I'D LIKE TO WELCOME YOU TO THE NIH COMMON FUND GLYCOSCIENCE PROGRAM'S ALL HANDS MEETING. THIS IS A REQUIREMENT OF THE PROGRAM. WE MEET ONCE A YEAR AND ALL THE PARTICIPATING INVESTIGATORS WITHIN THE PROGRAM UPDATE US ON THE WORK THAT THEY'VE ACCOMPLISHED OVER THE PAST YEAR. AS YOU WELL KNOW, THE COMMON FUND IS OUT OF THE OFFICE OF THE DIRECTOR OF NIH, AND THIS EFFORT IS DEVELOPING TOOLS, METHODS AND TECHNOLOGIES THAT CAN INTEGRATE GLYCOSCIENCE WITH THE BROADER SCIENTIFIC COMMUNITY. WE'VE HAD SEVERAL HUNDRED INVESTIGATORS WORKING ON THIS PROJECT OVER THE PAST FEW YEARS, AND THIS IS THE FINAL FUNDED YEAR OF THE PROGRAM, SO AT THIS POINT, MOST OF THE TOOLS AND TECHNOLOGIES ARE WELL-BEING DEVELOPED. I'D LIKE TO THANK ALL OF THE PARTICIPANTS IN THIS PROGRAM AND THEIR LABORATORIES WHO HAVE PUT SO MUCH EFFORT INTO MAKING THESE TOOLS AND TECHNOLOGIES AVAILABLE. AND I WOULD LIKE TO THANK ALL OF THE MEMBERS OF THE WORKING GROUP THAT HAVE BEEN PART OF THE NIH STAFF FROM THE VARIOUS INSTITUTES WHO HAVE BEEN INTEGRATING THIS WITH THEIR OWN INSTITUTES' EFFORTS. AT THIS POINT, I WOULD LIKE TO INTRODUCE DR. DOUG SHEELEY. HE IS OUR PROGRAM LEADER, COORDINATES THIS PROGRAM WITH THE COMMON FUND OFFICE OF STRATEGIC COORDINATION, DIVISION OF PROGRAM COORDINATION, PLANNING, STRATEGIC INITIATIVES OVER AT OD. DOUG, WOULD YOU LIKE TO SAY A FEW WORDS? >> ? IS DOUG MUTED? OKAY. I GUESS DOUG IS HAVING SOME PROBLEMS WITH HIS COMPUTER, SO WE'LL WAIT A FEW MINUTES FOR DOUG, BUT WHILE WE'RE WAITING, LET ME TELL YOU A LITTLE BIT ABOUT WHAT'S GOING TO HAPPEN. WE HAVE THIS PARTICULAR GROUP THAT ARE ON THE ZOOM, AND THEY'RE WITHIN THE MEETING, SO YOU CAN USE THE CHAT FUNCTIONS TO ASK QUESTIONS AND WHEN WE OPEN UP FOR DISCUSSION, YOU CAN BASICALLY TURN ON YOUR VIDEO AND ASK A QUESTION OR SIMPLY ASK A QUESTION BY UNMUTING YOURSELF. OUTSIDE OF THIS ROOM, THIS ZOOM ROOM, THIS PARTICULAR MEETING IS BEING PICKED UP AND WEBCAST TO THE BROADER SCIENTIFIC COMMUNITY. THEY HAVE THE PROGRAM, AND THEY CAN RELAY QUESTIONS TO US, EMAIL QUESTIONS TO US, WHICH THE MODERATOR CAN ASK OF THE SPEAKERS AT ANY TIME. YOU CAN ALSO GO BACK AND RE-WATCH THESE TALKS IF YOU NEED TO AT A LATER TIME SINCE THEY'LL BE UP ON THE NIH VIDEOCAST SITE. OUR MORNING SESSION IS GOING TO BE HEADED UP BY THE PEOPLE THAT ARE ESSENTIALLY DRIVING THE SUBGROUPS. THERE'S THREE ARMS TO THIS PROGRAM. THERE'S THE -- [THIS MEETING IS BEING RECORDED] THERE'S THE INFORMATICS ARM, THERE'S THE TOOLS ARM, AND THERE'S THE SYNTHESIS ARM. AND THESE THREE ARMS OF THE PROGRAM ARE ESSENTIALLY INTEGRATING THEIR EFFORTS TO PRODUCE MORE FOR THE BUCK, I GUESS YOU COULD SAY. THESE GROUPS OF INVESTIGATORS HAVE WORKED TOGETHER AS TEAMS TO FILL IN THE SPACES BETWEEN THE INDIVIDUAL PROJECTS, AND DEVELOPED TEAM PROJECTS THAT THEY ALSO ARE PRODUCING FOR THE PROGRAM. SO EACH OF THOSE SUBGROUPS HAS ELECTED A LEADER. NICI POHL IS HEADING THE SYNTHESIS WORK GROUP, CATHERINE GRIMES IS HEADING THE TOOLS AND TECHNOLOGIES SUBGROUP AND MIKE MIKE TIEMYER -- WHAT THEY'VE BEEN DOING AND WHAT THEY'VE ACCOMPLISHED OVER THE PAST FEW YEARS. THEN WE'LL TAKE A SHORT BREAK SO EVERYBODY CAN GRAB A CUP OF COFFEE AND WE'LL COME BACK AND WE'LL BEGIN WITH OUR TOOLS AND TECHNOLOGIES INVESTIGATORS, WHO WILL TELL US WHAT THEY'VE BEEN DEVELOPING FOR THE BROADER SCIENTIFIC COMMUNITY, AND HOW THESE CAN BE OBTAINED OR USED BY OTHER GROUPS. THEY MIGHT EVEN TELL US A LITTLE BIT ABOUT SOME OF THE GROUPS THAT HAVE BEEN USING THESE. WE HAVE ESSENTIALLY FUNDED I THINK 17 DIFFERENT LABORATORIES THAT WERE INTERESTED IN TOOLS AND TECHNOLOGIES THAT WERE BEING DEVELOPED WITHIN THE PROGRAM, AND WE GAVE THEM SUPPLEMENTS TO UTILIZE THESE TOOLS AND TECHNOLOGIES WITHIN THEIR OWN RESEARCH PROGRAM. THIS WAS A WAY OF ESSENTIALLY TRYING TO GET THE TOOLS AND TECHNOLOGIES INTRODUCED TO THE BROADER SCIENTIFIC COMMUNITY, BUT ALSO A WAY TO BETA TEST THESE TO SEE IF OTHER PEOPLE COULD UTILIZE THESE WHO WERE NOT EXPERTS IN GLOI COSCIENCE. GEICO GLYCOSCIENCE. SEVERAL OF THE INVESTIGATORS WILL BE TELLING US ABOUT THEIR WORK AND THEIR UTILIZATION OF THESE TOOLS AND TECHNOLOGIES. SO I THINK IT'S GOING TO BE A VERY EXCITING PROGRAM. WE'VE ALSO GOT A PROGRAM THAT SHADOWS THIS PROGRAM, MIRRORS IT, IN THE SBIR/STTR LINE, AND THAT PROGRAM HAS FUNDED WELL OVER TWO DOZEN SMALL BUSINESS ENTITIES WHO ARE INTERESTED IN THE GLYCOSCIENCES, IN MAKING TOOLS AND TECHNOLOGIES FOR THE GLYCOSCIENCES. AND TODAY WE'RE GOING TO HAVE A ROUNDTABLE DISCUSSION WITH SEVERAL OF THESE COMPANIES REPRESENTED. THEY'LL TELL US A LITTLE BIT ABOUT THEMSELVES, WHY THEY'RE INTERESTED IN GLYCOSCIENCE, WHAT THEY PRODUCE, AND WE'LL HAVE A DISCUSSION ABOUT HOW YOU CAN MOVE YOUR TOOLS AND TECHNOLOGIES INTO THESE COMPANIES, PARTNER WITH THEM, AND HOW YOU CAN OBTAIN THEIR RESOURCES IF YOU'RE INTERESTED. IS DOUG BACK ON YET? >> I'M HERE. >> GOOD. LET ME INTRODUCE DR. SHEELEY. AGAIN, HE'S OUR PROGRAM LEADER WHO COORDINATES US WITH THE OFFICE OF STRATEGIC COORDINATION DIVISION OF PROGRAM COORDINATION, PLANNING AND STRATEGIC INITIATIVES AT THE OFFICE OF THE DIRECTOR, NIH. DOUG, WOULD YOU LIKE TO SAY A FEW WORDS? >> THANKS. TIME FLIES WHEN YOU'RE HAVING FUN. I CAN'T BELIEVE IT'S BEEN SIX YEARS ALREADY. I WANTED TO THANK ALL OF YOU FOR BEING HERE AND THANK THOSE WHO WORKED SO HARD TO PUT THIS PROGRAM TOGETHER, PARTICULARLY PAMELA AND AMANDA, AND I'M SURE THAT WE'RE GOING TO HAVE SOME FUN OVER THE NEXT COUPLE OF DAYS LEARNING ABOUT WHAT'S GOING ON IN THE PROGRAM. ONE NOTE THAT I WANTED TO MAKE IS THAT AS THE PROGRAM DRAWS TO A CONCLUSION, WE'RE GOING TO BE EVALUATING ITS PERFORMANCE NOT SO MUCH TO GIVE EVERYBODY A GRADE AS TO UNDERSTAND WHAT'S GONE WELL AND WHAT ARE AREAS THAT WE CAN LEARN FROM AND MAKE ADJUSTMENTS TO IN FUTURE PROGRAMS, AND SO WE'LL BE SENDING OUT SOME INFORMATION AS THAT EVOLVES SO THAT YOU KNOW WHEN OR IF YOU MIGHT BE CONTACTED BY A THIRD PARTY WHO MIGHT BE INTERESTED IN ASKING YOU SOME QUESTIONS ABOUT THE WAY YOU'VE APPROACHED THE PROGRAM'S WORK. JUST A PRELIMINARY NOTE TO LET YOU KNOW THAT THAT WILL BE HAPPENING OVER THE NEXT YEAR OR SO. PAM, LET'S MOVE ON WITH THE PROGRAM. >> OKAY. WE HAVE A VERY PACKED PROGRAM, SO WE'D LIKE TO KEEP THINGS MOVING. EVERYBODY HAS A TIMER SO PLEASE NOTE YOUR TIMER. AND WE WILL TAKE A FEW QUESTIONS AT THE END OF EACH TALK. OUR FIRST TALK TODAY IS AUTOMATION OF GLYCAN AND GLYCAN BUILDING BLOCK SYNTHESIS. AND THIS TALK IS BEING GIVEN BY NICKI POHL, CHAIR OF OUR SYNTHESIS WORKING GROUP. NICKI? >> THANK YOU, PAM. IT'S A PLEASURE TO BE HERE TO START THIS MEETING OFF. ONE OF THE GREAT THINGS ABOUT THIS WHOLE COMMON FUND PROGRAM IS IT'S GIVEN ME A MECHANISM AND MY COLLEAGUES A MECHANISM TO REGULARLY TALK TO ONE ANOTHER AND TO WORK TOGETHER, AND AN INCENTIVE TO WORK TOGETHER. SO TODAY I'D LIKE TO TELL YOU A BIT ABOUT WHAT MY GROUP HAS BEEN DOING AS WELL AS SOME OF THE OTHER SYNTHESIS GROUPS AND I'D ALSO ENCOURAGE YOU TO TUNE BACK IN TOMORROW BECAUSE THERE'S A LOT OF REALLY COOL TALKS THAT MY COLLEAGUES ARE DOING ON SYNTHETIC CHEMISTRY. THIS ALL STARTED IN PART BECAUSE OF THIS NATIONAL ACADEMY OF SCIENCE REPORT THAT CAME OUT IN 2012, AND THIS IS REALLY GALVANIZING TO BRING THIS GROUP TOGETHER AND START THINKING ABOUT HOW DO WE DO A BETTER JOB AT SCIENTISTS IN DEVELOPING THE TOOLSNESS SIRE EE TO STUDY GLYCOMICS. WE HAVE GREAT TOOLS TO STUDY GENOMICS THAT MY LAB USES ALL THE TIME ALSO AS WELL AS TO STUDY PROTEINS AND PEPTIDES BECAUSE IN THOSE CASES, WE HAVE NICE WAYS OF ANALYZING THOSE BIOMOLECULES AND GREAT WAYS OF MAKING THEM. THERE'S STILL ROOM FOR IMPROVEMENT THERE ALSO, BUT IT'S REALLY THOSE TOOLS HAVE BEEN ABLE TO ALLOW US TO SEQUENCE THE WHOLE HUMAN GENOME AS WELL AS OTHER GENOMES, AS WELL AS LOOK AT PROTEINS IN THEIR INDIVIDUAL FORMS AND IN DOING STRUCTURE-BASED ACTIVITY RELATIONSHIP STUDIES. IT'S A LITTLE MORE CHALLENGING WITH SUGAR. SO THIS GROUP, BUT WITH THIS COMMON FUND, WAS CHARGED TO TRY TO COME UP WITH SOME TOOLS TO FILL IN THIS HUGE GAM THAT'S REALLY KEEPING -- HUGE GAP, INCLUDING ENZYMATIC WASTE OF WAYS, AS WELL AS THE ENTIRE DATA INFRASTRUCTURE BEHIND IT THAT YOU'LL ALSO HEAR ABOUT TODAY, AND OF COURSE EDUCATION THAT SCIENTISTS AND THE PUBLIC AT LARGE KNOWS WHAT GLYCO CAN DO AND WHAT THE NEW TOOLS ARE AVAILABLE TO STUDY ALL THE DIFFERENT FUNCTIONS THAT SUGARS HAVE IN BIOLOGICAL SYSTEMS. SO OF COURSE ONE WAY THAT WE OFTENTIMES GET GLYCANS IS FROM NATURAL SOURCES. BUT THAT CAN BE VERY CHALLENGING. THEY TEND TO BE MICRO HETEROGENEOUS MIXTURES, MEANING THERE'S MANY DIFFERENT VERSIONS OF THE MOLECULES THAT ARE VERY SIMILAR TO ONE ANOTHER, SO I'D LIKE TO START OUT WITH SHOWING THIS WORK BY RHEINHOLD'S GROUP WHERE THEY DISCOVERED MUCH EASIER WAYS OF RELEASING ON LARGER SCALE, GRAND SCALE, SOME OF THESE GLYCOCOMPOUNDS FROM NATURAL SOURCES THAT ARE HIGHLY PREVALENT IN NATURAL SOURCES, AND THEN BE ABLE TO GET STRUCTURAL CHARACTERIZATION AND CONFIRMATION OF THESE NATURAL GLYCANS. SO FOR A SMALL SEGMENT OF GLYCANS, THIS HAS TURNED OUT TO BE A FANTASTIC WAY OF GETTING SOME OF THESE DIFFERENT GLYCANS. OBVIOUSLY MANY OF THESE GLYCANS ARE NOT PRODUCED ON THE SCALE IN NATURAL MATERIALS, FOR EXAMPLE, SOME MICROBES OR EVEN IN OUR OWN BODIES, THEY'RE NOT PRODUCED ON A SCALE THAT YOU COULD GET KILOGRAMS OR GRAMS EVEN OF THESE GLYCANS WITHOUT HAVING HUGE AMOUNTS OF BIOLOGICAL SAMPLE. SO THEN, IF WE CAN'T GET ACCESS TO THESE, AND NATURAL SOURCES CAN ALSO BE STARTING MATERIALS FOR ENZYMATIC -- THEN WHAT DO WE HAVE? WELL, THERE HAVE BEEN SEVERAL BOTTLENECKS IN GETTING ACCESS TO THESE GLYCANS USED IN CHEMICAL SYNTHESIS. THE LENGTH AND REPRODUCIBILITY BE CHALLENGING, THE SOLID FAZE ME THODZ USED FOR COMMERCIAL SYNTHESIS OF DNA AND PEPTIDES ARE A LITTLE MORE PROBLEMATIC IN GLYCOSPACE AND WE'LL TALK MORE ABOUT THIS TODAY AND TOMORROW ALSO. MONITORING REACTIONS ON A SOLID PHASE CAN BE CHALLENGING AND THE AMOUNT OF BUILDING BLOCKS YOU NEED IN ORDER TO GET THESE REACTIONS TO WORK CAN BE CHALLENGING TO GET, ESPECIALLY FOR RELATIVELY RARE SUGARS AS ARE FOUND IN A LOT OF MICROBIAL CARBOHYDRATES. THERE ARE A LOT OF DITCH OF DIFFICULT LINKAGES TO MAKE, UNLIKE THOSE PEOPLE MAKING PEPTIDES OR DNA BACKBONES, SO WE'LL HEAR MORE ABOUT THAT TOMORROW ALSO, ABOUT DIFFERENT SYNTHETIC METHODS TO BE ABLE TO MAKE THESE GLYCO LINKAGES IN A WELL-DEFINED WAY. THEN OF COURSE WE NEED TO ACCESS THE BUILDING BLOCKS TO PUT TOGETHER THESE LARGER STRUCTURES, AND THEN OF COURSE PURIFICATION AND ANALYSIS OF PRODUCTS CAN ALSO BE QUITE CHALLENGING AND I'LL TALK VERY BRIEFLY ABOUT THAT AND YOU'LL HEAR MORE ABOUT THAT IN THE REST OF THE DAY. SO TRADITIONAL METHODS OF SYNTHESIS CAN BE HARD TO REPRODUCE. THE MANUAL TECHNIQUES THAT I ACTUALLY LOVE AS A SYNTHETIC CHEMIST TO DO CAN BE CHALLENGING TO PASS ON TO ANOTHER GENERATION OF CHEMISTS EVEN, LET ALONE IF YOU'RE COMING IN FROM A BIOLOGY OR AN EPIDEMIOLOGY STANDPOINT AND YOU JUST WANT THE COMPOUND, IT CAN BE CHALLENGING TO REPRODUCE THESE METHODS WITH ALL THESE SPECIALIZED TECHNIQUES. SO THE COMMON FUND SYNTHESIS GROUP HAS BEEN WORKING TO MAKE OLIGOSACCHARIDE SYNTHESIS EASIER THROUGH NEW CATALYSTS, NEW SYNTHETIC METHODS AND NEW AUTOMATION STRATEGIES. AND REALLY, WE SEE AUTOMATION ASCII TO AS KEY TO MAKING THESE PROTOCOLS FAIR. SOME OF YOU HAVE HEARD OF THIS, FINDABLE, ACCESSIBLE, INTEROPERABLE AND REUSABLE SCIENCE. WHAT WE SEE IS MOST DATA STILL LOCKED UP IN PDF FILES, BEHIND PAYWALLS SO THEY'RE NOT EASY TO READ BY HUMANS OR COMPUTERS, THEY'RE NOT EASILY SEARCHABLE. ACCESSIBILITY IS ALSO A CHALLENGE WITH A LOT OF OUR MANUAL PROTOCOLS AND TRADITIONAL PUBLISHING MODELS, SPECIFIC MANUAL TECHNIQUES THAT I MENTIONED, LITTLE IS OPEN SOURCE AND AUTOMATED METHODS ARE QUITE EX-PEN EXPENSIVE, TO FIND A MACHINE TO CARRY IT OUT. THEN INTEROPERABILITY. THERE'S RELATIVELY LITTLE DATA AND METADATA, THAT WORKS WELL WITH OTHER APPLICATIONS. THAT'S SOMETHING WE'RE DEFINITELY STILL WORKING ON AS A COMMUNITY. THEN HAVING THE SCIENCE BE REUSABLE, DATA AND METADATA ARE OFTEN NOT DESCRIBED WELL ENOUGH TO BE COMBINED IN MULTIPLE CONTEXTS AND TO BE EASILY REPLICATED. FOR THIS REASON, AUTOMATION IS REALLY, REALLY IMPORTANT. SO MY OWN LAB HAS BEEN WORKING ON A SOLUTION PHASE BASED OLIGOSAG RIDE SYNTHESIS APPROACH. WE BASICALLY