOR, PROTEIN RECEPTOR WITH PROTEOGLYCAN, WITH THE HEPARAN SULFATE. WHY THE VIRUS -- DOES MANY CASES THE VIRUS RECOGNIZE BOTH HEPARAN SULFATE OPT CELL SURFACE AND AT THE SAME TIME ALSO REQUIRES SPECIFIC PROTEIN RECEPTORS. IN THE OTHER CASE WE FIND THE BINDS WITH THE BACTERIA TOXIN BINDS WITH CELL SURFACE SULFATION GROUPS, LIKELY THROUGH JUST A CHARGING -- BUT ALSO INVOLVES LDLR AND RECEPTOR AND WE STILL DON'T KNOW THE MECHANISM, WE STILL DON'T KNOW'S THERE'S NO DIRECT BINDING BETWEEN THE TOXIN WITH THE PROTEIN RECEPTOR. WITH THE LTLR. BUT SOMEHOW THE LDLR IS INVOLVED AND IT'S IMPORTANT FOR THAT PROCESS. SO THERE'S A QUESTION HOW EXACTLY THE GAG ON THE CELL SURFACE, AND HEPARAN SULFATE ON CELL SURFACE SEEMS INTERACTING WITH CERTAIN PROTEINS OF THE CELLS THAT MAYBE MEDIATING THAT RECYCLE BETWEEN CELL SURFACE WITH THE INTERNAL ENDOSOME. >> THAT IS A GOOD POINT. ARE THERE TOOLS TO ADDRESS THIS AND GET TO THAT MECHANISM? I GUESS I'M ASKING M IN AND GEERT JAN ABOUT THAT. >> I DON'T KNOW. FOR US TO STUDY THE BIGGEST CONCERN FOR US WHEN WE STUDY THIS IS ALWAYS LIKE WHETHER WE ARE LOOKING AT SPECIFIC INTERACTION, WHETHER WE ARE -- WHETHER THE INTERACTION IS JUST DEGREE OF CHARTING INTERACTION OR IS SPECIFIC BIOLOGICAL FUNCTIONAL MEANINGS THERE? THAT'S ALWAYS A DIFFICULT QUESTION FOR US. >> I THINK IT WILL BE THE USEFUL THINGS FOR US IS THAT MOIETIES, THE INDIVIDUAL MOIETIES THAT BEING BY COMMUNITY, AS WELL AS HOW TO PUT THOSE SO WE CAN ACHIEVE MULTI-SAY LANT INTERACTIONS WITH THE TARGET PROTEINS WE ARE INTERESTED IN WHETHER IT IT'S TOXINS VIRUSES AND CELL SURFACE PROTEIN. THE INDIVIDUAL INTERACTION OFTEN TIME IS VERY LOW BINDING A FINTY BUT OVERALL ABILITY ABILITIEN -- BINDING AFFINITY ON CELL SURFACE IS HIGH SO THE QUESTION IS WHETHER WE HAVE A GOOD WAY TO REPLICATE RECAPITULATE THAT KIND OF OLIGOMERRIZE THE STATE IN VITRO. >> I ACTUALLY HAVE A QUESTION FOR UMESH. FOR THE LIBRARY YOU DESCRIBE THAT CAN PREDICT WHETHER A PROTEIN BINDS TO A GAG, HOW CAN WE USE THAT? AND >> I'M NOT SEEING UMESH IN OUR ROOM. HE MIGHT HAVE LEFT EARLY. >> APPEAL CONTACT HIM. CURIOUS ABOUT COUPLE OF CANDIDATES. >> DO YOU HAVE HIS EMAIL? >> I CAN FIND OUT. YEAH. >> OKAY. >> THANKS. >> SO ONE THING I WAS GOING TO SAY CERTAINLY THE BINDING UPTAKE OF THE VIRUS IN SARS COV-2 ALL THAT IS IMPORTANT TO UNDERSTAND THOSE MECHANISMS BUT I THINK THE OTHER OPPORTUNITY FOR GLYCOBIOLOGY HERE IS DO ANY TOOLS THAT HAVE BEEN MADE, CAN THEY INTERFERE WITH THE CYTOKINE STORM. BECAUSE EVENTUALLY THAT'S WHAT'S