TAKE A FLUOROCARBON TAG LIKE A LITTLE TINY PIECE OF TEFLON THAT WE ATTACH TO ONE PORTION OF OUR SUGAR, AND NOW WE HAVE A MACHINE THAT'S PROGRAMMED TO BE ABLE TO ADD THE NEXT BUILDING BLOCK IN SEQUENCE ALL TO CARRY OUT A SOLUTION-BASED REACTION TO THE NEXT BUILDING BLOCK STEP SO YOU CAN CHAIN-EXTEND AND THEN IT CARRIES OUT EXTRACTION TO PURIFY EVERYTHING THAT HAS THE -- THROUGH THE COMMON FUND AND EARLIER GRANTS ALSO, WE'VE BEEN ABLE TO COME UP WITH ROUTES TO VARIOUS RHAMNANS, AND WE'RE WORKING WITH OTHER PARTS OF THE OTHER INVESTIGATORS TO BE ABLE TO START AUTOMATING THEIR PROCESSES. UNFORTUNATELY THE PANDEMIC HAS PUT SOME OF THOSE THINGS ON HOLD BUT IT LOOKS LIKE WE'LL RESTART, WE'LL START HOSTING SOME PEOPLE LATER THIS MONTH, WHICH WE'RE QUITE EXCITED ABOUT TO COME UP JOINTLY WITH WAYS TO AUTOMATE THEIR OWN GLYCAN SYNTHESIS PROCESSES USING THE SOLUTION BASED AUTOMATION APPROACH. THROUGH THE COURSE OF THIS WORK ALSO, WE'VE WORKED WITH ANOTHER U01 FUNDED INVESTIGATOR AT TUFTS UNIVERSITY IN THINKING ABOUT HOW DO WE AUTOMATE PROCESSES THAT WERE DEVELOPED MANUALLY. SO WE'VE LEARNED QUITE A BIT MORE ON THE EVIDENT, HOW DO WE EFFORT, HOW DO WE GO ABOUT TAKING A MANUAL PROCEDURE AND PUTTING IT INTO AN AUTOMATION PLATFORM. SO FOR EXAMPLE, WITH THE NEW PROMOTOR THAT HELPS GLYCOSYLATE THESE GLYCOSOL DONORS, WHAT WE FOUND IS BY USING THE BASE HE ORIGINALLY PUBLISHED, WE ENDED UP GETTING NOT ONLY THE DESIRED PRODUCT BUT A SIGNIFICANT AMOUNT OF OTHER SIDE PRODUCTS THAT ARE NOT UNCOMMONLY SEEN IN GLYCOSYLATION REACTIONS. WHAT WE'VE FOUND IS THAT IF WE TAKE THIS BASE WE ALSO HAVE A LOT OF ORTHOESTER FORMATION. IF WE LEAVE THE BASE OUT ENTIRELY, WE GET A REACTION WHERE THE GROUP WE WANT TO REMOVE LATER ACTUALLY GETS TRANSFERRED. SO WHAT WE FOUND IS BY GOING WITH A COMPLETELY DIFFERENT BASE AND START HAVING THIS PROTON SPONG SOURCE, IT'S NICELY LIQUID, WHEN AUTOMATING PROCESSES, IT'S A LOT EASIER TO DO LIQUID HANDLING ON ALL THE OTHER AUTOMATION PLATFORMS. WE DON'T HAVE TO WORRY ABOUT INTERACTING WITH A SOLID RESIN BUT IT'S STILL MORE DIFFICULT TO DO SOLIDS HANDLING THAN LIQUID HANDLING EFFICIENTLY AND PRECISELY. SO WE HOSTED ONE OF HIS STUDENTS IN MY LAB IN INDIANA ALSO, WE WERE ABLE TO APPLY THAT CHEMISTRY TO BUILD UP THIS BETA GLUCAN CHAIN THROUGH A SERIES OF AUTOMATED CYCLES USING THE BENNETT LAB PROMOTOR. WE ALSO DISCOVERED WITH THIS SOLUTION BASE DURING THE COURSE OF THIS PROGRAM, WE WERE ABLE TO DO A VARIETY OF DIFFERENT AUTOMATION PROTOCOLS. THE NORMAL PROTOCOL THAT WE HAVE AUTOMATED, THE SO-CALLED NORMAL PROCEDURE. PROFESSOR SH MYTH AND CONSTANCE CAME UP WITH THIS TERMINOLOGY OR HIGHLIGHTED IT BASICALLY WHERE WE ADD THE DONOR AND THE CATALYST TOGETHER AND THEN ADD TO A SOLUTION OF OUR NUCLEOFILL. WE DISCOVERED WE HAD ALSO MORE SIDE PRODUCTS, SO THE BEAUTY OF HAVING A SOLUTION PHASE APPROACH IS THAT WE COULD ADD THE ACCEPTOR AND THE CATALYST TOGETHER AS OPPOSED TO IF THIS WAS ON A SOLID PHASE, AND THEN SLOWLY ADD THE ACTUAL GLYCOSOL DONOR. WHAT THIS ALLOWED US TO DO IS GET MUCH HIGHER YIELDS OF THESE REACTIONS TO FORM GLYCOKNEW ME TICKS, ALSO START SITE-SELECTIVELY INTRODUCING ATOMS THAT ALLOW THESE MOLECULES TO HAVE MUCH GREATER STABILITY IN THE PRESENCE OF THE ENZYMES THAT COMPLETE THE LINKAGES. WITH THE NORMAL PROCEDURE THAT WE HAD WORKED ON WITH CLAY BENNETT IN MY LAB, THE MOST COMMON PROCEDURE, THE MAIN PRODUCTS WERE THESE ORTHO ESTHER PRODUCTS OR ENTIRE DIMERIZATION PRODUCT. WHEREAS BY TUNING THE MACHINE TO DO THE INVERSE PROCEDURE, WE COULD GET THE DESIRED MAJOR PROJECTS. SO WE HAD A LOT MORE FLEXIBILITY WITH THIS METHOD THAN WE WOULD USING A SOLID PHASE PROTOCOL, ALTHOUGH THE SOLID PHASE PROTOCOL HAS ADVANTAGES TO IT WE'LL BE TALKING MORE ABOUT LATER. SO THIS WAY, WE CAN GO THROUGH AND SITE-SELECTIVELY INTRODUCE A FILE AT A PARTICULAR LOCATION AND STILL BE ABLE TO DO CHAIN EXTENSIONS AND SHOW THAT THIS IS STABLE IN THE PRESENCE OF THE OXYGEN. SO WE'VE NOT ONLY BEEN ABLE TO DEVELOP METHODS TO MAKE TRADITIONAL GLYCANS BUT WE'RE ALSO STARTING TO LOOK AT HOW TO MAKE GLYCOMIMETICS TO BE ABLE TO MAKE THESE MORE VALUABLE TOOLS. AS I SAID, WE'RE ALSO WORKING WITH OTHER PEOPLE NOT JUST CLAY BENNETT'S LAB IN TRYING TO CONVERT THEIR CHEMISTRY INTO AUTOMATION PLATFORMS, WE HAVEN'T BEEN ABLE TO WORK WITH EVERYONE YET IN THIS SHORT TIME, AND YOU'RE GOING TO HEAR A LOT MORE TOMORROW, I ENCOURAGE YOU TO GO TO THESE TALKS WITH MY COLLEAGUES WHO HAVE DEVELOPED BEAUTIFUL NEW CATALYSTS AND NEW METHODS TO MAKING THESE LINKAGES. ONE PERSON WHO WON'T BE SPEAKING TOMORROW THAT I THOUGHT I'D HIGHLIGHT AS AN EXAMPLE OF THE KINDS OF THINGS OUR GROUP HAS BEEN DOING IS THE WORK OF PROFESSOR ERIC JACOBSEN AT HARVARD, TO MAKE A UREA-BASED CATALYST THAT ALLOWED THE GROUP TO USE VERY MILD REACTION CONDITIONS AND DUAL ACTIVATION STRATEGY, AND GET CATALYST-CONTROLLED SELECTIVITY, SOMETHING THAT IS REALLY IMPORTANT IN BEING ABLE TO HAVE A LIMITED SET OF BUILDING BLOCKS TO BE ABLE TO MAKE A WIDE VARIETY OF DIFFERENT LINKAGES. THE LINKAGES OF BOTH ALPHA AS WELL AS BETA STERI CHEMISTRY. SO THIS IS JUST ONE EXAMPLE OF THE GROUP GETS TOGETHER EVERY OTHER MONTH AND TALKS ABOUT DIFFERENT PROBLEMS, AND INSPIRES US TO THINK ABOUT WHERE ARE THE HOLES STILL MISSING IN THE KINDS OF GLYCOSYLATION CHEMISTRY WE CAN CARRY OUT, AND THEN CAN WE PUT THESE INTO AUTOMATED PROCESSES. ONE OF THE INTERESTING THINGS TOO IS THE SAME MOTIVATION PLATFORM THAT WE USED WAS ALSO USED BY THE BOONS GROUP WITH ONE OF MY FORMER STUDENTS. ONE OF THE GREAT THINGS ABOUT THE U01 IS TRAINING A COHORT OF PEOPLE WHO KNOW HOW TO DO THIS CHEMISTRY. SO HE'LL BE TALKING MORE ABOUT THIS TOMORROW ALSO USES THE SAME IDEA OF AN AUTOMATED PLATFORM DOING THINGS IN SOLUTION BUT NOW HE'S DEVELOPED A NEW TAG THAT ALLOWS EVERYTHING TO BE MORE WATER-SOLUBLE, AND HE DEMONSTRATED HE COULD DO ENZYMATIC EXTRACTIONS AND THEREBY BUILD UP THESE REPETITIOUS CYCLES OF DIFFERENT GLYCANS. IN THIS CASE, THESE ARE DIVERSE -- SACCHARIDES. AND SO NOT ONLY DO WE HAVE A METHOD AUTOMATING USING CHEMICAL SYNTHESIS, BUT THERE'S ALSO PROMISE IN LOOKING AT ENZYMES IN THESE AUTOMATED PROCESSES. AND THE NIH HAS BEEN VERY INSTRUMENTAL IN BEING ABLE TO GET A LOT OF THESE -- SUPPORT THE PRODUCTION OF THESE ENZYMES AND THEIR DISTRIBUTION ACROSS LABS. SO THIS IS REALLY, REALLY EXCITING BECAUSE YOU CAN START TO SEE A LOT OF COMBINED CHEMICAL AS WELL AS ENZYMATIC PROCESSES TO ACCESS GLYCANS. -- AT GEORGIA STATE UNIVERSITY, ALSO USED A TRADITIONAL PEPTIDE SYNTHESIZER, AUTOMATED PEPTIDE SYNTHESIZER BUT THEN THEY WERE ABLE TO DEVELOP NEW METHODS WHEREBY THEY CAN COMBINE CHEMICAL AND ENZYMATIC METHODS TO DIVERSIFY O-GLYCANS AND O-GLYCOPEPTIDES TO A VARIETY OF DIFFERENT STRUCTURES. WE STILL DON'T HAVE EVERY ENZYME YOU CAN IMAGINE TO MAKE EVERY STRUCTURE YOU CAN SEE, BUT THIS HAS BEEN REALLY COOL TO WORK TOGETHER TO SEE WHERE ARE THE HOLES, WHERE DOES IT MAKE MOST SENSE TO MAKE THESE USING ENZYMATIC METHODS, AND WHERE IS IT THE CHEMICAL SYNTHESIS PEOPLE SHOULD FOCUS MORE EFFORTS ON. SO THERE'S BEEN A LOT OF ADVANCES IN MAKING THE O-GLYCANS AND HUMAN -- GLYCANS USING THIS SYNTHESIS METHOD AS WELL AS COMBINATION METHODS. THEY ALSO USED THIS CHEMICAL AND ENZYMATIC COMBINATION TO LOOK AT GLYCOLIPIDS. NOW THIS IS EVEN A KIT, SO YOU DON'T HAVE TO TRY TO GET ALL THESE THINGS TOGETHER YOURSELF, BUT YOU CAN NOW BUY A KIT THAT ALLOWS YOU TO CHEMICALLY START OUT WITH A TAG, THIS LIPID, AND THEN USING A SERIES OF ENZYMES AS WELL AS C18 SOLID PHASE EXTRACTION CARTRIDGES, YOU CAN BUILD UP YOUR GLYCOLIPID OF CHOICE SO YOU'RE NOT LIMITED NECESSARILY TO ONE COMPOUND YOU MIGHT GET FROM A COMPANY, BUT YOU CAN START USING THIS AS A STARTING POINT TO MAKE A VARIETY OF DIFFERENT GLYCOLIPIDS. SO A COMBINATION OF CHEMICAL AND ENZYMATIC SYNTHESIS, SAME SORT OF PROTOCOLS OF USING CHROMATOGRAPHY, AFFINITY CHROMING CHROMATOGRAPHY. SO IN PARALLEL, ONE OF THE CHALLENGES WE HAVE IS THE CHEMISTRY OF AUTOMATION PLATFORMS ARE QUITE EXPENSIVE INSTRUMENTS, SO ALEXA HAD THIS IDEA OF CAN WE COME UP WITH PERHAPS BETTER SOLID PHASE RESINS AND THEN USE AN OFF THE SHELF HPLC AND MODIFY THAT SO EVERYONE WHO HAPPENED TO HAVE AN HPLC COULD ALSO START MAKING GLYCANS. I WON'T SAY MORE ABOUT THIS UNTIL WE TALK AGAIN TOMORROW, BUT THERE'S ALSO BEEN A LOT OF IMPROVEMENTS THERE IN BEING ABLE TO ADAPT HPLC TO START AUTOMATED GLYCAN SYNTHESIS. WE'VE ALSO HOSTED STUDENTS IN MY LAB IN INDIANA TO HELP DEVELOP THESE TECHNOLOGIES IN PARALLEL. AND THIS IS JUST AN EXAMPLE OF SOME OF THE COMPOUNDS HIS GROUP HAS BEEN MAKING, THESE HUMAN LITTLE BEING OLIGOSACCHARIDES, ONCE AGAIN BY THIS SORT OF AUTOMATION PROCESS AND LOOKING AT TOOLS AND TECHNIQUES TO AUTOMATE THAT SYNTHESIS. ONCE YOU HAVE THAT HPLC, YOU CAN MODIFY IT BY A SLIGHTLY DIFFERENT WAY OF HAVING ANOTHER COLUMN AND PURIFYING THOSE COMPOUNDS. THIS IS A COLLABORATION, TO BE ABLE TO COME UP WITH REALLY GOOD WAYS OF PURIFYING THESE SYNTHETIC COMPOUNDS BEFORE WE DO PROTECT THEM, IDEALLY SO WE HAVE BETTER THAN 9 # 99% PURE 99% PURITY SO TH AT SMALL AMOUNT DOESN'T AFFECT ACTIVITY. BASICALLY BY PUTTING TWO COLUMNS IN A ROW, IT'S LIKE HAVING A SUPER LONG COLUMN, WE CAN EASILY CONVERT A REGULAR HPLC INTO A RECYCLING HPLC PROTOCOL BUT IN A METHOD THAT DOESN'T PUT THE COMPOUNDS THROUGH THE PUMPS THEMSELVES, SO THEREFORE, YOU DON'T HAVE TO WORRY ABOUT THE CHEMICAL ENTITIES OF THE COMPOUNDS THAT MAY DESTROY THE SEALS AND THE PUMP ITSELF. SO NOW WE HAVE ALSO A GOOD WAY -- THIS IS -- HPLCs ARE ALREADY AUTOMATED PROTOCOLS WE CAN SHARE, TO BE ABLE TO PURIFY OUR INTERMEDIATES OR FINAL COMPOUND TO THAT 99.5% PURITY THAT IS REALLY NEEDED FOR FOR ESPECIALLY IMMUNOLOGICAL STUDIES. SO WE'VE MADE IMMENSE PROGRESS, OF COURSE THERE'S ALSO A LOT OF DIFFERENT GLYCANS, FOR EXAMPLE ON MICROBIAL SURFACES THAT YOU'LL HEAR MORE ABOUT LATER TODAY ALSO, WE THINK ABOUT AN AUTOMATED -- DNA ONLY NEEDS 4 TO MAKE EVERY POSSIBLE UNMODIFIED STRUCTURE LIKE A FACTORY PRODUCTION LINE, BUT GLYCAN DIVERSITY IS MASSIVE. WE WOULD NEED AT LEAST HUNDREDS OF BUILDING BLOCKS JUST TO MAKE THE MAMMALIAN SUGARS, AND SO THE OTHER THING THAT WE'VE DONE IN THIS UO1 PROGRAM IS RE-THINKING HOW YOU LOOK AT SYNTHESIS. HISTORICALLY WE LOOKED INTO DNA AND PEPTIDES BECAUSE THAT'S THE CHEMISTRY THAT WORKED FOR THOSE BIOMOLECULES AND SAID OKAY, HOW DO WE BEST MIMIC THAT. WE REALLY WERE COMING TO THE CONCLUSION THAT'S NOT GOING TO WORK, WE NEED A DIFFERENT MODEL FOR GLYCANS JUST BECAUSE OF THE DIVERSITY. FOR THAT, I CONCEPTUALIZED THIS AS, WELL, AMAZON HAS LOTS OF BOOKS BUT THEY DON'T NEED EVERY ONE IN STOCK. THEY NEED A METHOD OF GETTING TO IT VERY QUICKLY OR PRINTING IT ON DEMAND, AND THAT'S EXACTLY WHERE WE'RE STARTING TO GO WITH GLYCAN SYNTHESIS, GETTING AWAY FROM THE TRADITIONAL FACTORY MODEL TO ONE OF HOW DO WE VERY QUICKLY MAKE THAT BUILDING BLOCK ON DEMAND SO YOU CAN GET ACCESS TO IT AND PUT IT INTO YOUR SYNTHESIZER. THE REALITY IS, THIS IS NOT YET AUTOMATED. WE'VE AUTOMATED THE STEPS TO PUT THE BUILDING BLOCKS TOGETHER AND DO -- BUT THE SYNTHETIC STEPS TO MAKE THE BUILDING BLOCKS NOW ARE A MAJOR TIME SINK FOR CHEMISTS AROUND THE WORLD STILL. SOME OF THESE ARE COMMERCIALLY AVAILABLE BUT A VERY, VERY LIMITED SET OF BUILDING BLOCKS IS COMMERCIALLY AVAILABLE. THE VAST MAJORITY, WE HAVE TO START FROM SCRATCH, MAKING THROUGH THESE OLD-FASHIONED MANUAL METHODS THAT CAN BE FUN, CRYSTALS, COOL COMPOUNDS, BUT IT REALLY SLOWS DOWN OUR ABILITY TO ACCESS GLYCANS. AND SO THE IDEA IS HOW DO WE DO THIS ON DEMAND. HAVE A NEW PARADIGM. IDEALLY WE HAVE A MUCH CHEAPER SYNTHESIZER AS OPPOSED TO MORE LARGE AND COMPLEX SYNTHESIZER WHERE WE'RE PUTTING THE ENTIRE GLYCAN TOGETHER. IDEALLY WE CAN HAVE AUTOMATED PROTOCOLS THAT ANYTHING THAT WE DEVELOP AS CHEMISTS COULD EASILY BE TRANSFERRED TO A CONTRACT SYNTHESIS FIRM, FOR EXAMPLE, OR IN YOUR OWN LAB IF YOU HAVE A FUME HOOD AND SOME HPLC SET UP SO YOU CAN REPRODUCE EVERY CHEMICAL STEP AT THE DESIRED SCALE JUST IN TIME TO ACTUALLY US OOO THE BUILDING BLOCK. SO THE SCALING ISSUE BROUGHT US TO FLOW PROCESSES, CONTINUOUS PROCESSES AS OPPOSED TO THE BATCH PROCESSES I'VE TALKED TO YOU ABOUT UNTIL NOW, WHERE WE'RE MAKING A CERTAIN DEFINED SCALE. HERE WITH FLOW PROCESSES, IF WE JUST FLOWED