KILLING THE PATIENTS. SO ARE ANY OF THESE GAGS BEING MADE, SPECIFIC GAGS COULD ANY OF THOSE POSSIBLY HAVE VERY GOOD EFFECT IN BLOCKING SOME OF THE MECHANISMS THAT ARE DRIVING THE STORM? ONE INDICATION THIS MIGHT BE USEFUL IS MOST OPHTHETI PATIENTS ODE OF THESE PATIENTS ARE BEING TREAT WITH HEPARAN FOR COAGULATION PROBLEMS BUT THAT IS ALSO HAVING EFFECT ON CYTOKINE STORM SO JUST ANOTHER ASPECT TO THINK ABOUT. WITH SOME OF THE TOOLS YOU GUYS MAY -- >> ARE THERE SPECIFIC TOOLS YOU ARE THINKING OF? >> YEAH, I'M THINKING ABOUT THE DIFFERENT GAGS, DIFFERENT HEPARAN GAGS, THINGS JIM LUI HAS MADE OR SOME OF THE OTHERS. THEY MAY BE ABLE TO INTERFERE WITH CYTOKINE INTERACTIONS WITH THEIR RECEPTORS. PARTICULARLY IF YOU HAVE GAG FRAGMENTS NOT ANTI-COAGULANT, TO BE GIVEN IN A REASONABLY HIGH DOSE. >> RIGHT. SO YOU MIGHT WANT TO TALK TALK TO JIN LUI AND LINDA WILSON AND GEERT JAN BOONS, ALL ARE MAKING COMPOUNDS IN THE COMMON FUND AND IF THERE'S SOMETHING SPECIFIC, CERTAINLY GLYCAN THERAPEUTICS IS COMMERCIALIZING THE LIBRARYING COMING OUT OF THE COMMON FUND SO THEY MIGHT ALSO GOOD PLACE TO START IF YOU ARE TRYING TO GET LARGE AMOUNTS OF THESE M.. >> IF WE PARTICULAR CYTOKINES, THAT ARE MEANINGFUL WE CAN SCREEN OUR MICROARRAYS THAT HAVE DEFINE SULFATION MOTIFS TO LOOK FOR SULFATED MOTIFS THAT ARE SPECIFIC FOR THOSE CYTOKINES THAT ARE MOST IMPORTANT, THOSE COULD BE USED TO POTENTIALLY ANTAGONIZE THE INTERACTIONS. SO I THINK THAT WOULD BE REALLY EXCITING. >> I WILL SECOND TO LINDA, IT IS POSSIBLE WE CAN SYNTHESIZE IN PROBABLY GOOD TO DO SAME THING. WE CAN SYNTHESIZE MANY DIFFERENT HEPARAN STRUCTURE WITHOUT ANTI-COAGULANT ACTIVITY. THE POSSIBILITY IS TOO LARGE. WE ARE NOT QUITE SURE. WHICH WE THOUGHT WE SHOULD GO AFTER. THAT'S THE COMPLEXITY OF THE PROCESS. >> SO THAT -- FOR US THAT WAS PART OF THE MOTIVATION IN GOING WITH A VERY WELL DEPINED TETRA SACCHARIDE LIBRARY WHICH WE ARE HOPING TO COMPLETE IN A COMPREHENSIVE WAY WHERE WE HAVE COVERED ALL THE POSSIBLE SULFATION MOTIFS. WE ARE NOT YET AT 3-0 SULFATION BUT WE ARE TACKLING IN, 2 AND 6 SULFATION AND HOPING TO HAVE ALL THE POSSIBLE SULFATION MOTIFS SO ONE COULD SCREEN FOR SPECIFIC CYTOKINES OF INTEREST TO FIND MINIMAL EPITOPES THAT BIND WITH HIGH AFFINITY AND SPECIFICITY FOR THOSE CYTOKINES. AND ONCE MINIMAL EPITOPES ARE FOUND THEY CAN BE ELABORATED TO LARGER POLYMERS OR LONGER HEPARAN STRUCTURE THAT LACK ANTI-COAGULANT ACTIVITY. YET HAVE MULTI-SAY LANT PROPERTIES OR HAVE LONG LONGER CHAINS THAT ARE NEEDED FOR FULL