CHEMICALS IN THE TUBES LONG ENOUGH, THEN WE CAN MAKE AS MUCH COMPOUND AS THE TIME IT TAKES AND THE REACTION IS ONLY HAPPENING IN VERY SMALL SUBSECTION IN THAT TUBING. SO TO THIS END, MY LAB HAS BEEN WORKING ON A PYTHON-BASED PROGRAM THAT'S OPEN SOURCE THAT CONTROLS THIS TINY LITTLE $35 RASPBERRY PIE, AND THAT ALLOWS TO YOU CONTROL THE SYRINGE PUMPS AND THE HPLC PUMPS THAT MANY CHEMISTS AND BIOLOGISTS TOO ALREADY HAVE SITTING AROUND OR YOU CAN BUY THEM ON THE USED MARKET, AND THEN YOU DON'T NEED TO ACTUALLY CONTROL EVERYTHING YOURSELF, BUT YOU CAN GET A PROGRAM THAT THEN CAN BE SHARED FROM LAB TO LAB TO LAB IN A MUCH MORE COST-EFFECTIVE WAY. WE ALSO SEE THIS AS A GOOD WAY OF EDUCATING A MUCH LARGER GROUP ON AUTOMATED CHEMISTRY THAN YOU WOULD GET JUST USING THE MORE EXPENSIVE FULL HPLC SETUPS. THE NICE THING IS THIS ALSO SPECIFIES THE APPARATUS. UNLIKE IN MY NORMAL LARGE SCALE APPARATUS, I CAN GIVE YOU THE CODE IF YOU HAVE THE SAME APPARATUS, IF YOU DON'T, YOU HAVE TO REPROGRAM YOUR OWN APPARATUS. HERE, WE TELL YOU EXACTLY HOW TO BUILD IT FROM STANDARDIZED PARTS THAT YOU CAN MIX AND MATCH, AND THEN WE HAVE A SETUP THAT ALLOWS US TO GO FROM THERE AND PROGRAM THE ACTUAL CHEMISTRY. SO MY GROUP STARTED WITH WHAT ARE THE REACTIONS WE NEED TO GET DONE IN FLOW PROCESSES TO MAKE THESE BUILDING BLOCKS AND UNFORTUNATELY MANY OF THEM RELIED ON SOLID PHASE REAGENTS THAT MAKE IT MUCH MORE DIFFICULT. SO THIS IS WORK THAT WAS CARRIED OUT TO LOOK AT FOR EXAMPLE THE BENZYL REACTION, THE ONE EVERYONE USES FOR BLOCKING CARBOHYDRATE HYDROXYL GROUPS UNTIL THE END OF A SYNTHESIS AND THE TRADITIONAL WAYS OF DOING THIS WILL OFTENTIMES INVOLVE SOMETHING LIKE SODIUM -- CAME UP WITH A CLEVER WAY OF ADAPTING A COLUMN WITH BARIUM OXIDE AND FLOWING THIS THROUGH TO BE ABLE TO GET THESE BENZYLATIONS AND FOUND A WAY OF MAKING THIS MUCH MORE OFFICIALLY THAN WHAT WAS ALREADY IN THE LITERATURE THROUGH THE PROCESS OF THINKING WILL ABOUT HOW DO WE AUTOMATE THESE PROCESSES AND PUT THEM INTO FLOW VERSUS BATCH PROCESSES. SINCE THEN, WE ALSO HAVE BEEN WORKING WITH CLAY BENNETT'S LAB AT TUFTS TO REALLY DEVELOP THIS INTO A WIDE VARIETY OF DIFFERENT BUILDING BLOCK STRATEGIES THAT I CAN'T TELL YOU ABOUT ALL THE EXCITING WORK THAT'S GOING ON THERE, I'M QUITE HAPPY ABOUT HOW THIS HAS GONE IN THE LAST FEW YEARS, BECAUSE IT LOOKS LIKE WE FIND THEY'RE NOW ABLE TO PRODUCE A WIDE VARIETY OF BUILDING BLOCKS ON DEMAND. NOT ONLY THAT, BUT WE'VE BEEN WORKING TOGETHER TO CONCATENATE STEPS, FOR EXAMPLE, SET UP A LARGER FLOW SYSTEM AND DO MULTIPLE STEPS SO YOU CAN TAKE SOMETHING LIKE COMMERCIALLY AVAILABLE L-RHAMNAL AND MAKE THIS PROTECTED BUILDING BLOCK IN NOT MUCH TIME AT ALL COMPARED TO WHAT IT NORMALLY TAKES, AND NOT ONLY THAT, IT TAKES UP VERY FEW -- THE BEAUTY OF THE FLOW SYSTEMS, WE DISCOVERED THAT WE DIDN'T ACTUALLY NEED THE ACTIVE HEATING AND COOLING AND WE HAD MUCH SHORTER REACTION TIMES THAN WAS THE CASE WHEN THESE WERE DEVELOPED IN BATCH PROCESSES. THE INITIAL WORK WAS JUST PUBLISHED LAST YEAR AND WE'RE WORKING ON THE NEXT PAPERS THAT WILL EXPAND THIS INCLUDING SOME MICROBIAL BUILDING BLOCKS. SO WE'RE GETTING TO THE POINT WHERE WE CAN START BEING ABLE TO AUTOMATE NOT ONLY THE BUILDING BLOCKS -- NOT ONLY THE GLYCAN SYNTHESIS BUT ALSO PUTTING THE BUILDING BLOCKS TOGETHER. PART OF THIS USING THIS LESS EXPENSIVE OUGHT MAKE AUTOMATION SYSTEM BESIDES THE TRAINING ASPECT AND THE ABILITY TO SPREAD THIS MUCH MORE WIDELY IS THE ABILITY TO ALSO HAVE EVERYTHING IN AN OPEN SOURCE FORM THAT ALLOWS YOU TO MAKE EVERYTHING ACCESSIBLE. AND SEARCHABLE. SO MY LAB HAS DEVELOPED AN INTERFACE THAT ALLOWS YOU TO HAVE NOT ONLY THE CODE BUT ALSO ALLOWS YOU TO HAVE EVERYTHING ELSE YOU WOULD EXPECT TO SEE IN A NORMAL NOTEBOOK. WE'RE ALSO STARTING TO PUT ENTRY KEYS AND ENTRY STRINGS IN THERE, THE FORMAT THAT COMPUTERS USE TO SEARCH FILES SO YOU CAN HAVE THESE PROTOCOLS OF PDF FILES AND YOU CAN SEARCH AND FIND IT QUITE EASILY. WE'RE WORKING TO PUT THIS INTO A REPOSITORY SO OUR SYNTHESIS PROCEDURES ARE NOT ONLY SCALABLESE LEGAL FROM USING FLOW CHEMISTRY, BUT NOW THEY'RE FINDABLE THROUGH THIS MECHANISM, AND THEY'RE ACCESSIBLE AND POTENTIALLY INTEROPERABLE AND ALL OF THIS IS BASED ON OPEN SOURCE PLATFORMS FOR SHARING THAT INFORMATION. SO WITH THAT, I WANT TO FORESHADOW SO THE WORKING GROUP -- SOME OF THE THINGS YOU'LL HEAR ABOUT LATER IN THE DAY, SOME OF THE WORK I'VE BEEN TALKING ABOUT TODAY AND YOU'LL HEAR ABOUT LATER TODAY IS BEING TRANSLATED INTO COMPANIES SUCH AS THESE. WE'VE ALSO WORKED TOGETHER IN MULTIPLE TIMES IN COMING WITH REVIEW ARTICLES, SPECIAL REVIEW ISSUES LIKE THIS VERSION OF CHEMICAL REVIEWS THAT I HELPED EDIT EARLIER DURING THIS TIME, TO BE ABLE TO SPREAD THE WORD ABOUT GLYCAL. SO WITH THAT, I'D BE HAPPY TO TAKE SOME QUESTIONS. THANK YOU. >> THANK YOU, NICKI. WE HAVE TIME, SO PLEASE UNMUTE YOURSELF AND ASK QUESTIONS IF YOU HAVE THEM. I'LL START. NICKI, ARE ALL OF THE METHODOLOGIES PUBLISHED NOW FOR PRODUCING THE AUTOMATED SYSTEM OR INDIVIDUAL METHODS? >> MANY OF THEM ARE BUT IT'S BEEN SLOWED DOWN, TO BE HONEST, WITH THE PANDEMIC. SO WE'RE STILL IN THE PROCESS OF PUBLISHING A LOT OF THESE PROTOCOLS AND GETTING THINGS OUT THERE. BUT YOU SAW, I PUT IN PUBLICATIONS WHERE THINGS ARE PUBLISHED FOR A LOT OF THESE DIFFERENT METHODS, BUT THE WORK IS STILL ONGOING TO GET THOSE OUT THERE. AND AS I SAID, WE'VE HOSTED MULTIPLE PEOPLE IN MY LAB FROM TANG'S LAB AND ALEXA'S LAB, WE'RE GOING TO HOST SOME PEOPLE FROM LINDA SHAI'S LAB NEXT WEEK. IT'S BEEN A SLOWDOWN BECAUSE OF THE PANDEMIC RESTRICTIONS TO ACTUALLY HOST PEOPLE AND THEREBY FINISH SOME OF THE WORK THAT -- WE'VE BEEN SENDING COMPOUNDS BACK AND FORTH BUT WE HAVEN'T ACTUALLY GOTTEN TO WORK TOGETHER ON THE ACTUAL AUTOMATION PLATFORM. BECAUSE PART OF THIS IS ALSO MAKING SURE THAT A WIDE VARIETY OF SCIENTISTS FEEL COME COMFORTABLE USING AUTOMATION PLATFORMS BY SEEING IT IN USE IN A LAB. >> THANK YOU. DOUG, DID YOU HAVE A QUESTION? I SEE YOUR HAND UP. >> YEAH. THANKS, NICKI. THAT WAS A GREAT OVERVIEW OF ALL THESE THINGS AND I CAN'T WAIT TO HEAR THE OTHER TALKS. I WONDER, EVEN THOUGH IT'S KIND OF EARLY DAYS FOR SOME OF THESE LATER METHODS YOU WERE TALKING ABOUT, WHAT DO WE KNOW ABOUT UPTAKE IN THE COMMUNITY OF PEOPLE WHO AREN'T DEVELOPERS OF THESE METHODS? WE'VE HAD GREAT COOPERATION BETWEEN THE PEOPLE WHO ARE WORKING ON THESE TECHNIQUES, AND WE'VE BUILT FASTER AND BETTER BECAUSE OF THAT INTERACTION, BUT WHAT CAN WE SAY AT THIS POINT ABOUT HOW OTHER FOLKS HAVE DECIDED TO TAKE THESE THINGS UP? >> WELL, YOU'LL HEAR SOME OF THESE FROM THE -- TALKS LATER IN THE DAY. WHAT I SEE, THERE'S CERTAINLY SLOWLY OTHER CHEMISTS WITHIN THE SYNTHETIC COMMUNITY ARE STARTING TO THINK SERIOUSLY ABOUT AUTOMATION, WHICH IS ALREADY A CHANGE, WHICH IS GOOD, AND THIS WILL HELP NOT JUST GLYCAN SYNTHESIS BUT MAKING A LOT OF DIFFERENCE -- MORE REPRODUCIBLE. THE NEXT STEP IS TO BE ABLE TO HAVE MORE COMPANIES FEEL COMFORTABLE, FOR EXAMPLE, DOING A QUOTE, SO THE IDEA IS NOT NECESSARILY THAT A BIOLOGIST IS GOING TO RUN OUT THERE AND USE THIS AUTOMATION TO MAKE SOMETHING, BUT BECAUSE IT'S AN AUTOMATED PROCESS, IT'S GOING TO BE -- THERE AREN'T GOING TO BE AS MANY THINGS YOU HAVE TO TROUBLESHOOT SO THAT YOU CAN REPRODUCE IT, AND THAT MEANS THAT A CONTRACT SYNTHESIS FIRM IS GOING TO BE ABLE TO REPRODUCE THE SYNTHESIS OF THESE AUTOMATED PROCESS MUCH MORE RELIABLY AND MORE QUICKLY, THEREFORE LESS EXPENSIVELY, THAN IT WOULD BE IF THESE ARE STILL MANUAL PROCESSES. SO THAT'S WHERE I REALLY SEE THINGS GOING, IS THAT MORE OF THESE AUTOMATED PROTOCOLS ARE OUT THERE IN THE LITERATURE AND MORE PEOPLE FEEL COMFORTABLE USING AUTOMATION, THERE'S A LOT OF AUTOMATION IN THE INDUSTRY, FRANKLY, COMPARED TO AN ACADEMIC -- IF YOU HAVE AN AUTOMATED PROCESS WHERE EVEN THE SMALLEST THING HAS TO BE SPECIFIED, IT CAME TO ME WHEN JUST STARTING AUTOMATION OVER 15 YEARS AGO NOW, THE STUDENT MENTIONED THE FACT THAT YOU SHOULD PROGRAM THE NEEDLE TO DELIVER THE REAGENT ABOVE THE SURFACE OF THE SOLUTION RATHER THAN GO INTO THE SOLUTION AND THEN RELEASE THE -- IN THIS CASE, IT ACTUALLY WOULD CHANGE THE OUTCOME OF THE REACTION. AND THAT LEVEL OF DETAIL IS NOT SOMETHING YOU'D EVEN THINK TO WRITE IN THE MANUAL PROCEDURE, WHEREAS IF YOU HAVE AN AUTOMATED PROCEDURE, ALL THAT LEVEL OF DETAIL IS IN THERE SO IT BECOMES MUCH FASTER TO BE ABLE TO REPRODUCE PROCEDURES ON THE -- THAT YOU WANT. BECAUSE I DON'T SEE BIOLOGISTS STARTING TO HAVE LITTLE SYNTHESIS OPERATIONS IN THEIR -- EXCEPT WE DO HAVE SOME THAT I DIDN'T TALK ABOUT, SOME ONE-STEP 40 SESS PROCESSES TO MAKE GLYCOSYLATED AMINO ACIDS, SOMETHING YOU COULD SEE ADOPTING MORE WIDELY, BUT A LOT OF THIS WILL BE IN THE SENSE OF HAVING CONTRACT SYNTHESIS FIRMS BE ABLE TO MAKE SOMETHING THAT YOU WANT MUCH MORE QUICKLY AND MUCH MORE CHEAPLY SO IT DOESN'T TAKE HALF THE YEAR AND TENS OF THOUSANDS OF DOLLARS TO MAKE A SINGLE COMPOUND, BUT IT CAN BE DONE WITHIN A MONTH BECAUSE THE VARIABLES HAVE ALL BEEN SPECIFIED. >> THANKS, NICKI. THAT'S REALLY INTERESTING. >> DO WE HAVE ANY OTHER QUESTIONS? RAJA, DID YOU HAVE A QUESTION? >> YEAH. THE SLIDE WHERE YOU SHOWED THE JUPYTER NOTEBOOK, THAT'S GREAT. SO I HAVE A QUESTION. SO UP SO ALL THE CODE, YOU SAID YOU ARE GOING TO PUT ON GITHUB, DO YOU HAVE STEP BY STEP INSTRUCTIONS HOW TO USE THE CODE, LIKE A README WITH EXAMPLE FILES AND SO ON, THAT WOULD ALSO BE REALLY HELPFUL. >> THAT'S A GREAT POINT. SO WE'RE ALSO DEVELOPING AN UNDERGRAD LAB THAT USES THE BASIC IDEA OF THE JUPYTER NOTEBOOK AND -- BOOK CODE FOR -- JOURNAL OF MEDICAL EDUCATION IS WHERE WE'LL SUBMIT IT, WE HAVEN'T BEEN ABLE TO REPLICATE IT BECAUSE OF ALL THE LABS THAT HAVE GONE ONLINE. MALLORY IN MY LAB HAS BEEN PUTTING TOGETHER VIDEOS TO SHOW PEOPLE HOW TO DO SOME OF THESE THINGS BECAUSE YOU'RE RIGHT, PART OF OF THIS IS ALSO BEING ABLE TO PUT ALL THE METADATA AROUND IT SO THAT THAT DATA ACTUALLY MEANS SOMETHING. AND WE'RE ACTIVELY WORK ON THAT. ALSO WE TALKED TO -- GLYGEN WILL MAKE THIS FINDABLE THROUGH THESE LARGER DATA INTERFACES. >> YEAH, THAT WILL BE GREAT. THANK YOU. >> NICKI, YOU MENTIONED CHEMICAL REVIEWS. ARE ALL THE METHODS GOING TO BE LIKE ON A DETAILED PROTOCOL WHERE SOMEBODY COULD JUST REPRODUCE IF THEY HAD IT, OR -- >> SO RIGHT NOW, THE THINGS THAT WE HAVE IN THE PDF FILES ARE ANY PERSON TRAINED IN THE ART SHOULD BE ABLE TO REPRODUCE THESE THINGS. WHAT WE'RE WORKING TOWARD IS BEING ABLE TO USE THE JUPYTER NOTEBOOK INTERFACE WHERE EVERYTHING IS AUTOMATED SUCH THAT YOU DON'T NEED TO BE A TRAINED CHEMIST ANYMORE, YOU CAN PUT PARTS TOGETHER LIKE BUILDING BLOCKS, LIKE LE LEGOS THAT I PLAYED WITH AS A KID, BE ABLE TO MAKE YOU SOLUTIONS, GET YOUR COMPOUND AND USE AN AUTOMATED PROTOCOL AN YOUR HPLC ALSO ALL THERE. THOSE PROTOCOLS, WE'RE STILL HAVING TO DISCUSS, HOW DO WE PRESENT THESE. THE CHEMICAL LIT TEURL IS NOT -- HASN'T ADAPTED YET TO THE NEW REALITY OF GOING TOWARD AUTOMATION BECAUSE RIGHT NOW THE WAY YOU PRESENT THINGS ARE PDF FILES, YOU CAN START -- YOU'RE GOING TO SEE WHETHER WE CAN START ADDING SOME OF THESE OTHER FILES -- FILE TYPES AND SUPPORTING INFORMATION BUT FRANKLY THINGS THAT ARE SUPPORTING INFORMATION ARE HARDER TO FIND. THEY'RE NOT AS ACCESSIBLE AS THINGS IN A REPOSITORY SOMEWHERE. SO THAT'S STILL SOMETHING THAT WE AS A COMMUNITY HAVE TO WORK ON AND WORK OUT THE BEST WAYS OF SHARING THESE PROTOCOLS. THE -- EVERYTHING SPECIFIED EXACTLY HOW LONG IS THE TUBING, WHAT'S THE TUBING MADE OUT. INCREDIBLE LEVEL OF DETAIL FROM WHAT WE'VE BEEN PUBLISHING SO FAR. >> THANKS. THAT'S VERY HELPFUL. AND PEOPLE WITHIN YOUR GROUP ARE DEVELOPING METHODS TO MODIFIED THESE COMPOUNDS OR ARE THEY JUST THE COMPOUNDS AND CAN THEY SULFATE THEM OR PHOSPHORYLATE THEM OR -- >> YEAH, SO WE HAVE PROTOCOLS TO DO SULFATION AND WE'RE GOING TO BE WORKING WITH LINDA SHAI WILSON'S GROUP TO SEE IF WE CAN APPLY SOME OF THESE TO THE GLYCOAMINO GLYCANS SHE'S WORKING ON. NEXT WEEK HER STUDENT WILL ACTUALLY BE PHYSICALLY IN BLOOMINGTON IF ALL GOES WELL, AND WE ALSO STARTED LOOKING AT THESE GLYCO -- TO BE ABLE TO SITE-SELECTIVELY -- LIKE SULFUR BECAUSE THEY'RE KNOWN TO BE MORE STABLE AGAINST GLIE KOAS NAISES. AND I ALLUDED TO WORK THAT CLAY BENNETT AND I HAVE BEEN WORKING ON IN MAKING MOWR BUILDING MORE