ACTIVITY. BUT THE TETRA SACCHARIDE LIBRARIESES IS A WAY TO FULLY SCREEN WE HOPE ONCE FULLY COMPLETED, TO FULLY SCREEN THE MOTIFS AND WE ARE ALMOST HALFWAY THROUGH THE SYNTHESIS OF COMPOUND AND WORKING TOWARD THE COMPLETE 256 COMPOUND LIBRARY. >> EXCELLENT. ONE OF THE THINGS WE HOPE COMES OUT OF THIS WORKSHOP IS THAT YOU HAVE COLLABORATIONS. AND CERTAINLY I WOULD ENCOURAGE Y'ALL TO GET IN CONTACT WITH EACH OTER, YOU HAVE EACH OTHER'S EMAIL ADDRESSES. AND TRY TO SET UP THESE COLLABORATIONS. ANYBODY ELSE WANT TO PUT FORWARD ANY OTHER TOOLS PEOPLE WOULD LIKE TO ADDRESS? >> I HAVE ONE QUICK QUESTION. IT'S POSSIBLE THAT SOME OF THIS INFORMATION IS ALREADY THERE ON THE COMMON FUND WEBSITE. BUT COULD YOU HAVE MAYBE COMPREHENSIVE LIST OF TOOLS WHICH IS AVAILABLE FOR EVERYBODY TO START UP SO WE KNOW WHAT TOOLS ARE OUT THERE, UP TO DATE SO WE CAN REACH OUT TO PEOPLE? >> CERTAINLY, WE HAVE ALL INVESTIGATORS ALONG WITH THE ABSTRACTS OF THEIR FUNDED WORK UP ON THE WEBSITE. BUT WE CAN MAKE A CHEAT SHEET. WHO CAN CONTACT THEM DIRECTLY. WE ARE ACTUALLY WORKING ON THAT RIGHT NOW. >> THAT WOULD BE GREAT. THANK YOU. >> HAPPY TO SEND THAT OUT TO THE PARTICIPANTS HERE. DOES ANYBODY ELSE HAVE SOMETHING THEY WOULD LIKE TO DISCUSS, TOOLS THEY WOULD LIKE TO SEE MADE? COLLABORATIONS, RESOURCES THAT ARE INTERESTING? (INDISCERNIBLE) (INDISCERNIBLE) >> WE ARE NOT ABLE TO UNDERSTAND YOU. YOUR VOICE IS REALLY GETTING GARBLED. YOU MIGHT WANT TO TYPE IT INTO THE CHAT AND THEN I CAN REPEAT IT. I DID HAVE A QUESTION FOR HANNES. ALL THESE ANTIBODIES THAT YOU HAVE, ARE THEY AVAILABLE FOR PEOPLE TO USE? WHERE DOES THAT STAND RIGHT NOW? >> WELL, WE HAVE SENT OUT PLASMIDS FOR THE VERSION AS MUCH AS WE HAVE. AS I SAID WE HAVE ALMOST ALL ALL OF THEM, WHAT WILL BE CHALLENGING GOING FORWARD AT LEAST AT THIS STAGE IS TO DISTRIBUTE MUCH PROTEIN. JUST BECAUSE THIS IS QUITE LABOR INTENSIVE TO PRODUCE AT LEAST FOR DISTRIBUTION. BUT THE CDNAs WE HAVE DISTRIBUTED SEVERAL TIMES ALREADY. WITH SIMPLE MTA. WE ARE PLANNING TO DO THAT IN THE FUTURE TOO. >> ARE YOU HOPING TO COMMERCIALIZE THIS ANTIBODIES? BECAUSE THE VERSIONS OF THEM FOR FLOW CYTOMETRY WOULD BE USEFUL. >> I AGREE. SOME POINT I DON'T THINK OUR PLANS THAT DEVELOP IN THIS REGARD, BUT I THINK IF IT HOLDS TRUE WHAT WE ARE SEEING AND HOW USEFUL THEY ARE, IT WOULD BE APPROPRIATE AND IMPORTANT TO FIND SOME WAY OF GETTING THEM MADE AND MADE AVAILABLE BY SOMEONE. SO THERE HAVE BEEN DISCUSSIONS BUT WE HAVEN'T MADE CONCRETE PLANS ON HOW TO DO THAT. >> WE HAVE A QUESTION FOR DR. LUO. CAN SHE TALK ABOUT REGENERATION OF NEURON, IS SHE ABLE TO DIRECT GROWTH IN THE REPAIR PROCESS? >> CAN YOU REPEAT THAT QUESTION AGAIN? IT'S NOT TYPED UP. IS THAT FOR ME? >> YES, IT JUST CAME IN BY EMAIL. >> I SEE. CAN YOU REPEAT THAT AGAIN. >> SURE. COULD YOU TALK A LITTLE MORE ABOUT REGENERATION OF NEURONS AND ARE YOU ABLE TO DIRECT GROWTH IN THE REPAIR PROCESS? >> SO FAR OUR DATA WITH ISP, WHAT WE ARE SAYING, IS INCREASED NEWLY FORMED NEURONS. MIGRATING TOWARDS INJURY AREA. AND THE ISP WAS GIVEN BY SUBCUTANEOUS INJECTION. THEY HAVE A TAD PEPTIDE MEDIATING THEIR PENETRATION TO CNS. WE HAVE NOT TRIED LOCAL INJECTION TO GUIDE THEM IF THAT'S A QUESTION. NOT SURE WHETHER I UNDERSTOOD IT. WE HAVEN'T TRIED TO BE ABLE TO GUIDE THEM TO WHERE THEY NEED TO GO YET. WE ARE JUST SAYING A GENERAL INCREASE NUMBER AND MIGRATION. >> THANK YOU. WE GOT A QUESTION IN FOR GEERT JAN. IN TERMS OF THE LIBRARIES THAT YOU GENERATED, COULD YOU TAKEOR TALK A LITTLE BIT ABOUT SACCHARIDE LENGTH AND SULFATION PATTERNS FOR BINDING SARS COV-2? >> YES. WHAT WE FOUND IS THAT IT IS FIRST OF ALL LENGTH DEPENDENT, AT LEAST A HEXA SACCHARIDE IS REQUIRED FOR BINDING AND A LITTLE BETTER. AND A RELATIVELY HIGH SULFATION PATTERN ON THE AMEAN C-2 AND C OF-6 SULFATE. WHAT WE ALSO OBSERVE IS THAT -- IF YOU LOOK AT THE RBD, THE BINDING IS RELATIVELY LOW AFFINITY. HIGH MICROMOLAR BUT IF YOU LOOK AT INTACT SPLICE I'S NANOMOLAR RANGE. COUPLE OF LABS HAVE IDENTIFIED MULTIPLE SITES ON THE VIRUS WHERE HEPARAN SULFATE CAN BIND. WORKING HYPOTHESIS IS WHAT I WAS REFERRING TO, THERE'S WEAK INTERACTIONS ON MULTIPLE SAY LANT LEVEL BUT NOT COVALENTLY POTENT. THE RBD BINDS REALLY COMPARED TO ACE, SO WHAT REALLY ALLOWINGS THROUGH -- BINDING TO HEPARAN SULFATE. MIGRATE THROUGH THE HELIX AND THEN MORE POTENTLY BIND THE ACE 2 FOR EVENTUAL FUSION WITH THE CELL MEMBRANE. I HOPE THAT ANSWERS QUESTION. >> EXCELLENT. YOU CAN ALL TURN YOUR CAMERAS ON NOW IF YOU WILL LIKE. WE HAVE A QUESTION FROM MIKE GELB. WHERE IS THE LIST OF GAG FRAGMENTS STRUCTURES THAT ARE NOW AVAILABLE TO GET SAMPLES FOR RESEARCH EITHER FROM THE COMMON FUND OR FROM GLYCAN THERAPEUTICS. (INDISCERNIBLE) >> MIKE YOU ARE STILL GARBLED. BUT LINDA JIN LIU? >> I'M HERE. YES, WE DO HAVE SUGARS ARE LISTED ON THE WEBSITE. AND IF IT'S MOST OF THEM ARE IN STOCK SO ONCE THE -- WE RECEIVE THE ORDER WE CAN DELIVER WITHIN A WEEKS OR SO. WE APPROXIMATELY CLOSE TO 100, IF ALL SIDE OF THE CATALOG, LET ME KNOW WHAT IS IT. WE CAN TALK ABOUT IT TO TRY