BUILDING BLOCKS THAT ARE MICROBIAL SO THEY'RE MORE DEOXYGENATED FROM WHAT I'VE SHOWN THUS FAR. SO THE U01 HAS BUILT A FANTASTIC FOUNDATION TO START GOING INTO A WIDE VARIETY OF GLYCAN SPACE BEYOND WHAT WE'VE BEEN ABLE TO DO SO FAR. >> THAT'S REALLY EXCITING. THANK YOU VERY MUCH. SO THAT'S WHAT THE CHEMISTRY GROUP HAS BEEN DOING AND YOU'LL HEAR ABOUT THE INDIVIDUAL EFFORTS WITHIN THAT GROUP DURING THE NEXT TWO DAYS. CATHERINE GRIMES IS GOING TO TELL US WHAT THE TOOLS GROUP HAS BEEN UP TO. CATHERINE IS THE CHAIR OF THE TOOLS WORKING GROUP, AND SHE'LL TELL US ABOUT NEW TOOLS AND TECHNOLOGIES FOR TBLIE GLYCOSCIENCE. CATHERINE? >> THANKS. SORRY, I'M TRYING TO GET MY TIMER SO I CAN SEE IT. SO I'M A PROFESSOR AT THE UNIVERSITY OF DELAWARE. I'VE BEEN LUCKY TO LEAD A DYNAMIC SUBGROUP EXTREMELY INTERESTED IN DEVELOPING GLYCOBIOLOGY TOOLS FOR NON-EXPERT. I'M EXCITED TO TELL BUT SOME OF THE WORK THAT OUR TEAM HAS DEVELOPED, THE MAJOR AREAS WE'RE WORKING ON AND FOR THE COMPANIES IN THE AUDIENCE OR PERHAPS LISTENING LATER ON, HOPEFULLY EXCITE YOU ABOUT OUR WORK TO WORK WITH US TO HOPEFULLY COMMERCIALIZE SOME OF OUR TOOLS AND ASSAYS. I THINK THE WORST NIGHTMARE IS ALL OF OUR WORK OVER THE NEXT FIVE OR SIX YEARS ENDS UP IN THE BACK OF OUR MINUS 80 FREEZERS AND NOT IN THE HANDS OF SCIENTISTS WHO NEED THESE TOOLS. NICKI DID A BEAUTIFUL JOB HIGHLIGHTING HOW HER SUBGROUP IS WORKING TO MAKE SYNTHETIC METHODS USABLE. THIS SUBGROUP IS THE TOOL BUILDERS. THE OUR COLLEAGUES HAVE RUN INTO ROAD BLOCKS AND CAN'T PROCEED WITH INTERESTING BIOLOGICAL QUESTIONS AS THE TOOLS AND ASSAYS SIMPLY DON'T EXIST. OUR GROUP HAS WORKED HARD NOT ONLY TO DEVELOP THE TOOLS AND TECHNOLOGIES BUT THEN TO ASSURE THAT THEY'RE IN THE HANDS OF THE, QUOTE-UNQUOTE, NON-EXPERT TO EU THEM. USE THEM. WE HAVE A GROUP OF 27 ACTIVE PRIMARY INVESTIGATORS AND THOSE THAT -- WHOSE FUNDING HAS ENDED STILL PARTICIPATE. WE MEET EACH MONTH TO DISCUSS OUR PROGRESS AND HELP EACH OTHER THINK CREATIVELY ABOUT HOW BEST TO ADAPT OUR TOOLS FOR THE NON-EXPERT. WE'RE EXCITED TO HAVE THE OPPORTUNITY TO TELL YOU ABOUT OUR WORK AS THE PROGRAM UNFOLDS OVER THE NEXT FEW DAYS, AND THE PURPOSE OF THIS TALK IS TO INTRODUCE SOME OF THE TOOL DEVELOPERS AS A WHOLE AND SET THE STAGE FOR SOME OF OUR PROGRAMMING. THE WORK THAT WE DO CAN BE DIVIDED INTO A VARIETY OF BINS. SOME OF THESE TOOLS OVERLAP BETWEEN BINS. WE WORKED TO DEVELOP MASS-SPEC APPROACHES FOR CHARACTERIZING AND QUANTIFYING GLYCANS. WE HAVE NEW METHODS AND TOOLS TO PROFILE GLYCANS IN COMPLEX MIXTURES. AND HAVE STARTED TO PRODUCE GLYCAN BINDING PROTEINS FOR DETECTING AND ENRICHING GLYCANS H A SUBSET OF OUR INVESTIGATORS HAVE NEW MECHANISMS TO DETECT CHANGES IN THE BIOSYNTHESIS OF GLYCANS AND OTHERS ARE WORKING ON BIOINFORMATICS TOOLS FOR STUDYING GLYCANS. WE HAVE DEDICATED CHEMICAL BIOLOGY TOOLS FOR PATHWAYS AND TOOLS TO MANIPULATE GLYCAN LEVELS. MANY OF OUR INVESTIGATE INVESTIGATORS ARE HERE TO HIGHLIGHT THAT'S PRORNLGS AND THEIR PROJECTS. -- THE PRIMARY GOAL OF PROFESSOR LI'S WORK IS TO DEVELOP SEVERAL VERSATILE MASS DETECTED MULTIPLEX TAGS FOR HIGH-THROUGHPUT QUANTIFICATION OF GLYCANS AND GLYCOPEPTIDES IN COMPLEX BIOLOGICAL SAMPLES USING MASS SPEC AND ION -- SHE HAS DEVELOPED A SERIES OF MS1 AND MS2 PROBES FOR QUANTIFICATION OF BIOLOGICAL SAMPLES, REALLY AMAZING. COLLECTIVELY, DR. LI AIMS TO DEVELOP NOVEL ENABLE TOOLS AND WILL GENERATE COST-EFFECTIVE AND NOVEL MASS SPEC LABELING REAGENTS FOR ROBUST SENSITIVE AND ACCURATE GLYCAN ANALYSIS WITH ENHANCED QUANTITATIVE PERFORMANCE AND STRUCTURAL ELUCIDATION CAPACITIES. METHODS ALLOW GLYCAN STRUCTURES AND SITES OF ATTACHMENTS TO BE DISCERNED FOR COMPLEX BIOLOGICAL SAMPLES FOR BOTH N AND O GLYCANS ALIKE. A MAJOR BREAKTHROUGH IS THE METHOD DOES NOT REQUIRE TRUNCATION OF THE GLYCANS ALOING RICH INFORMATION TO REMAIN INTACT DURING ANALYSIS. IN ADDITION, THIS METHOD THEY HAVE TERMED ISOTOAP TARGETED DPLIE COPROTEOMICS ENRICHES GLYCOPROTEINS -- ISOTAG ALSO MITIGATES THE NEED FOR -- AND COMPUTATIONAL TIME. ALSO THEY HAVE SINCE BEEN ABLE TO TRANSFER THE ISOTAG METHOD TO INTERESTED LABS WHO HAVE NO PRIOR GLYCOBIOLOGY EXPERTISE WHO ARE THEN ABLE TO SUCCESSFULLY PERFORM ANALYSIS ON THEIR SAMPLES OF INTEREST. WITH THESE BARRIERS TO LARGE SCALE IMPLICATION OF GLYCOPROTEOMICS SUBSTANTIALLY REDUCED, THEY HAVE MADE ISOTAG ACCESSIBLE TO THE BROADER SCIENTIFIC COMMUNITY AS A STANDARD SERVICE, OFFERED BY MS CORES ACROSS THE COUNTRY. IN ADDITION, THESE TOOLS ARE AVAILABLE SO YOU CAN PURCHASE THEM. PROFESSOR RICHARD JAKE AND COLLEAGUES AIMED TO DEVELOP RAPID AND REPRODUCIBLE GLYCAN PROFILING METHODS FOR MOST AVAILABLE BIOLOGICAL SAMPLES. SERUM, PLASMA, CELL LINES, IMMUNE CELL SUBSETS. THE KEY TO THE METHODS ARE THAT THEY ARE SLIDE-BASED AND DO NOT REQUIRE EXTENSIVE PROCESSING, THEY ONLY USE MINIMAL AMOUNTS -- ADOPTING ANTIBODY ARRAYS ALLOWS -- FROM THE BIOLOGICAL SAMPLES. ANY CULTURED CELL LINE CAN BE EVALUATED USING MASS SPECTROMETRY. THE NEXT STEPS ARE TO DEVELOP WORKFLOWS FOR NON-ADHERENT CELLS ALLOWING THE MOLECULAR -- WHICH HE HAS DEVELOPED TO LOOK FOR THESE CHANGES. HE'S LOOKING FORWARD TO TELLING YOU MORE ABOUT THIS TECHNOLOGY IN HIS TALK TODAY. PROFESSOR ZAIA WANTS TO ELIMINATE BOTTLENECKS. ANALYTICAL METHODS WERE USING LCMS ARE MATURE AND AVAILABLE TO BIOMEDICAL SCIENTISTS WITH ACCESS TO THE MASS SPECTROMETRY INSTRUMENTS. HE AND HIS TEAM HAVE DEVELOPED THREE SOFTWARE PROGRAMS FOR GLYCOSCIENCE MASS SPECTROMETRY THAT HE NOW PROPOSES TO ENGINEER AND DISSEMINATE AS A SUCCESSFUL SOLUTION FOR BIOSCIENTISTS THEY'VE DEVELOPED SOFTWARE THAT YOU OOOS MODERN GLYCAN REPRESENTATION STANDARDS AND COMMUNICATES WITH THE INTERNATIONAL REPOSITORY AND THEY HAVE BEEN VERY SUCCESSFUL IN DEVELOPING GAG FINDER SOFTWARE INTO A COMPLETE SOLUTION FOR SEQUENCING. THE GOAL OF DR. YANG'S TEAM IS TO CREATE A METHOD FOR CHARACTERIZING GLYCOSYLATION FEATURES BASED AROUND FLOW CYTOMETRY. EMPLOYING GLYCAN-SPECIFIC REAGENTS, LECTINS AN ANTIBODIES, BY COMBINING THE DETECTION ELEMENTS INTO A MULTIPLEXED ARRAY, AN ANALYSIS CAN BE OBTAINED IN MINUTES ON A BASIC -- THE APPROACH WHICH SHE CALLS GLYCOSENSE CAN BE THOUGHT OF AS A SOLUBLE 3D ANALOG ON A 2D MICRO ARRAY AND OFFERS SEVERAL UNIQUE AND IMPORTANT ADVANTAGES INCLUDING EASE OF USE, HIGH SENSITIVITY, THE BENEFITS OF EQUILIBRIUM BINDING KINETICS. SHE WORKS WITH COMPANIES THAT HAVE EXPRESSED INTEREST IN HER TECHNOLOGY TO BETA TEST THE KITS THAT HER COMPANY HAVE IS DEVELOPING. PROFESSOR SONG AND HIS TEAM ARE DEVELOPING AN ALTERNATIVE TO A TRADITIONAL MICRO ARRAY MUCH LIKE LORI. NEXT GENERATION GLYCAN MICRO ARRAY ENABLED BY NEXT GENERATION SEQUENCING. MIXTURE OF GLYCANS -- COATED WITH THESE OLIGONUCLEOTIDE SEQUENCES. THEY WILL SELECT SPECIFIC BOUND GLYCANS WITH DNA CODES. THE CODES CAN THEN BE DECODED USING POWERFUL TECHNOLOGY TO EE ELUCIDATE THE RELATIVE BINDING SPES SPECIFICITIES OF BINDING STRUCTURES TO THE PROTEINS. THE SEQUENCE READS WILL BE CONVERTED TO BINDING SPECIFICITIES OF THE PROTEINS. THEY EXPECT THIS NEW APPROACH WILL SOLVE THE PROBLEMS OF CAPACITIES THROUGHPUT, EASY ACCESSIBILITY AND AFFORDABILITY. IT WILL GREATLY LOWER THE THRESHOLD FOR THE COMMUNITY TO STUDY GLYCOPROTEIN INTERACTIONS. -- TO ALLOW THOSE NOT FAMILIAR WITH GLYCAN ARRAYS TO ANALYZE THEIR DATA. HE CALLS THE METHOD ON SHIFT GLYCAN MODIFICATION AND PROBING -- WHICH IS ACCEPTABLE TO NON-SPECIALISTS, USABLE WITH LOW SAMPLE VOLUMES AND EFFECTIVE IN PROVIDING EFFECTIVE MEASUREMENTS OVER MANY SAMPLES. HIS TEAM HAS PREVIOUSLY DEMONSTRATED -- AND GLYCOPROTEINS FROM BIOLOGICAL SAMPLES, AS WELL AS ALGORITHMS AND SOFTWARE FOR INTERPRETING THE DATA. THEY HAVE RECENTLY ADDED ENZYMATIC ALTERATIONS OF THE CAPTURED GLYCANS FOLLOWED BY LECTIN ANTIBODY PROBING AND AUTOMATED INTERPRETATION OF THE DATA. THE PROAT KROLLS PROTOCOLS ARE STRAIGHTFORWARD, COSTS CAN BE KEPT LOW BECAUSE OF THE MINIATURIZED SCALE AND INTERPRETATION BE MADE READILY UNDERSTANDABLE THROUGH THE SOFTWARE WHICH IS REALLY NICELY AVAILABLE ON HIS WEBSITE WHICH I SHOWED HERE. PROFESSOR XI HAS DEVELOPED THE GLYCAN ARRAY WHICH CONTAINS JUST OVER 500 GLYCANS. LMGA IS A TECHNOLOGY THAT MEETS ALL THE REQUIREMENTS FOR LARGE SCALE BINDING ANALYSIS. THE TEAM'S PILOT STUDY WHICH USED 202 GLYCANS DEMONSTRATED THE FEASIBILITY OF USING LMGA FOR USING MANY GLYCAN BINDING PROTEINS, ANTIBODIES, NATURAL GLYCAN ANTIBODIES IN SERUM AND PLAZ MA. UP TO 500 GLYCANS IN A SINGLE ASSAY OVER 1,000 SAMPLES PER DAY PER TECHNICIAN. THEY HAVE SUCCESSFULLY DEMONSTRATED THAT LMGA HAS VERY HIGH SENSITIVITY AND SPECIFICITY, HAS A RAPID TURNAROUND TIME AND REQUIRES SMALL VOLUME SAMPLE. THESE ATTRIBUTES RENDER THE PLATFORM ACCESSIBLE BY ALL BIOLOGISTS -- FOR CLINICAL TESTING. -- DERIVED FROM BACTERIAL GLYCANS. THE MAIN GOAL IS TO EXPAND THE PLATFORM WITH CLINICALLY RELEVANT MICROBES AND MICRO FLOOR WA AND COUPLE THESE WITH THE GENOMIC SEQUENCE TO FACILITATE DIRECT COMPARISON WITH MORE COMMONLY USED MICROBIAL GENOMIC SEQUENCING APPROACHES. IT'S REALLY FASCINATING. THIS TEAM HAS FOUR LABS. HANS BOLO, STEVEN ALMO AND ZHENG COLLABORATE TO STUDY HEPARIN SULFATES AND PLAY IMPORTANT ROLES FOR THE DEVELOPMENT AND PHYSIOLOGY. CELLS EXPRESS -- WITH HIGH SPECIFICITY MODIFICATION PATTERNS ON THEIR SURFACE. IN THIS PROJECT, THE TEAM IS TAGGING AND ENGINEERING 36 PREVIOUSLY ISOLATED SINGLE CHAIN VARIABLE FRAGMENT ABT ANTIBODIES TO DESCRIBE CELLS BY WAY OF THEIR GLYCOHIGH PRESSURE GLYCO TYPE. -- THAT COULD NOT BE ISOLATED BY EXISTING TECHNOLOGIES. STEFFAN AND PAUL ARE DEVELOPING TOOLS TO STUDY IN THE MICROENVIRONMENT. ADHESIONS OF ORAL STREPTOCOCCI -- FOR DETECTING AND LOCALIZING GLYCAN RECEPTOR STRUCTURES IN A VARIETY OF APPLICATION. THEY'VE ALSO IDENTIFIED UNKNOWN -- BINDING PROTEINS THAT EXHIBIT NOVEL BINDING SPECIFICITIES. THE OVERALL GOAL IS TO EXPAND AND GREATLY DIVERSIFY THE TOOLBOX BY HARVESTING NOVEL SERINE-RICH PROTEINS OF OTHER MAMMALSS THAT EXPRESS VIKINGLY DIFFERENT TERMINI. PROFESSOR VARKI -- THE DIVERSE ARRAY OF TERMINAL SIALIC ACIDS. THE TEAM AIMS TO DEVELOP A SYSTEMATIC APPROACH FOR IDENTIFICATION, TRACKING -- THEY'VE COLLECTED, CURATED AND OPTIMIZED A WELL-DEFINED SET OF RECOMBINANT SOLUBLE STABLE TAG SILO GLYCAN RECOGNIZING PROBES OR SGRBs, WHICH CAN EVENTUALLY BE USED AS A PRACTICAL TOOL BY THE NON-EXPERTS. THE GOAL IS TO DEFINE A SET OF SGRPs THAT CAN BE MADE AVAILABLE TO ANY BIOSCIENTIST INVESTIGATOR WHO CAN PROFILE THE DIVERSITY IN BIOLOGICAL SAMPLES. THE FINAL OUTCOME WHICH WOULD BE CROSS VALIDATED AND FURTHER OPTIMIZED IS A SIMPLE AND RELIABLE TOOLKIT TO TRACK -- IN BIOLOGICAL SAMPLES. THIS WORK WILL BE SIMULTANEOUSLY ACQUIRE BASIC KNOWLEDGE VALUABLE TO INVESTIGATORS WHO GENERATE PROBES DEVELOPS SWELS THOSE WHO STUDY THE BIOLOGICAL SYSTEMS THEY ORIGINATE FROM. THE GOAL -- INTERROGATING THE FUNCTION OF MICROBIAL GLYCANS AND THE MICROBIAL SPECIES UPON WHICH THEY'RE PRESENTED. THEY GENERATE M GAPs FROM 20 HUMAN -- WHICH MEDIATE DEFENSE THROUGH GLYCAN ENGAGEMENT. THE TEAM HAS BEEN SUCCESSFUL IN PRODUCING AND CHARACTERIZING MGAPS IN A REPRODUCIBLE MECHANISM AND THEY AIM TO DISTRIBUTE KITS TO PROBE TRUCK TOUR AND FUNCTION. PROFESSOR CUMMINGS, HERE THEY HAVE USED NEW TECHNOLOGIES TO GENERATE SMART ANTIGLYCAN REAGENTS, WHOSE ANTIGLYCAN SPECIFICITIES ARE DEFINED. THESE WILL BE CREATED OR THESE ARE CREATED FROM BOTH MIRRORING SINGLE CHAIN FRAGMENTS AND -- RECEPTORS OR VLRs. THESE HIGHLY SPECIFIC AND TARGETED REAGENTS WILL ALLOW RESEARCHERS TO TBIN MAPPING THE HUMAN GLIE COMB ATLAS WHICH WILL COMPLETE INFORMATION ABOUT GLYCAN EXPRESSION. PROFESSOR I AND PROFESSOR AI AND HIS TEAM -- GLYCOPROTEINS WITH LIPIDS AND SECONDARY METABOLITES. THE NEW TECHNOLOGY THAT HIM AND HIS TEAM ARE DEVELOPING FEATURES GENETICALLY ENCODED -- TO INTRODUCE INTERACTIONS -- AND EXTENDED PEPTIDE -- TO ENHANCE AFFINITY AND SPECIFICITY AND A CIRCULAR -- RED FLUORESCENT PROTEIN DOMAIN FOR FLUORESCENT READOUTS. THEY ARE PROVIDING THE PLASMIDS THAT THEY SYNTHESIZE AND MAKE FOR GENETICALLY ENCODED DETECTORS THROUGH ADDGENE. ISOENZYME-SPECIFIC CELL-BASED -- OF GALNAC ACTIVITY. HIGH-THROUGHPUT SCREENING TO IDENTIFY ISOFORM SPECIFIC SMALL MOLECULE -- MEDIATING -- PROMISES TO BE TRANSFORMATIVE IN THIS AREA OF GLYCOBIOLOGY RESEARCH, AND POTENTIALLY THE