TO FIND SUBSTITUTE STRUCTURE FOR THE RESEARCHERS. WE CAN DIESES CUSS WHAT -- DISCUSS WHAT THE ACTUAL NEED IS. WE CAN DO THAT. >> ANYONE WHO IS INTERESTED IN AS WE MAKE THEM MORE ADDING TO THE LIST, SO FEEL FREE TO EMAIL ME IF YOU ARE INTERESTED AND I CAN TELL -- I CAN SEND YOU OUR UPDATE LIST OF COMPOUNDS THAT WE HAVE. >> EXCELLENT. (INDISCERNIBLE) >> YOU ARE STILL BREAKING UP. MIKE, YOU ARE STILL BREAKING UP, YOU MIGHT WANT TO TRY TO PHONE IN. HANNES, ARE YOU GOING TO USE -- TO DISTRIBUTE YOUR PLASMIDS? >> CAN YOU SAY THAT AGAIN? >> ARE YOU GOING TO USE ADDGENE.ORG TO DISTRIBUTE YOUR PLASMIDS? >> I MEAN, THAT IS A POSSIBILITY BUT I'M ALSO HAPPY TO SEND THEM TO BE HONEST. IT WOULD BE UNITE AN EXPENSIVE UNDERSTOOD TAKING FOR PEOPLE TO ORDER THEM FROM A ADDGENE AT 60-DOLLAR AS PLASMID; YOU WOULD SPEND $1,000 ON 30 PLAIDS MIDS AT LEAST. IF YOU THINK IT WOULD BE ADVANTAGEOUS WE MAY, I DON'T KNOW. BUT AS I SAID WE WE ARE HAPPY TO DISTRIBUTE THEM IF SOMEBODY SENDS ME AN EMAIL. YOU MAY ALSO -- I WAS PRETTY RESERVED EARLIER. WE MAY BE ABLE TO SEND A FEW PROTEIN ALLOQUATS AS WELL. WE JUST -- IT DEPENDS HOW MUCH WE HAVE. AND WE DON'T HAVE EVERY ANTI-BODY QUITE YET. WE HAVE MORE -- SOME ARE EASIER TO PURIFY THAN OTHERS SO WE HAVE MORE ALLOQUATS OF SOME THAN OTHERS SO IF SOMEBODY WANTS TO TRY SOMETHING, WE PROBABLY CAN SEND A FEW ALLOQUATS. BUT WE PREFER TO AT THIS STAGE AT LEAST, TO STICK MOSTLY TO THE PLASMIDS. >> THANK YOU, WE ARE GETTING CLOSE TO END OF TIME HERE. WE HAVE TWO MINUTES. DOES ANYBODY WANT TO MAKE ANY FINAL COMMENTS? IF NOT, I WILL SEND OUT AN EMAIL TO ALL OF YOU SO THAT YOU CAN TALK TO EACH OTHER AND SET UP COLLABORATIONS, GET LISTS OF COMPOUNDS, ARRAYS, WHAT NOT. AGAIN, I CAN'T THANK YOU ALL ENOUGH. I THINK IT'S REALLY IMPORTANT FOR PEOPLE TO REALIZE THE BREADTH OF SCIENCE GOING ON ACROSS NIH AND GAGS. I MEAN LITERALLY EVERY INSTITUTE HAS SOMEBODY WORKING ON THESE MOLECULES. IT'S NOT A LOT OF PEOPLE AT ANY ONE INSTITUTE. BUT WHEN YOU ALL COME TOGETHER, YOU CAN SEE THE PATTERNS FOR THESE MOLL DUALS AND HOW THEY -- MOLECULES AND HOW THEY FUNCTION. BE IT FOR VIRUSES OR FOR METASTASIS. BUT THE CELL BIOLOGY IS REALLY IMPORTANT, SIGNALING MOLECULES ARE REALLY IMPORTANT. I THINK THERE ARE LESSONS TO BE LEARNED HERE. SO I WANT TO THANK YOU ALL FOR TAKING THE TIME TO SPEND THIS DAY DOING THIS EXERCISE. AND I HOPE YOU GOT SOMETHING OUT OF IT AS WELL. WE ARE ONE MINUTE AWAY. ANY LAST COMMENTS? ALL RIGHT. THANK YOU ALL VERY MUCH. HAVE A GREAT DAY. >> THANK YOU.