CLINIC. ROB WOODS AND HIS TEAM ARE OBSESSED WITH ASSURING GLYCANS FOUND ON PROTEIN STRUCTURES ARE SEARCHABLE AND -- CORRECTLY ON THE PROTEIN DATA BANK. THEY HAVE DEVELOPED A NUMBER OF TOOLS AVAILABLE FOR USE AND HIS TEAM HAS RUN MANY DEMOS TO USE THE PROGRAMS HIM AND HIS TEAM HAVE DEVELOPED. HE REALLY GETS EXCITED ABOUT THESE TOOLS. GAGs ARE HIGHLY HETEROGENEOUS -- EACH OF WHICH MAY BIND TO PROTEINS IN HIGHLY SELECTIVE OR PLASTIC NON-SELECTIVE MANNER. UNFORTUNATELY, IT'S DIFFICULT TO IDENTIFY SEQUENCES THAT RECOGNIZE PROTEINS SELECTIVELY. IT'S ALSO DIFFICULT TO PINPOINT THE PREFERRED SITE OF BINDING. WITHOUT THESE KEY PIECES OF INFORMATION, IT'S DIFFICULT TO DEVISE, DESIGN AND DEVELOP GAG SEQUENCINGS SEQUENCES THAT COULD SERVE AS DRUGS. THE DESAI TEAM HAS DEVELOPED A CVLS ALGORITHM THAT DOES PRECISELY THAT. IT IDENTIFIES NEEDLES IN THE HAYSTACK OF HUNDREDS OF THOUSANDS OF GAG SEQUENCES AND FOR THE NON-GLYCOSCIENTIST RESEARCHERS, IT ANSWERS QUESTIONS SUCH AS WHETHER TARGETED PROTEINS FIND GAGs AND IF THEY DO, WHAT IS THE SITE OF BINDING, WHAT IS THE -- WHAT RESIDUES OF THE PROTEINS ARE IMPORTANT FOR BINDING? AND FINALLY, WHAT IS THIS RELATIVELY BINDING AFFINITY TO THE PARTICULAR GAG SEQUENCES? THE CVLS TOOL WILL SOON BE AVAILABLE ONLINE. ACCESSIBLE AND EFFECTIVE METHODS TO MONITOR THE LEVELS AND INTERACTION PARTNERS, REPRESENT A CRUCIAL LINK BETWEEN METABOLIC STATE AND CELLULAR SIGNALING. HOWEVER, CELL-BASED METHODS TO CHARACTERIZE LEVELS AND INTERACTION PARTNERS OF THESE MOLECULES REMAIN INADEQUATE. KOHLER AND HER TEAM COLLABORATE TO DETECTIVE INTERCELLULAR LEVELS AND ARE DEVELOPING CROSS LINKING SUGAR TECHNOLOGIES ON THE CARBOHYDRATE TO MAP NOT ONLY BINDING PARTNERS BUT ALSO BINDING SITES WITH THESE GLYCANS. LAURA MAHALL AND HER TEAM ARE STUDYING -- ONE OF THE MOST DIVERSE AND ABUNDANT POST TRANSLATIONAL MODIFICATIONS. SHE HAS PIONEERED THE MICRO RNA PROXY HYPOTHESIS WHICH STAKES IF A MICRO RALPH NADER DRIVES A SPECIFIC BIOLOGICAL PHENOMENON, THE TARGETS WILL HAVE THE SAME BIOLOGICAL PHENOTYPE. SHE AND HER TEAM HAVE DEVELOPED A HIGH-THROUGHPUT EXPERIMENTAL PLATFORM TO ANALYZE MICRO RNA TARGET INTERACTIONS, MICRO R4R. THEY'VE UTILIZED THE IS IS TEM TO ANALYZE -- WITH BETA 3 -- OR B3GLCT. -- UNDERPINS THE CONGENITAL ORDER. THIS ASSAY IS READILY ADAPTABLE TO OTHER TRANSFERASES IMPROVING THE PROXY HYPOTHESIS. SHE CAN READILY SHARE IT, SWAP OUT WHICH GENE YOU'RE ENCODING FOR. DR. WANG AND HIS TEAM ARE DEVELOPING A MUTANT CELL LIBRARY THAT WILL LEAD TO THE CHARACTERIZATION AND VALIDATION -- OF HEPARAN SULFATE IN A CELLULAR CONTEXT. THEY'RE USING CHRIS CRISPR/CAS THAT INCLUDES ALL THE MAJOR HEPARAN -- TO ENABLE SYSTEMATIC INTERROGATION OF HEPARAN SULFATE IN A CELLULAR CONTEXT. DEVELOPMENT OF CELL LIBRARY THAT CONTAINS ALL THE -- TO EXTEND AND VALIDATE THE BIOCHEMICAL FINDINGS IN A CELLULAR CONTEXT IS ESSENTIAL FOR BETTER UNDERSTANDING OF THE BIOLOGICAL FUNCTIONS IN VITRO AND IN VIVO. THESE EFFORTS ARE ANTICIPATED TO RESULT IN A NOVEL, PREHEN SIEVE, EASY TO USE -- IN A CELLULAR CONTEXT AND A DOOL SET TOOL SET -- GENE TARGETING REAGENTS. THE AVAILABILITY OF THESE REAGENTS IS EXPECTED TO GREATLY ACCELERATE CURRENT HEPARAN SULFATE STUDIES. PROFESSOR WANG AND HIS TEAM ARE DEVELOPING TOOLS TO ANALYZE INTACT GAG CHAINS BY DEVELOPING SINGLE MOLECULES SEQUENCER FOR ANALYSIS OF POLYSACCHARIDES USING RECOGNITION TUNNELING NANOCOURSE DEVICES CURRENTLY UNDER DEVELOPMENT FOR THIS $1,000 GENOME PROJECT AS A TEM LATE PLATE. THEY TEMPLATE. NANOPORT SIGNALS PRODUCED BY GAG -- AND SETS THE STAGE FOR SEQUENCING OF INTACT GAG CHAINS BY RT DEVICES. AND LANCE WELLS AND CRIST WEST -- ROLE GLYCOSYLATION PLAYS PLAYS -- GLYCOSYLATED GENES. THEY AIM TO EX-TEND TO OTHER PARASITES AS IS NOW POSSIBLE TO PLAN APPROACHES TO RIGOROUSLY TEST THE PROPOSED ROLES OF GLYCANS GENETICALLY. AND IN ORDER TO EXPLORE THE ROLE OF -- ON GLYCOPROTEINS IN HUMAN DISEASES, PROFESSOR WOO AND HER TEAM HAVE DEVELOPED AN APPROACH FOR TARGETING. THE METHOD USES A NANOBODY FUSE TO COAL LEVELS ON THE DESIRED TARGETED PROTEIN CELLS. -- ON THE TARGETED TRANSCRIPTION FACTORS REVEAL FUNCTION IN PROTEIN STABILIZATION AND REGULATION OF PROTEIN-PROTEIN INTERACTIONS. TARGETED O-GL. LCNACYLATION -- THIS WORK WAS RECENTLY PROPERTIED IN NATURE CHEMICAL BIOLOGY. IN ORDER TO EXPLORE THE ROLE -- SORRY. PROFESSOR ZAHAR AND HER TEAM ARE DEVELOP AGO EXRE MEN TRI SET OF TOOLS FOR STUDYING. HER TEAM AIMS TO GENERATE INEXPENSIVE CHEMICAL GENETIC TOOLS THAT PROBE THE ROLE OF O-GLCNAC. THEY HAVE -- IN AN ORGANELLE SPECIFIC MANNER. THESE TOOLS ARE AVAILABLE VIA CONSTRUCTS AND THROUGH DEVELOPMENT OF THE CORRECT MOUSE MODELS. IN ADDITION TA WORKING ON HER OWN PROJECTS, PROFESSOR ZACHARA HAS BEEN MY REAL PARTNER IN CRIME IN PROMOTING THE REAL WORK OF THIS NIH COMMON FUND SUBGROUP. I'LL QUICKLY MENTION TWO OF OUR MAJOR EFFORTS TO BROADCAST THIS TOOL'S SUBGROUPS DURING THE PAST YEAR. NATASHA AND I WORKED WITH DR. JESSICA LAWYER TO ESTABLISH A SPECIAL ISSUE ON GLYCOSCIENCE, THE GLYCOSCIENCE COLLECTION. IN TOTAL, OUR GROUP HAS PUBLISHED 11 NEW DETAILED METHODS IN 2021. WE ARE WORKING WITH JESSICA AND HER TEAM TO ENSURE THESE ARTICLES ARE BEING A SESSIBLE TO ACCESSIBL E TO THE PUBLIC AND THAT EXTENDED TABLE OF CONTENTS WILL COLLATE ALL THE GLYCOSCIENCE METHODS FROM PREVIOUS PROTOCOL ISSUES. THIS WILL BE AN ESSENTIAL STEP IN ASSURING THE WORK OUR GROUP IS DEVELOPING IS ACCESSIBLE. AND PROFESSOR KIESSLING AND ENCOURAGED MANY OF OUR MEMBERS AND ENGAGED MANY OF US TO SUBMIT DURING OUR MONTHLY MEETING, SO WE'RE LOOKING FORWARD TO SEEING THAT SPECIAL ISSUE AS WELL. IN ADDITION, WE HAVE BEEN GOING TO SOCIETY MEETINGS TO HIGHLIGHT YOUR WORK. WE'VE WORKED WITH ASBMB TO HAVE SPECIAL WORKSHOPS IN WHICH WE HIGHLIGHTED THE GROUP'S WORK AND ANSWERED THE QUESTIONS TO THOSE INTERESTED IN USING TOOLS. WE HAVE ACTIVELY WORKED TO ENSURE YOUR TOOLS ARE VISIBLE AND USABLE BY THE NON-SPECIALIST. I REALLY HOPE THAT OUR TOOLS AND METHODS ARE TAKEN UP BY THE COMMUNITY AND MADE ACCESSIBLE TO COMMERCIALIZATION. AS I MENTIONED AT THE BEGINNING OF MY TALK, IT'S DR. RENO'S WORST NIGHTMARE FOR ALL OF OUR AMAZING METHODS TO END UP IN A DEEP FREEZER. I LOOK FORWARD TO ALL THE UPDATES AND HOPE THIS WAS USEFUL AND EMPOWERS TO SEE THE IMPACT OF OUR TEAM'S WORK. THANKS. >> THANK YOU, CATHERINE. THAT WAS A REALLY NICE SUMMATION OF THE MANY TOOLS COMING OUT OF YOUR GROUP, AND I WANT TO THANK YOU FOR LING THAT GROUP UP. IT'S REALLY BEEN FUN COMING TO YOUR SESSIONS AND LISTENING TO THE TALKS. I LIKE THE WAY EVERYBODY CRITIQUES EACH OTHER AND HELPS OUT. IT'S REALLY BEEN A NICE WAY TO MOVE FORWARD. HAVING THOSE METHODS PUBLISHED IS REALLY KEY, SO I'M GLAD THOSE ARE GOING FORWARD AND THANKS TO LAURA FOR PUTTING HER JOURNAL INTO PRACTICE FOR US TOO. DO WE HAVE ANY QUESTIONS? FOR CATHERINE? YOU'RE GOING TO HEAR A LOT MORE ABOUT ALL OF THESE METHODS THAT ARE STILL ON THE BOOKS. SOME OF THESE HAVE COMPLETED AND FOLKS HAVE ACTUALLY MOVED ON, BUT A LOT OF THESE ARE STILL ON THE BOOKS AND YOU'LL HEAR ABOUT THEM TODAY. SO IF WE DON'T HAVE ANY OTHER QUESTIONS, I'D LIKE TO MOVE FORWARD BECAUSE THE GLYGEN PROJECT IS REALLY HITTING STRIDE RITE NOW. AND MIKE TIEMYER AND RAJA MAZUMDER ARE GOING TO TELL US ABOUT WHAT'S HAPPENING. >> GOOD MORNING, EVERYONE. THANK YOU, PAM. SO MIKE AND I ARE GOING TO DO 30 MINUTES EACH SO THE FIRST HALF OF THE TALK, I'M GOING TO PRESENT AN OVERVIEW FROM THE DATA SIDE AND THEN MIKE WILL SHOW THE FUN STUFF, WHAT YOU CAN DO ON OUR WEBSITE ON THE GLYGEN WEBSITE WITH ALL THE DATA THAT HAS BEEN COLLECTED. ALL RIGHT. SO THE DATA TEAM AND COLLABORATIONS, ROBELL AND I CO-LEAD -- BUT WE WORK VERY CLOSELY WITH SEVERAL OTHER PEOPLE FROM GEORGETOWN UNIVERSITY, NATHAN AND DARREN AND KAREN AND -- FROM UNIVERSITY OF DELAWARE, VIJAY AND PENG SU FROM NIH, EVAN, PAUL AND TERRANCE MURPHY, MARIA MARTIN AND HER COLLEAGUES FROM SOKA UNIVERSITY, KIYOKO, GRIFFITH UNIVERSITY MATTHEW, AND SWISS INSTITUTE OF BIOINFORMATICS, FRED REEK AND CATHERINE. SO BEFORE I GO INTO WHAT WE HAVE BEEN DOING, I JUST WANT TO MEX A FEW HOUSEKEEPING THINGS. SO GLYGEN WILL BE OFFERING FOUR CONCURRENT DEMONSTRATION AND DISCUSSION SESSIONS ON WEDNESDAY MORNING, WHICH IS TOMORROW. EACH SESSION WILL BE REPEATED TWICE SO YOU'LL HAVE OPPORTUNITY TO ATTEND TWO SESSIONS OF YOUR CHOICE. SO SESSION ONE AND TWO AND THREE ARE DEMOS, AND THEN SESSION FOUR ARE DROP-IN OFFICE HOURS, AND I'LL EXPLAIN WHAT EACH OF THESE SESSIONS ARE GOING TO DO OR TRY TO DO. SO SESSION 1 IS GLYGEN NEW FEATURES TUTORIAL, SO IF YOU'RE UNFAMILIAR WITH WHAT GLYGEN CAN DO, THIS IS THE SESSION YOU WANT TO JOIN. ALSO YOU WANT TO GET AN OVERVIEW OF NEW FUNCTIONALITIES, DATA AND FEATURES, THEATION THAT'S SESSION ONE IN THE MAIN MEETING ROOM AND ALSO VIDEOCASTED THROUGH THE WEBSITE. SESSION TWO IS GLYGEN TOOLS: BIOSYNTHETIC PATHWAYS, CANONICAL RESIDUES, MOTIF MATCHING AND ADVANCES IN SUBSUMTION. GLYCAN STRUCTURES, MOTIVES, AND ALSO THE KNOWN STRUCTURE BROWSER, SO GLYCAN ONTOLOGY STRUCTURE BROWSER. SESSION THREE IS DATA REPOSITORY FOR GLYCAN ARRAY DATA, IF YOU WANT TO LEARN ABOUT THE REPOSITORY, WHAT IS ITS CURRENT STATUS, FEATURES AND FUNCTIONALITIES. SESSION FOUR IS DROP-IN OFFICE HOURS,, I THINK MIKE AND A FEW OTHERS WILL BE THERE SO YOU CAN DROP IN AND ASK QUESTIONS. SO ANOTHER IMPORTANT THING IS, WE ARE DOING THIS USE CASE COLLECTION DRIVE, SO WE HAVE BEEN DOING THIS FROM TIME TO TIME AND THIS IS ONE OF OUR DRIVES IN THE MONTH OF JULY WHERE WE WANT FROM YOU, USE CASES. SO IT'S QUITE SIMPLE ACTUALLY. SO WHAT YOU NEED TO DO IS YOU NEED TO GO TO THIS TINYURL.COM. YOU CAN TAKE A SCREEN SHOT IF YOU WANT TO REMEMBER THIS URL, GLYGEN3. IF YOU CAN'T FIND THE URL, PUT IT IN THE CHAT BOX, MIKE OR I WILL SEND YOU THE ACTUAL LINK. SO ONCE YOU GO THERE, WE WANT TO HEAR FROM YOU SO YOU CAN SAY AS A GLYCOBIOLOGIST, I WANT, SO AS A -- WHO YOU ARE, WHAT IS YOUR INTEREST, WHAT DO I WANT TO DO? AND THEN SO THAT IT WILL HELP YOU BUILD A BETTER GENOME-BASED DIAGNOSIS. SO THESE USE CASES HELPED UTION OR HELP US BUILD THE RESOURCE, THE GLYGEN RESOURCE, WHICH IS DATA INTEGRATION AND DISSEMINATION PROJECT FOR CARBOHYDRATE AND GLYCOCONJUGATE-RELATED DATA. THIS IS A NATIONAL PROJECT FUNDED BY NIH, AND THESE ARE THE FOUR AIMS THAT WE START OFF WITH, AND WE ARE STILL FINISHING. SO AIM ONE IS TO INTEGRATE DATA INTO, AIM TWO IS IMPLEMENT A WEB PORTAL, AIM THREE DEVELOP AND INTEGRATE CRITICAL GLYCOINFORMATICS INFRASTRUCTURE, AND AIM FOUR IS TO OUTREACH EAND GAUGE WITH THE AND ENGAGE WITH THE COMMUNITY. SO THERE ARE THREE MAJOR TASKS. TASK ONE IS DATA COLLECTION, TASK TWO IS DEVELOPING THIS USER-FRIENDLY INTERFACE FROM THE DATA WE HAVE COLLECTED AND THE TASK THREE IS HOW EASY IS THE DATA AVAILABLE TO ANYBODY WHO WANTS TO RE-USE IT. SO BEFORE I GO MORE INTO IT, SO WE CAN -- THERE'S A LOT OF THINGS ONE NEEDS TO DO, BUT WE HAVE TO FOCUS FROM YEAR ONE, WHAT IS IT WE WANT TO DO FIRST? SO YEAR ONE, IT FOCUSED ON HUMAN AND MOUSE DATA. SO WHEN WE TALK ABOUT DATA HERE, I WILL EXPLAIN WHAT DOES IT MEAN, LIKE WHEN I SAY IS IT GLYCANS, IS IT PROTEINS IN HUMAN AND MOUSE, SO IT'S ALL IMPORTANT. THEN YEAR TWO, WE ADDED RAT, YEAR THREE, WE SYNTHESIZED GLYCANS, AND OF COURSE SARS-COV1 AND COV2 AND YEAR FOUR, DROSOPHILA, SO THAT'S WHERE WE'RE AT. SO DATA IN GLYGEN IS BROKEN DOWN INTO THREE MAJOR CATEGORIES. SO WE HAVE THE PROTEIN, WE HAVE THE GLYCAN, AND THEN WE HAVE THE GLYCOPROTEIN/SITE, SO POSITION IN THE GLYCAN WHEN IT IS KNOWN. NOW ONCE WE HAVE THESE THREE MAJOR CATEGORIES, YOU CAN ADD A VARIETY OF INFORMATION AROUND EACH OF THESE DATA POINTS. SO FOR PROTEINS, I CAN HAVE VARIATION INFORMATION, DISEASE, FUNCTION, EXPRESSION, SEQUENCE -- PATHWAY AND MORE. AND IT COMES UNDER A VARIETY OF PLACES THAT WE COLLABORATE WITH. GLYCAN CAN HAVE -- AND SO ON. AND WE FOCUS OUR ID SPACE -- SO ONCE YOU HAVE ALL OF THIS INFORMATION, THEN THESE ARE THE FOUR WAYS YOU CAN ACCESS THIS DATA. SO MIKE WILL BE TALKING ABOUT THE GLYCAN PORTAL ON HOW YOU CAN DO SEARCHES IN THERE, AND I WILL TALK A LITTLE BIT ABOUT THE DATASET COLLECTION. SO WHEN YOU COLLECT DATA, THEY COME IN AS FILES, SO IT'S A VERY INFORMATIC CONCEPT, SO WE NEED TO TRACK THESE FILES, WE NEED TO TRACK EVERY ROW IN THESE FILES, WE NEED TO TRACK THESE IMAGES, SO WE USE BIOCOMPUTER OBJECTS AND I'LL TALK A LITTLE ABOUT IT, HOW WE CAN TRACK THEM. SO WE HAVE VERSION NUMBERS, WHICH MEANS IF YOU -- IT TELLS HOW YOU GOT IT, WHAT WE DID WITH IT, AND ALL OF THIS GOES INTO TRIPLE STORE -- AND THAT'S HOW THE FRONT END GETS ALL THE INFORMATION. WE ALSO HAVE SPARQL END POINTS FOR THOSE WHO ARE MATHEMATICIANS AND ALSO APIs, APPLICATION PROGRAMMING INTERFACES. SO THESE ALLOW YOU TO ACCESS THE DATA. SO THE DATA COMES IN -- HERE I'M SHOWING A FEW TABLES, WE HAVE -- EACH OF THESE DATASETS HAVE BEEN CAREFULLY LOOKED INTO, RULE-BASED CURATION HAS BEEN APPLIED TO THEM FOR QC PURPOSES AND THAT'S HOW WE KNOW THAT WHAT WE ARE PROVIDING IN THE GLYGEN FOR THEM IS THE BEST OF OUR ABILITY AND THIS LEADS TO THE FRONT ENDS. HERE'S AN EXAMPLE, IF YOU CLICK ON ONE OF THESE TABLES, THIS IS THE KIND OF INFORMATION YOU'LL SEE. SO FOR EXAMPLE, YOU'RE INTERESTED IN GETTING THE HUMAN PROTEINS, ALL OF THE HUMAN PROTEINS. SO YOU CAN COME TO GLYGEN AND IN THIS CASE, WE WORKED WITH MARIA MARTIN FROM EBI AND PRET WHO HELPED US WORK WITH -- SO THERE'S AN EE QI SYSTEM WHERE SYSTEM WHERE PEOPLE ARE TALKING TO EACH OTHER TO PRODUCE THIS FINAL PRODUCT. ANY SCRIPT THAT WAS USED TO THEN FURTHER WORK WITH THE DATA, ANY KIND OF ADDITIONAL INFORMATION THAT WAS ADD, HOW TO USE THIS TYPE OF DATA. IS THERE ANY ERROR ASSOCIATED WITH IT, AND WHAT KIND OF PARAMETERS ARE USED TO DEFINE, WHAT IS THE CANONICAL SEQUENCE, WHAT IS THE ISOFORM AND SO ON. SO IN TERMS OF GLYCAN DATA EXPANSION IN YEAR FOUR, -- ADDITIONAL KEYWORD SYNONYMS, ADDITION OF SEMANTIC NAMES, TYPES AND SUBTYPES, TO -- ADDITION OF NEW SITES, TISSUE CELL EXPRESSION INFORMATION, NEW PUBLICATION FROM NCFG, ADDITION OF REACT TOMORROW -- FOR COMMONLY USED GLYCAN TERMS. FOR PROTEIN, WE ALSO WORKED ON ADDITION OF DISEASE DESCRIPTION SYNONYMS, ADDITION OF CROSS-REFERENCES PHOSPHORYLATION, PRO-ANNOTATIONS, AND NEW SPECIES DROSOPHILA FOR YEAR FOUR. SO FOR THE PROTEIN CENTRIC DATASETS YEAR FOUR IN PROGRESS, THESE ARE THE LIST OF ITEMS IN PROGRESSOR ALREADY HAS BEEN ADDED AND IN YEAR FIVE, WE PLAN TO ADD MORE. SO THE LIST OF MOTIVES, SO IF YOU GO TO BETA.GLYGEN.ORG LIST OF MOTIVES AT THE URL, YOU CAN SEE THE LIST OF MOTIFS THAT HAVE BEEN COLLECTED AND YOU CAN SEE THE LIST HERE, AND OF COURSE IF YOU HAVE ANY COMMENTS OR QUESTIONS HAS A FEEDBACK MECHANISM. OR YOU CAN CONTACT US AND YOU CAN PROVIDE US WITH FEEDBACK. SO HERE ARE THE TWO LINKS THAT WILL BE SHOWN IN MORE DETAIL DURING THE DEMO, THE SANDBOX AND THE RELATED GLYCAN LINK SO FOR THIS PARTICULAR GLYCAN, WHICH USES A -- YOU CAN GET THE ACCESSION, MASS, COMPOSITION, GLYCAN TYPE AND SUBTYPE, AND WITHIN THE SPACE, THERE IS INFORMATION ABOUT -- IF YOU PUT THIS LINK, #MOTIVES AFTER THAT, YOU #MOTIFS AFTER THAT, YOU WILL SEE THE MOTIFS IN THE STRUCTURE. IT TELLS YOU WHICH ARE THE GLYCANS WHICH ARE SUBSUMED BY OTHERS. AND AGAIN, IT TAKES YOU TO ANOTHER PAGE WHICH NATHAN MAINTAINS AND YOU CAN THEN EXPLORE THE SUBSUMTION IN MORE DETAIL. FOR EXPRESSION, THERE IS INFORMATION COMING FROM GLYCOCONNECT FROM FREDERIQUE'S GROUP, AND WE COLLECT THAT INFORMATION WHERE IT SHOWS CELL LINES, THIS IS THE EXPRESSION INFORMATION AND EACH OF THESE ROWS AS YOU CAN SEE HAS A PUBMED LINK WHICH IS EVIDENCE LINK WHICH MEANS THAT THE INFORMATION WAS COLLECTED FROM THIS PARTICULAR PAPER. PHOSPHORYLATION, WE HAVE A COLLABORATION WITH ITT AND MET WITH CATHY MU AND CECILIA, SO WHAT THEY DO IS THAT THEY PROVIDE PHOSPHORYLATION, THE PROTEIN, THE GENE, THE RESIDUE, AND ALSO THE -- BECAUSE CATHY IS ONE OF THE PIs AND THEY ALSO MAP TO PUBMED IDs AND THIS INFORMATION. IN CASES WHERE THERE IS NO -- LINK, SOME OF THE INFORMATION MIGHT BE MISSING BUT NONETHELESS IT INFORMATION THAT CAN BE OF USE TO THE COMMUNITY. THIS IS A REALLY WELL USED INTERFACE. THE SEQUENCE. SO ONCE YOU GO TO A PROTEIN, YOU CAN TAKE A LOOK AT THE SEQUENCE AND IN THERE WE HAVE THESE BOXES WHERE YOU CAN CLICK OR UNCLICK. AND SAY I WANT TO SEE ALL THE O LINKED SITES, I WANT TO SEE THE SEQUON, I WANT TO SEE PHOSPHORYLATION, I WANT TO SEE A PARTICULAR STRETCH OF OF AMINO ACID. THIS WAY -- THIS IS A SMALL PROTEIN, BUT FOR LARGE PROTEINS, IT'S REALLY USEFUL TO QUICKLY EXPLORE THE PROTEIN SEQUENCE AND KNOW WHAT KIND OF INFORMATION IS AVAILABLE IN TERMS OF SITE AND GLYCOSYLATION AND PHOSPHORYLATION FOR THAT PROTEIN. FOR THE GLYCAN LIGANDS -- NOW YOU CAN HAVE THAT INFORMATION WITHIN GLIE JEFNLT FOR PROTEIN THIS A PROJECT RUN BY CATHY AND DARREN NATALI AND FOR PROTEIN ONTOLOGY, WE ARE MODELING THE INFORMATION FOR SPECIFIC PROTEINS IN THE GLYCOSYLATION SITE USING PROTEIN ONTOLOGY. SO IT PROVIDES A DESCRIPTION, IT PROVIDES ADDITIONAL INFORMATION, WHICH CAN LINK OUT TO ADDITIONAL RESOURCES OR PAPERS WHICH PROVIDES THIS TYPE OF INFORMATION. AND ONCE YOU HAVE THIS, YOU CAN THEN LOOK ACROSS ORGANISMS TO UNDERSTAND HOW THE DIFFERENT GLYCOSYLATION SITES FOR EXAMPLE ARE DIFFERENT OR IF THEIR FUNCTION IS DIFFERENT BECAUSE OF THE DIFFERENT SITES THAT ARE IMPACTED. SO ADDITIONAL UPDATES INCLUDE CROSS-REFERENCES, LINK-UPS TO -- UPDATES, NEW GLYCAN TYPES AND SUBTYPES, AND FROM THE DATABASE, NEW PUBLICATIONS ASSOCIATED WITH GLYCAN MOTIFS. SO IN A LITTLE BIT MORE DETAIL FROM UNICARBKB, WE HAVE 64 SITES, ALSO COLLABORATION WITH UNIVERSITY OF DELAWARE, WE HAVE AUTOMATIC LITERATURE MINING, WE EXTRACT INFORMATION FROM PAPERS AND THEN WE DETECT WHICH SITES ARE GLYCOSYLATED AND IF THERE'S ADDITIONAL INFORMATION WE CAN PROVIDE DIRECTLY TO THE USERS FROM THE GLYCAN WEBPAGES. THIS IS SCALABLE, SO ALL OF THIS IS MANUALLY DONE, THE ONE YOU SEE ON THE LEFT, BUT AUTOMATIC LITERATURE MINING ALLOWS US TO SCALE THINGS AND EVENTUALLY EVEN ALLOW US TO USE SOME OF THE MACHINE LEARNING TOOLS AND THINGS LIKE THAT TO CONNECT DIFFERENT PIECES OF INFORMATION THAT IS COMING FROM PAPERS. GLYCONET IS MANUALLY CURATED BY FREDERIQUE AND CATHERINE SO WE HAVE THE FOLLOWING DATASETS FROM THERE. UNIPROT, WE HAVE BEEN CONTINUALLY PULLING FROM THEIR FT LEANS WHICH ARE THE LINES BUT THEY TO HAVE AN OWE TAIGS ASSOCIATED ANNOTATIONS FROM RELATED SITES AND THE EVIDENCE FOR THAT INFORMATION. ADDITIONAL ADDITIONS ARE THE O-GLCNAC DATABASE. THIS IS A NEW THING WHICH CAME FROM THE LITERATURE MINING DATA WHICH IS A GLYCAN DICTIONARY. SO THE GLYCAN DICTIONARY IS EXTREMELY IMPORTANT FOR US TO DO LITERATURE MINING BUT ON THE OTHER HAND, IT ALSO ALLOWS US TO TO -- WHEN A USER DOES A FREE TEXT SEARCH, WHAT KIND OF TEXT IS THAT PERSON WRITING. SO I'LL EXPLAIN A LITTLE HOW THIS WORKS. SO YOU HAVE THE TERM AND THEN YOU HAVE THE TERM IN A SENTENCE SO IN THIS PAPER THERE'S A SENTENCE WHERE THE TERM EXISTS AND THERE ARE MULTIPLE PAPERS WHICH ALSO HAVE THE TERM, WE HAVE A DEFINITION, WE HAVE A CROSS-REFERENCE, SYNONYMS, DISEASE ASSOCIATION IF THERE IS ANYTHING THAT WE HAVE, WIKIPEDIA IF IT HAS ARTICLE, SEVERAL OF THESE HAVE WIKIPEDIA ARTICLES BUT I WISH THERE WERE MORE WIKIPEDIA ARTICLES FOR SOME OF THEM. MAYBE SOME OF THE COMMITTEE MEMBERS CAN ADD CONTENT IN WIKIPEDIA. IT STILL ALLOWS THE NOVICE TO GO AND CHECK THINGS OUT AND WIKIPEDIA STILL IS ONE OF THE SOURCES THAT PEOPLE END UP FIRST WHEN THEY DO NOT KNOW WHERE TO GO. THIS IS A WORK IN PROGRESS. IF THERE'S A TERM THAT IS MISSING, PLEASE LET US KNOW AND WE WILL ADD IT. TWO OR MORE PAPERS TO MAKE IT INTO THIS LIST. WE HAVE BEEN MAPPING APIs, THESE ARE EXTREMELY USEFUL IF YOU ARE MAINTAINING YOUR OWN RESOURCE WHERE YOU ARE TRYING TO GET INFORMATION FROM GLYGEN PROGRAMMATICALLY. SO IN TERMS OF CONNECTING RESOURCES, WE HAVE A PAPER IN REVIEW. THIS PAPER IS , WE ARE WORKING TOGETHER TO MAKE THE INFORMATION THAT WE ARE HARMONIZING AND INTEGRATING MAKING SOME OF THAT ALSO AVAILABLE DIRECTLY TO -- BECAUSE USERS, THEY HAVE A LARGE USER BASE SO WE WANT TO MAKE SURE THAT USERS HAVE INFORMATION THAT DON'T NEED TO ALWAYS COME TO GLYGEN, THEY CAN ACTUALLY HAVE THAT INFORMATION DIRECTLY FROM OTHER RESOURCES. SO AGAIN THIS IS PART OF THE SAME STORY -- PUB CHEM HAS BEEN DEVELOPING THESE PAGES, PLEASE CHECK IT OUT AND IN PUBCHEM HUB AND SEE -- AND PROVIDE AGAIN FEEDBACK TO US DIRECTLY TO PUB CHEM. THEY ALSO HAVE PROTEIN PAGES AND WE ALSO HAVE BEEN POPULATING THESE PROTEIN PAGES WITH INFORMATION AND LINKS BACK TO GLYGEN SO IF PEOPLE END UP ON A PROTEIN PAGE THEY STILL HAVE INFORMATION WHICH WILL AT LEAST GET THEM STARTED. WE ARE ALSO WORKING CLOSELY WITH UNIPROTKW AND B AND FROM OUR PAGES WE LINK BACK TO THEM. THIS IS AN EXAMPLE WHERE IN THE FT LINE OR THE FEATURE LINE PTM DATABASES IT SHOWS HOW MANY SITES, HOW MANY GLYCANS AND SO ON. SO AGAIN, THE SAME THING, -- RELATED TO CHEMICAL COMPOUNDS SO WE ARE WORKING WITH THEM TO HELP INTEGRATE SOME OF THE INFORMATION THAT WE HAVE WITHIN GLYGEN INTO CHEBI SO IT CAN BE USED WITHIN THEIR ONTOLOGY. SO NEW DATA IS COMING TO GLYGEN. THIS IS NOT THE ENTIRE LIST BUT THIS IS SOME OF THE LIST THAT WE ALREADY HAVE PLANS WITHIN THE NEXT SEVERAL MONTHS. SO THE SUMMARY OF WORK TO BE DONE IN YEAR FIVE, SO WE WILL HAVE NEW SPECIES DATA INTEGRATION OF NEW DATASETS, AND OF COURSE MAINTAINING WHAT WE ALREADY HAVE, BACKEND AND DATA FOR COMMON FUND DATA, WE WILL BE WORKING WITH THE COMMON FUND DATA SYSTEM AS PART OF THEIR INITIATIVE TO INTEGRATE GLYCAN INFORMATION AND DATA WITH OTHER TYPES OF INFORMATION SUCH AS METABOLOMICS, PROTEOMICS, AND A VARIETY OF OTHER TYPES OF DATA USING WHAT IS CALLED A C2M2 DATA MODEL. WE'LL BE ALSO DEVELOPING NEW APIs. WE ARE ALWAYS INTERESTED IN CROSS-REFERENCING TO OTHER RESOURCES, SO THESE CROSS-REFERENCES ARE EXTREMELY USEFUL BECAUSE THE CROSS-REFERENCES NOT ONLY ALLOWS USERS SO IDENTIFY WHICH RESOURCES HAS INFORMATION FOR A PARTICULAR ENTRY, BUT IT CAN ALSO HELP IDENTIFY INFORMATION. FOR EXAMPLE, IF I GIVE ALL THE HUMAN PROTEINS AND I SAY FIND ME ALL THE CROSS-REFERENCES FOR PDB, THEN I CAN EASILY KNOW WHICH OF THE PROTEINS HAS CRYSTAL STRUCTURES IN GLYGEN OUT OF ALL THE HUMAN PROTEINS. SO THIS CROSS-REFERENCE IS A POWERFUL TOOL ALSO TO ASK QUESTIONS ABOUT THE ENTIRE DATABASE. AND THEN THE NEW PUBLICATIONS, NEW DATA COLLABORATIONS, WE ARE OPTIMIZING SEARCH RESULTS, SORTING AND FILTERING AND SO ON. SOME OF THEM YOU WILL SEE ALREADY IN PROGRESS AND WE'RE LOOKING FOR FEEDBACK. ADDITION OF TOOLS FOR GLYCOPROTEOMICS RESEARCH, EXPANSION OF OUTREACH ACTIVITIES, IMPLEMENT FEEDBACK AND SUGGESTIONS. WE ARE REALLY INTERESTED IN GETTING SOME USE CASES FROM THIS GROUP SO WE CAN LOOK AND SEE IF THEY ARE ALREADY IMPLEMENTED OR WE CAN TELL YOU THIS IS ALREADY IMPLEMENTED, THIS IS THE TYPE OF SEARCH YOU NEED TO DO, OR IF THEY'RE NOT IMPLEMENTED, THEN WE CAN PUT IT IN OUR THINGS TO DO AND PRIORITIZE THEM WITHIN THE NEXT YEAR. ADDING INFORMATION FOR TOP 20 AND 50 SELECTED GLYCOPROTEINS. SO THIS WILL ALLOW, BECAUSE PLANE OF THE USERS COME WITH SOME GLYCOPROTEINS THAT THEY HAVE BEEN WORKING ON, SO IDENTIFYING THE LIST ON TOP, 20 OR 50 GLYCOPROTEINS, MAKING SURE THE INFORMATION IS COMPREHENSIVE FOR THOSE PROTEINS WITHIN OUR RESOURCE IS ALSO ONE OF THE THINGS THAT WE WANT TO DO. SO THAT WAY, WHEN USERS COME IN, MAJORITY OF THE USERS WILL FIND INFORMATION WHICH THEY HAVE BEEN LOOKING FOR. SO THAT'S ALL FROM MY SIDE. I THINK AFTER MIKE'S TALK, WE ARE TAKING QUESTIONS, SO I'M GOING TO STOP SHARING MY SCREEN. OKAY, THAT'S GREAT. >> SORRY, RAJA, I TOOK IT FROM YOU. THANKS FOR THE INTRODUCTION. TO ALL THE WORK THAT'S BEEN GOING ON IN TERMS OF ACQUIRING DATA, ORGANIZING DATA AND MAKING DATA ACCESSIBLE. I JUST WANT TO REITERATE THAT WE REALLY -- I JUST WANT TO REITERATE AND BEG THAT WE REALLY NEED YOUR HELP. THIS IS AB IMPORTANT AN IMPORTANT YEAR FO R GLYGEN AS WE LOOK FOR SUSTAINABILITY INTO THE FUEL TOUR WITH OTHER FUNDING SOURCES. THESE USE CASES REALLY HELPED GLYGEN AT THE BEGINNING THAT PUT ITSELF ON A TRACK THAT MADE ITSELF USEFUL. THAT'S WHAT WE'RE LOOKING AT, INPUT FROM YOU TO HELP US WRITE OUR RENEWAL, WRITE OUR NEXT FUNDING PROPOSAL, SO THAT IT'S CLEARLY RESPONSIVE TO THE NEEDS OF THE COMMUNITY. SO -- BY THE WAY, I'M NOT CONFLICTED EXCEPT I HOPE GLYGEN SUCCEEDS. I'M GOING TO TALK ABOUT SOME OF THE IMPROVEMENTS ON THE PORTAL OVER THE LAST YEAR AND SHOW A FEW EXAMPLES AND MY GOAL HERE IS TO JUST SORT OF WHET YOUR APPETITE TO GO TO SOME OF THE -- ONE OR TWO OF THE SESSIONS TOMORROW MORNING TO GET A LITTLE MORE DEEPER VIEW OF WHAT SOME OF THESE FEATURES DO, WHAT GLYGEN CAN DO TO HELP YOU. SO I'LL SHOW YOU A FEW THINGS, BUT I'M NOT GOING TO TRY AND BE EXHAUSTIVE. JUST SHOW YOU SOME INTERESTING THINGS THAT WE'VE DEVELOPED. SO I WORK CLOSELY WITH THE INTERFACE TEAM HERE AT THE UNIVERSITY OF GEORGIA. RENE RANZINGER, AND WILL YORK WHO INSPIRES US AND DRIVES US FORWARD IN MANY WAYS, AND I'LL BE SHOWING YOU BRIEFLY SOME OF WHAT HE'S BEEN WORKING ON IN HIS OWN PLAYGROUND. THEN OF COURSE THE PEOPLE THAT IMPLEMENT EVERYTHING THAT WE TALK ABOUT, A LOT OF THIS IS THEIR WORK, AND SANDEEP WHO I DON'T HAVE A PICTURE OF WHO'S BEEN HIGHLY INVOLVED IN THE GLYCAN ARRAY REPOSITORY AND DEVELOPING THAT. WHICH RENE WILL TALK ABOUT TOMORROW IN HIS BREAKOUT SESSION, SO PLEASE ATTEND TO LEARN ABOUT THE GLYCAN ARRAY REPOSITORY. SO I'M GOING TO HIT ON THESE ITEMS. I WANT TO TALK A LITTLE AT THE BEGINNING ABOUT OUR USE BASE, WHO'S ACCESSING GLYGEN, WHAT WE KNOW ABOUT THAT. AND HOPEFULLY SHOW YOU WE'RE GROWING AND MATURING, AND THEN TALK ABOUT SOME OF THE PROGRESS IN YEAR FOUR IN TERMS OF -- IMPROVEMENTS. THEN FINISH UP BY KIND OF STEPPING YOU THROUGH IN SORT OF A DEMONSTRATION MARKED FOR IDENTIFICATION SOME OF THE NEW FEATURES OF GLYGEN AND THE KINDS OF QUESTIONS YOU CAN NOW ASK ABOUT IN TERMS OF GLYCOPROTEINS AND GLYCOCONGRESS CONGREGATES IN GENERAL. GOING BACK TO JANUARY OF 2020, AND LOOKING FORWARD TO JUST THE LAST MONTH OR SO, WE'VE BEEN EXPERIENCING INCREASED GROWTH AND INTEREST IN GLYGEN, WHICH IS GREAT. WE'VE GONE UP ABOUT 20 FOLD OVER THE LAST YEAR IN TERMS OF USERS PER MONTH, AND CORRESPONDINGLY IN TERMS OF PAGE VIEWS, AND WE RECOGNIZE AS MANY OTHER PEOPLE DID THAT DURING THE PANDEMIC IT WAS A GREAT TIME TO PIVOT INTO DEPO DEMOS AND WEBINARS, AND THAT'S THAT'S BEEN AN IMPORTANT COMPONENT OF DRIVING INCREASED TRAFFIC AND IT SORT OF FEEDS ON ITSELF. SO A GOOD GROWTH MODE AT GLYGEN FOR REACHING OUT TO THE COMMUNITY, SO HOW ARE PEOPLE GETTING TO US? WELL, IT'S PRETTY BALANCED IN TERMS OF PEOPLE DIRECTLY COMING TO GLYGEN OR SEARCHING THROUGH SOME SEARCH ENGINE AND GETTING THERE OR BEING REFERRED FROM OTHER DATABASES AND WE'RE PARTICULARLY PROUD OF THE BALANCE BUT ALSO THAT A LOT OF OUR PEOPLE ARE COMING FROM REFERRALS, AND IF YOU ASK ABOUT WHAT SORTS OF PLACES PEOPLE ARE COMING FROM, IT'S PARTICULARLY USEFUL OR GRATIFYING TO SEE THAT PEOPLE ARE COMING TO GLYGEN, ARE BEING REFERRED TO GLYGEN FROM MAINSTREAM RESORES IN THE BIOMEDICAL RESEARCH COMMUNITY. SO WE'RE DUE TO OUTREACH THAT WE'VE MADE ALL ACROSS GLYGEN, WE'RE BEGINNING TO BECOME CONNECTED IN A WAY THAT PEOPLE FIND US WHETHER THEY WANT TO FIND US OR NOT. THAT WE'VE BECOME THE GO-TO FOR PEOPLE TO GET INFORMATION ABOUT GLYCOSYLATION OF THE PROTEIN OR THE GLYCOCONGREGATE THAT THEY MIGHT BE STUDYING. SO WE'VE HAD SOME SUCCESS IN DISSEMINATING THROUGH PUBLICATIONS, BOTH COLLABORATIVELY WITH PEOPLE THAT HAVE PROVIDED DATA, IN PARTICULAR, FOR THE O-GLCNACOME, WE'VE HAD A JOINT PUBLICATION, WE'VE ALSO HAD SOME PUBLICATIONS DESCRIBING SOME OF THE INFORMATIC APPROACHES, WE HAVE A PUBLICATION DESCRIBING HOW INFORMATION KNOWS BETWEEN OTHER RESOURCES AND GLYGEN THAT RAJA MENTIONED AND THIS IS JUST RESUBMITTED AFTER SOME RELATIVELY MINOR RESCISSION VISIONS REVISIONS. SO WE'RE REACHING OUT THROUGH PUBLICATIONS, ATTENDING MEETINGS, OF COURSE, AS OFTEN AS WE CAN AS SITUATIONS ALLOW. WE HAD A BOOTH AT THE LAST GLYCOBIOLOGY MEETING TO INTERACT WITH FOLKS AS MUCH AS WE COULD. WE'VE INCREASED OUR ACTIVITY ON SOCIAL MEDIA. RELEASING VIDEOS AND POSTER TALKS AND TWEETING AND TRYING TO ENGAGE WHEREVER WE CAN IN TERMS OF BRINGING THEM TO GLYGEN AND UNDERSTANDING WHAT WE DO. SO THAT'S THE USAGE STATISTICS. AND I'LL JUST KIND OF HIT SOME HIGHLIGHTS IN TERMS OF IMPROVEMENTS. THE FIRST THING I'D POINT OUT IS WE'VE ALWAYS HAD THE GOAL OF GETTING A RELEASE OUT EVERY THREE MONTHS OR SO AND THAT WAS TRUE THIS YEAR AS WELL. SO FIRST RELEASE OF THIS PAST YEAR WAS 1.7, IN OCTOBER. RAJA SHOWED YOU SOME OF THESE THINGS, ESPECIALLY RELATED TO PROTVISTA, SITE VIEWS FOR MUTAGENESIS DATA, SO THAT WAS A USEFUL RELEASE, AND THEN 1.8, IN APRIL, WE RELEASED A MAPPER WHICH ALLOWS YOU TO MAP GLYCAN IDENTITIES ACROSS OR -- GLYCOPROTEIN IDENTITIES ACROSS DIFFERENT NAME SPACES, AS WELL AS UPGRADING CERTAIN ASPECTS OF OUR COMPOSITION SEARCHES TO MAKE THEM MORE USER-FRIENDLY. THEN THE SORT OF, I THINK, THREE VERY IMPORTANT COMPONENTS IN THIS LAST RELEASE, SITE SEARCH, SUPERSEARCH AND SAND BOX. YOU CAN HEAR ABOUT THESE IN MORE DETAIL IN SESSION ONE AND TWO TOMORROW MORNING. I'M GOING TO FOCUS BRIEFLY IN SORT OF A DEMO MODE TO TALK A LITTLE BIT ABOUT SUPERSEARCH AND WHAT WE CALL OUR GLYGEN SAND BOX. YOU CAN HEAR MORE ABOUT THESE TOMORROW AS YOU'RE INTERESTED. IT'S CURRENTLY IN BETA, IT'S ACCESSIBLE ONLINE AND PROVIDES A LOT OF NICE NEW FEATURES SUCH AS THE ABILITY OF THE USER TO FILTER THE RESULTS, FOR THOSE BITS THAT YOU'RE MOST INTERESTED IN, WHETHER IT'S SPECIES OR DIFFERENT SORTS OF EXPRESSION PARAMETERS OR EVEN MASS OR WHAT KIND OF GLYCAN, VERY USEFUL WAY TO GET THE DATA ORGANIZE ED THE WAY YOU WANT TO. RAJA MENTIONED THE EXPRESSION DATA WE NOW HAVE INCORPORATED. AS WELL AS OR SORTS OF PTNs AND HOW THEY MIGHT CORRELATE WITH GLYCOSYLATION AND SOME OTHER FEATURES YOU CAN HEAR ABOUT TOMORROW IN THE SESSION ONE AS YOU'RE INTERESTED. THE SORT OF KEY ACHIEVEMENTS WILL BE THAT WE MAINTAIN THIS ROBUST SCHEDULE -- NEW COMPONENTS, NONE OF THEM WERE TRIVIAL RELEASES, THEY ALL HAD SOMETHING NEW TO ADD. ONE OF THESE MAJOR INCORPORATED CHANGES IN OUR FRAMEWORK WHICH WAS PRETTY MUCH INVISIBLE TO EVERYBODY BUT VERY IMPORTANT FOR GLYGEN GOING FORWARD IS TO CHANGE THE FRAMEWORK TO SOMETHING THAT'S MORE SUSTAINABLE AND IS MORE BROODILY BROADLY USED IN THE WEB COMPLEUBT. IN IN THE WEB COMMUNITY, THAT'S THE REACT FRAMEWORK. WE'VE ALSO ENHANCED SEARCH OPTIONS IN VERY IMPORTANT WAYS. EXPANDED GLYCAN TYPES, AND INCORPORATED SORT OF SOFTLY AT THIS POINT GNOME ONTOLOGY AND THE SAND BOX WHICH I'LL SHOW YOU IN A LITTLE MORE DETAIL. SO THOSE ARE VERSION IMPROVEMENTS. IF YOU HAVEN'T BEEN TO GLYGEN IN THE PAST YEAR, SHAME ON YOU. I HOPE YOU'LL GO LOOK. BUT IF YOU WERE HERE A YEAR AGO, YOU PROBABLY WOULD HAVE RECOGNIZED THAT AT THAT POINT, THERE WERE SORT OF A CORSET OF TWO OR THREE CARDS THAT YOU COULD CLICK ON AND SEARCH. THERE WAS A GLYCAN SEARCH AND A PROTEIN SEARCH. IN THE LAST YEAR, WE'VE ADDED SITE SEARCH, WHICH IS A VERY POWERFUL WAY TO LOOK AT GLYCOSYLATION IN A VERY HIGH RESOLUTION WAY OF PARTICULAR GLYCOSYLATION SITES ON PROTEINS DIRECTLY, AND WE'VE AT THE SUPERSEARCH, WHICH I'LL TELL YOU A LITTLE MORE ABOUT, THE GLYGEN MAPPER I MENTIONED AND IF YOU SCROLL DOWN ON THE HOME PAGE, THERE'S ADDITIONAL FEATURES, THE GNOME BROWSER STRUCTURE, WHICH YOU CAN HEAR MORE ABOUT TOMORROW IN SESSION TWO IF YOU'RE INTERESTED IN, FROM NATHAN. AND NATHAN HAS ALSO DEVELOPED A LIST OF MOTIFS IN CONSULTATION WITH A LOT OF EXPERTS THAT HE CAN REACH OUT TO AND TRY AND HELP ORGANIZE THE MOTIF LIST, SO THIS LIST DESCRIBES ALL THE MOTIFS THAT COVER GLYCOPROTEIN GLYCANS AND -- SINGLE LIPID GLYCANS AS WELL, A BROAD RANGE OF MOTIFS THAT WE ALL RECOGNIZE. THIS DEFINES GLYCAN STRUCTURES -- GLYCAN FUNCTIONS MORE DEEPLY WITHIN GLYCAN. THERE'S ACCESS TO THE SANDBOX, AND I'LL SHOW YOU WHAT THE SANDBOX CAN OFFER IN AN EXAMPLE. SO THESE SEARCHES ATIVANS IN, ADVANCES IN SEARCHES HAVE LARGELY BEEN DRIVEN BY THINKING ABOUT HOW PEOPLE COME TO GLYGEN AND WANT TO ACCOMPLISH SOMETHING BUT THEY DON'T REALLY KNOW ANYTHING ABOUT -- SO YOU THINK OF A SCENARIO WHERE SOMEONE MAY HAVE READ A PAPER LIKE THIS, THEY'RE NEURAL STEM CELL PEOPLE, INTERESTED IN NOTCH SIGNALING. THEY KNOW THINGS CAN BE GLYCOSYLATED BUT THEY HAVE NO IDEA WHAT LEWIS X IS BUT THIS REALLY POINTS OUT THAT LEWIS X IS HIGHLY REGULATED, BUT CERTAINLY ON GLYCOPROTEINS, THE AMOUNT OF LEWIS X ON STEM CELLS IS MUCH HIGHER THAN IT IS ON DIFFERENTIATED NEURONS. THIS STUDY GOES ON TO INDICATE -- MIGHT LEAD PEOPLE TO THINK WHAT'S LEWIS X, WHAT PROTEINS ARE MODIFIED WITH LEWIS X AND ARE THEY INVOLVED IN ANY WAY WITH DISEASES OR MORE MALL MENTAL FUNCTIONS? WELL, YOU CAN COME TO GLYGEN AND HOW WOULD YOU LEARN MORE ABOUT LEWIS X? I'M GOING TO SHOW YOU A LITTLE ABOUT WHAT WE CALL SUPER SEARCH. IT LOOKS A LITTLE LIKE SPAGHETTI BUT IT'S ACTUALLY QUITE SIMPLE. IT'S A WAY FOR US TO OFFER CONCEPTS THAT GLYGEN HAS DATA FOR, IN A WAY THAT ONE CAN BEGIN TO LOOK AT THE CONNECTIONS BETWEEN DIFFERENT CONCEPTS. SO I'M JUST GOING TO -- SO RIGHT NOW, THIS IS SHOWING WHAT EXISTS IN OUR DATABASE IN TERMS OF WE HAVE 33,715 GLYCANS, AND THEY'RE IN NINE DIFFERENT ORGANISMS INCLUDING SOME VIRUSES, AND WE HAVE 64,000 PROTEINS. THOSE PROTEINS HAVE 435,000 GLYCOSYLATION SITES. AND I'M JUST GOING TO CLICK ON THIS ADVANCED VIEW HERE AND THAT ADDS SOME ADDITIONAL INFORMATION ACROSS THE EDGES HERE. SO 64,200 PROTEINS, 53,000 OF THOSE HAVE GLYCOSYLATION SITES, SOME OF THESE PROTEINS ARE ALSO ENZYMES THAT BUILD GLYCANS, FOR INSTANCE, THAT YOU CAN SEE ARE ALSO HIGHLIGHTED HERE. SO NOW ONE CAN THINK ABOUT, FRENS, CLICKING UP HERE ON THE MOTIF CONCEPT, ONE GETS A SEARCH BOX HERE. I'M INTERESTED IN A MOTIF CALLED LEWIS X, SO LET'S ADD IN LIEU IS IN LEWIS X, YOU CAN SEE ONE MOTIF IS SHOWN, THE LEWIS X MOTIF THAT WE IDENTIFIED. THAT MOTIF IS ASSOCIATED WITH 1,649 GLYCANS. SOME OF THOSE HAD BEEN MAPPED TO 12 GLYCOSYLATION SITES ASSOCIATED WITH SEVEN PROTEINS. SO NOW ONE CAN EXPLORE A LITTLE MORE DEEPLY WHAT THOSE PROTEINS ARE AND WHAT THOSE SITES ARE AND WHAT'S THE GLYCAN THAT CARRIES THAT MOTIF? NOT JUST THE MOTIF BUT WHAT'S THE FULL GLYCAN? SO LET'S JUST START BY TAKE THE ONE ON THE TOP OF THE LIST, WHICH OUR RESULTS PAGE IS TELLING US THERE'S A POSITION 95 ON THIS PARTICULAR PROTEIN, WHICH IS INTERFERON ALPHA 14 -- 95 THERE IS, IN FACT, A LEWIS X-CONTAINING GLYCAN STRUCTURE. SO YOU CAN NOW LOOK A LITTLE MORE CAREFULLY AT OTHER ASPECTS OF WHAT WE KNOW ABOUT THIS GLYCOPROTEIN. FOR INSTANCE, RAJA SHOWED YOU THE SEQUENCE PANEL AND NOW YOU CAN SEE THAT AT RESIDUE 95, THIS GLYCOSYLATION SITE WE KNOW HAS A LEWIS X STRUCTURE. THERE IS A MUTATION THAT RESULTS IN THE LOSS OF THAT SEQUENCE, CONVERTED TO ASIRINE, ACTUALLY ASSOCIATED WITH COLORECTAL CANCER. SO NOW YOU'VE GONE FROM THINKING THAT LEWIS X MIGHT BE IMPORTANT TO THOSE THAT HAVE LEWIS X ON THEM, HERE'S A LIST OF PROTEINS THAT HAVE MAPPED LEWIS X STRUCTURES, AND YOU CAN CLICK AND WE JUST LOOKED AT INTERFERON ALPHA 14 BUT YOU COULD EXPLORE SOME OF THESE OTHERS AND ASK WHETHER THEIR GLYCOSYLATION SITES ARE INVOLVED IN IDENTIFIED DISEASES OR THERE ARE SINGLE NUCLEOTIDE VARIATIONS OUT THERE THAT MIGHT IMPACT THE GLYCOSYLATION OF THOSE PARTICULAR SITES. SO THAT'S A SIMPLE USE OF OUR SUPERSEARCH. I'M GOING TO SHOW YOU ANOTHER USE IN A SECOND THAT'S A LITTLE BIT MORE COMPLEX, BUT I WANTED TO KIND OF TAKE A SIDE TRIP INTO THE SANDBOX BECAUSE WE'RE TALKING ABOUT GLYCAN STRUCTURES HERE, WE WERE JUST LOOKING AT INTERFERON ALPHA 14 AND WE WERE LOOKING AT THIS GLYCAN -- 95 THAT HAS THIS LEWIS MOTIF. SO OF COURSE WITHIN GLYGEN, IF YOU SEE A GLYCAN, YOU CAN ALWAYS ACCESS MORE INFORMATION ABOUT THAT GLYCAN BY CLICKING ON THE SESSION NUMBER, WHICH TAKES YOU TO A DETAILED PAGE FOR THE GLYCAN AND HERE YOU CAN SEE THIS IS THE GLYCAN THAT WE WERE STUDYING, AND NOW THIS YEAR, WE'VE ADDED LINKS TO SANDBOX, YOU CAN ALSO GO TO GNOME HERE, WE HAVE SIMILAR COMPOSITION, DIFFERENT TOPOLOGIES, AND YOU CAN LEARN MORE ABOUT GNOME TOMORROW IN SESSION TWO OR YOU CAN GO TO THE SANDBOX BRIEFLY AND WE WILL BE AVAILABLE TOMORROW AS WELL TO TALK ABOUT HOW THE SANDDOCK BOX SAND BOX WORKS. WILL HAS SPENT CONSIDERABLE AMOUNT OF TIME OVER THE PAST COUPLE YEARS AS HAVE OTHERS COMING UP WITH WAYS TO DESCRIBE CANONICAL RESIDUES WITHIN GLYCAN STRUCTURES, SO EACH ONE OF THESE RESIDUES NOW HAS AN IDENTITY AND YOU CAN SEE THOSE HIGHLIGHTED IN THIS TABLE. THAT ALLOWS WILL TO MAYBE BIOSYNTHETIC PATHWAYS BASED ON HOW THOSE RESIDUES GET ADDED, AND SO YOU CAN GO AND CLICK -- I'M NOT GOING TO DO THIS, BUT YOU CAN LOOK AT ALL THE ENZYMES THAT ARE ASSOCIATED WITH THE BIOSYNTHESIS OF THIS GLYCAN. YOU CAN GO TO GNOME AND SEE GLYCANS HAVE SIMILAR STRUCTURES. I'M JUST GOING TO HIGHLIGHT THIS FEATURE, SHOW PATHWAYS, WHERE WE'LL SET UP A WAY STARTING WITH A LIKELY PRECURSOR, INITIAL PRECURSOR FOR THE BIOSYNTHESIS OF THIS GLYCAN, WHICH IS DOWN HERE AT THE END OF THE PATHWAY, AND HIGHLIGHTING LIKELY PATHWAYS FROM PRECURSORS TO PRODUCTS ALL THE WAY THROUGH THE BIOSYNTHESIS. SO WE'RE JUST GOING TO TAKE ONE STEP THROUGH THIS, AND YOU CAN SEE NOW WE'VE ADDED ANOTHER TO THE PRECURSOR STRUCTURE TO TAKE ANOTHER STEP DOWN THIS PATHWAY. THESE ARE PREFILLED WITH THE MOST LIKELY STEPS, BUT A USER CAN COME IN HERE AND CLICK THESE OFF OR CLICK ON A DIFFERENT GLYCAN AND TRY AND DRIVE THE PATHWAY THROUGH DIFFERENT STRUCTURAL POSSIBILITIES. SO IT'S A FUN THING TO EXPLORE, BUT YOU CAN RENDER THIS PATHWAY WITH ALL THE INFORMATION ASSOCIATED WITH ENZYMES AND YOU CAN SEE MORE ABOUT THAT TOMORROW. SO THAT'S SANDBOX AND WE'RE GOING TO INCREASINGLY HARD INCORPORATE THE COMPONENTS INTO GLYGEN AND MAKE USE OF THE POWER THAT THESE CANONICAL RESIDUES PROVIDE FOR UNDERSTANDING GLYCAN STRUCTURES THAT HAVE BEEN PREVIOUSLY DESCRIBED AND HIGHLIGHTING NEW GLYCAN STRUCTURES THAT MIGHT NOT FIT CANONICAL RULES BUT MIGHT BE IMPORTANT FOR THE FUTURE OF GLYCOBIOLOGY. SO I'M GOING TO END UP NOW WITH SORT OF ANOTHER BRIEF DEMONSTRATION, WE'RE GOING TO ENTER INTO THE SITE -- TRYING TO UNDERSTAND WHY GLYCOSYLATION SITES WITHIN THE DATASET. SO I'M GOING TO PROPOSE A SITE SEARCH WHERE WE ARE GOING TO NAME A RESIDUE, ASPARAGINE, THAT WE WANT TO CHECK OUT GLYCOSYLATION, SO WE WANT TO LOOK AT GLYCOSYLATED ASPARE JEANS BUT WE ALSO WANT THEM TO BE ASSOCIATED WITH SINGLE NUCLEOTIDE VARIATIONS. SO SHOW ME ALL THE END LINK GLYCOSYLATION SITES IMPACTED BY CHANGE IN THE ASPARAGINE RESIDUE -- THAT'S A PRETTY POWERFUL SEARCH IF YOU THINK ABOUT IT. SO YOU DO THAT SEARCH AND YOU CAN SEE THERE ARE 9,681 SITES ASSOCIATED WITH 3,731 PROTEINS THAT HAVE THESE VARIATIONS AT ASPARAGINE SITE. YOU CAN LOOK A LITTLE MORE CLOSELY, WE'LL TAKE THE TOP ONE OFF THE LIST WHERE THE RESULTS ARE SUGGESTING AT POSITION 281, THERE IS A -- THIS PROTEIN, WE CAN GO JUST LIKE WE DID BEFORE, GO TO SEQUENCE DESCRIPTION AND, IN FACT, IF WE LOOK AT RESIDUE 281, THERE IS A SINGLE SEQUENCE -- SINGLE NUCLEOTIDE VARIANT THAT RESULTS IN THE LOSS OF THAT GLYCOSYLATION SITE CHANGE. SO THAT'S JUST SORT OF A RAPID OVERVIEW. I HOPE THAT I'VE SORT OF ENGAGED YOU ENOUGH THAT YOU WILL PARTICIPATE IN THE BREAKOUTS TOMORROW. AND LEARN MORE ABOUT THESE NEW FEATURES THAT WE'VE IMPLEMENTED OVER THE YEAR, AND AGAIN, JUST TRYING TO HIGHLIGHT WHAT I THINK WE'VE ACCOMPLISHED THIS YEAR, WE'VE BEGUN WITH OUR NEW SEARCH OPTIONS, BEGUN TO LET PEOPLE ANSWER QUESTIONS THAT REALLY CAN'T BE ANSWERED ANYWHERE ELSE VERY EASILY, AND THAT'S FROM THE SEARCHES LIKE SUPER SEARCH, LIKE THESE MECHANISMS THAT WE'VE DEVELOPED, AND ALSO WE'RE ALWAYS TRYING TO ENHANCE THESE FUNCTIONALITIES SO THEY'RE NOT JUST ACCESSIBLE BY TRAINED GLYCOSIGN PACIFIC SCIENTISTS BUT PEOPLE CA N ENTER IN USING CONCEPTS THAT MIGHT BE MORE FAMILIAR WITH THEM. WE'VE EXPANDED THE DATA TYPES AND DATA VOLUME AND THIS IS A NEVER ENDING NOT STRUGGLE BUT IT'S A NEVER ENDING MISSION, AND WE HOPE -- WE CONTINUALLY WANT TO HOPE THAT WE CAN TURN TO THE COMMUNITY TO GET YOUR DATA, PUBLISHED DATA, AND TO HONEST, THE GLYCOMICS AND THE GLYCOPROTEOMICS FIELD, THIS IS GOING TO REQUIRE A BIFT A BIT OF A SOCIETAL CHANGE. MY LAB, WHICH DOES A LOT OF GLYCOMICS AND GLYCOPROTEOMICS IS JUST TURNING THE CORNER, I THINK, IN THIS CHANGE THAT WE NEED TO AS A COMMUNITY ADOPT. I WOULD LOVE TO BE IN A ROOM AND ASK PEOPLE TO PUT THEIR HANDS UP AND ASK THEM HOW MANY OF YOU IN YOUR LAST GLYCOMICS PAPER SUM MANAGEMENT TRI TABLE BULL A GLYCAN 2 -- ASSOCIATED WITH EACH OF THE 89 GLYCANS ON YOUR DATASET? IT TAKES TIME, BUT GLYGEN CAN HELP YOU DO THAT. IT'S ALMOST DESIGNED JUST TO HELP YOU ASSIGN GLYTOUCAN NUMBERS TO YOUR DATASETS. WHEN WE HAVE DATASETS WITH THAT KIND OF ANNOTATION, WE CAN INCORPORATE THAT VERY NICELY INTO GLYGEN. RIGHT NOW WE WORK WITH PEOPLE TO MAKE THAT HAPPEN. BUT EXPANDING OUR DATASET IS SO IMPORTANT TO THE USABILITY OF THIS RESOURCE, AND WE CONTINUE TO REACH OUT. ONE OF THE OTHER ACHIEVEMENTS THAT WE'VE SORT OF LAID THE FRAMEWORK FOR BIOSYNTHETIC PATHWAY MAPPING WITHIN TBLIE GENERAL AS WELL GLYGEN AS WELL USING SAND BOX FEATURES THAT I SHOWED YOU. AND I WILL JUST BRIEFLY TOUCH ON SOME OF THE THINGS WE'RE THINKING ABOUT. RAJA MENTIONED SOME OF THESE FOR THE IN EX-YEAR, ONE OF OUR HIGHEST PRIORITIES IS TO MAKE SURE EVERYTHING WORKS REALLY WELL, AS WE GO INTO WRITING APPLICATIONS FOR FUTURE FUNDING, SO WE WANT EVERYTHING TO WORK AS SMOOTHLY AS POSSIBLE. IN FACT, EFFORT WILL BE PUT INTO THAT AS OPPOSED TO ADDING MORE FUNCTIONS, QUITE HONESTLY. AND WE'VE ALREADY BEGUN INCORPORATING DROSOPHILA. THROUGH YOUR USE CASES THAT YOU MIGHT PLEASE PROVIDE US, WE ARE GOING TO EXPLORE OTHER SPECIES. WE'VE BEEN THINKING ABOUT BACTERIAL GLYCOSYLATION, OF COURSE, BUT WE'RE OPEN TO ALL KINDS OF SUGGESTIONS AND WE'RE ALWAYS EXPANDING PHENOTYPES, GLYCAN BINDING INTERACTIONS, GAG INTERACTIONS, FINDING AS MUCH AS WE CAN ABOUT GLYCOSYLATION SITES. RAJA MENTIONED OUR ALIGNMENT WITH THE COMMON FUND DATA ENVIRONMENT. WE THINK THAT'S GOING TO PROVIDE -- ACCESS TO ALL SORTS OF BIG DATASETS, AND IT WILL BE INTERESTING TO SEE HOW THAT WORKS, AS WELL AS BRINGING UP, STANDING UP THE GLYCAN ARRAY DATA REPOSITORY THAT RENE CAN TELL BUT TOMORROW IN ABOUT TOMORROW IN SE SSION THREE. RAJA MENTIONED THE NEW ANNOTATIONS WE'RE ADDING AND I MENTIONED SOME OF THE FILTERING OPTIONS AS WELL AS BEGINNING TO INCORPORATE BIOSYNTHETIC DATA IN TERMS OF PATHWAYS, NOT JUST IN TERMS OF ENZYME RISKS. SO I'LL END THERE, AND WE CAN ENTERTAIN ANY QUESTIONS AND RAJA MENTIONED ALL OF OUR COLLABORATORS THAT OF COURSE YOU CAN'T DO ANYTHING WITHOUT THESE INTERACTIONS, THEY'VE BEEN VERY POWERFUL IN ADVANCING GLYGEN. I WILL STOP SHARING. >> THANK YOU, MIKE AND RAJA. DO WE HAVE QUESTIONS FOR EITHER OF THEM? I GUESS I'LL START. THERE IS A HUGE AMOUNT OF DATA THAT'S BEING TAKEN FROM DIFFERENT DATASETS AND THEN ESSENTIALLY NEW DATASETS DEVELOPED FROM THOSE THROUGH YOUR TOOLS. ARE THOSE BEING SAVED, ARE THERE DATA REPOSITORIES THAT NEED TO BE MAINTAINED? WHAT'S THE FUTURE FOR ALL OF THIS DATA? >> YOU'RE TALKING ABOUT RAW DATA, RIGHT? >> RIGHT. >> LIKE REAL DATA. AND OF COURSE GLYGEN'S MISSION IS TO TRY AND PRESENT KNOWLEDGE TO THE COMMUNITY, AND WHERE THERE'S A PATHWAY BACK TO THAT RAW DATA, WE SHOULD THEN TRY TO HIGHLIGHT IT. IF JOE IS ON, JOSIAH, HE CAN TALK ABOUTING IT JUST AS MUCH AS I CAN, ABOUT GLYCOPOST IS ONE OF THE PRIMARY PLACES FOR GLYCOMICS DATA AND PROTEOMIC DATA. THESE THINGS NEED TO BE SUPPORTED AND EXPANDED. >> YEAH, AND YOU GENERATE NEW DATASETS WITH YOUR TOOLS, SO ARE ANY OF THOSE HOUSED ANYPLACE? I MEAN, DO THEY HAVE BROADER FUNCTIONS THAT THEY NEED TO BE HOUSED, OR CAN THEY JUST BE REGENERATED EACH TIME? >> THEY CAN CERTAINLY BE REGENERATED, SO SOME OF THOSE, WE HAVE SOME PRECANNED SEARCHES TO GET PEOPLE UP AND GOING, BUT THEY'RE ESSENTIALLY RESEARCHED EACH TIME. BUT IT'S AN -- THERE ARE USE CASES, COMMON USE CASES THAT ARROW PEOPLE TO GENERATE THAT QUICKLY. >> IN DATA IN GLYGEN.ORG, SO OF COURSE ALL THAT DATA, PAM, CAN BE DOWNLOADED, IT'S NOT REALLY RAW, BUT OF THE TABLES, AT LEAST, AND IMAGES CAN BE DOWNLOADED BY ANYBODY. YOU CAN DO A SEARCH AND YOU CAN DOWNLOAD. WE ALSO HAVE CREATED NEW TABLES BECAUSE LET'S SAY THE FRONT END, WE DO NOT HAVE A FUNCTIONALITY AND A USER IS REQUESTING FOR A VERY SPECIFIC SEARCH, SO WE CAN ON THE BACK END WRITE SCRIPS AND WE CAN GENERATE A TABLE OR PIECES OF INFORMATION, WHICH WE THEN PUT IT BACK AS COMMUNITY REQUESTED BUT WE DON'T UPDATE IT ON THE REGULAR MONTHLY CYCLE. THAT'S BEYOND OUR ABILITY. BUT WE WILL GENERATE IT AT ONE TIME WHEN A USER HAS REQUESTED IT AND WE WILL KEEP IT THERE, IT DOESN'T GO ANYWHERE, IT STAYS THERE. >> RIGHT. SO ALL OF THIS DATA THAT YOU'RE DRAWING FROM IS IN DATA REPOSITORIES. WHAT'S THE STABILITY OF THOSE REPOSITORIES? ARE THEY PRIVATE OR ARE THEY GOVERNMENT-SUPPORTED OR -- >> SO WHEN WE PULL IN DATA FROM ANY PLACE, WE ALSO MAKE A COPY OF THE DATA ON OUR SERVERS. SO EVEN IF THE REPOSITORY GOES AWAY, WE STILL HAVE THE INFORMATION THAT IS FUELING GLYGEN. AND I THINK THAT IS THE KEY. AND THEN EVERYBODY THAT WE GET DATA FROM, WE ALSO HAVE AN AGREEMENT, WE HAVE THIS LICENSE CALLED CC BY 4.0, WHICH MEANS WHATEVER YOU GIVE US, WE WILL SHARE WITH OTHERS, AND WHEN WE SHARE WITH OTHERS, THEY CAN DO WHATEVER THEY WANT TO DO WITH IT, THEY DON'T NEED TO TELL US, THEY CAN SELL IT, THEY CAN USE IT IN THEIR RESEARCH, SO IT'S A VERY PERMISSIVE LICENSE. SO WE TAKE CARE OF THESE TWO MAJOR THINGS WHICH IS IMPORTANT FOR SUSTAINABILITY OF ANY WORK OF THIS TYPE. ONE IS WE HAVE THE DATA IN FORMAT THAT PEOPLE CAN USE, AND AND THEN WE ALSO HAVE THEM UNDER VERY PERMISSIVE LICENSE, WHICH IS RIGHT THERE ON THE WEBPAGE SO THAT PEOPLE DON'T HAVE TO ASK US QUESTIONS, MIKE OR ME OR ANYBODY, HEY, CAN I DO THIS, CAN I DO THAT? YOU CAN DO THAT, WHATEVER YOU WANT WITH IT, AND I THINK THAT'S VERY -- WE MADE AN AGREEMENT THAT ALL THE PARTICIPANTS WILL DO THE SAME. THAT WAS A BIG STEP FORWARD AS EARLIER, YOU KNOW, WE WERE TALKING ABOUT FAIR, FINDABLE, ACCESSIBILITY, RE-USABILITY, SO THIS LICENSE IS ALSO A REALLY, REALLY IMPORTANT PART OF SHARING DATA, SO WHEN PEOPLE ARE GENERATING DATA, MAYBE THEY CAN ALSO THINK ABOUT HOW THEY WANT TO SHARE IT, NOT JUST PEOPLE FIND IT, BUT IS IT -- WHAT KIND OF LIE SANS THEY ARE USING, AND I'LL BE HAPPY TO TALK ABOUT A WORKSHOP WE DID A COUPLE OF YEARS BACK IN CAMBRIDGE ON LICENSING DATA. IT WAS A REALLY INTERESTING WORKSHOP, AND SINCE THEN, THERE ARE LOTS OF RESOURCES WHO HAVE BEEN USING THESE VERY PERMISSIVE LICENSES. YOU CAN SEE THAT IN THE LAST TWO YEARS, THINGS ARE JUST CHANGING SO QUICKLY. >> I AGREE. AND I THINK IT'S REALLY IMPORTANT BECAUSE WE SAW THIS WITH THE SARS COV2, WHERE PEOPLE WERE USING DATA, PULLING IT DOWN AND DECORATING THE SPIKE PROTEIN WITH IT, AND THOSE ARE ESSENTIAL FOR VACCINE DEVELOPMENT. SO YOU COULD THINK ABOUT, YOU KNOW, OTHER CASE USES WHERE PEOPLE ARE GOING TO BE LOOKING TO SEE WHAT'S GOING ON HERE FOR BIOTHERAPEUTICS, AND THAT'S A HUGE MARKET. BUT PEOPLE DON'T WANT TO GET TIED UP IN LICENSING. SO HAVING IT OPEN ACCESS IS DEFINITELY IMPORTANT. WHAT ARE YOUR PLANS FOR TAKING THIS FORWARD? NSF HAS RE-AUTHORIZATION COMING UP, AND CERTAINLY THERE'S -- THE NSF FOR THE FUTURE ACT THAT HAS, YOU KNOW, INPUT FROM CONGRESS THAT SUGGESTS THAT DATA REPOSITORIES ARE IMPORTANT, AND NEED TO BE FUNDED. SO ARE PEOPLE LOOKING AT THAT? >> SO WE HAVE NOT BEEN LOOKING SPECIFICALLY AT RUNNING A DATA REPOSITORY. IT'S KIND OF ON OUR LONG WISH LIST, IF THERE WOULD BE VALUE FOR US TO TAKE THAT ON, BUT IT WOULD TAKE A DIFFERENT FUNDING MODEL THAN WE CURRENTLY HAVE. WE HAVE BEEN LOOKING AT OTHER MERCK NISMS, FUNDING MECHANISMS AT NIH. WE HADN'T THOUGHT ABOUT NSF, THAT MIGHT BE WORTH LOOKING AT. U24 MECHANISM IN PARTICULAR, KNOWLEDGE BASED FUNDING MECHANISMS, AND THAT'S OUR CURRENT TARGET FOR THE COMING FALL. TO GET AN APPLICATION IN THERE. >> DOUG, DO YOU HAVE A QUESTION? >> I DON'T HAVE A QUESTION, I DO HAVE A COMMENT. I THINK MIKE'S POINT IS AN IMPORTANT ONE FOR US TO KEEP FRONT OF MIND. THE ORIGINAL GOAL HERE WAS NOT TO BECOME THE REPOSITORY FOR ALL THE RAW DATA THAT PEOPLE ARE DEVELOPING. ACROSS GLYCOSCIENCE OR EVEN FOR A PARTICULAR PLATFORM, FOR INSTANCE, MASS SPECTROMETRY, WHICH IS ONE I'M MOST INVOLVED WITH. REALLY, THE IDEA HERE IS TO BE ABLE TO HARMONIZE AND INTEGRATE KNOWLEDGE ACROSS THE FIELD, AND WITH THE EXISTING WELL-REGARDED KNOWLEDGE BASES IN GENOMIC AND PROTEOMIC DATA. I THINK THAT IT'S IMPORTANT TO KEEP OUR EYE ON THE BALL, SO TO SPEAK, AND NOT GET SUCKED INTO PROVIDING REPOSITORIES FOR FIRST LINE ANALYTIC DATA, SOME OF WHICH HAVE GOT PERFECTLY ADEQUATE HOMES ELSEWHERE. >> OF COURSE THEN THERE IS THIS ONE EXCEPTION TO THAT RULE AND THAT IS THE GLYCAN ARRAY DATA REPOSITORY WHICH WE HAVE BEEN WORKING WITH THE COMMUNITY TO TRY AND ESTABLISH. I THINK IT'S KIND OF A UNIQUE CASE THAT PROVIDES A REAL VALUABLE ADVANTAGE TO GLYGEN TO HAVE ACCESS TO THAT DATA, TO TRY AND INTEGRATE BINDING DATA WITH STRUCTURES AND OTHER GLYCOPROTEIN ASSOCIATED DATA. AND THAT ALSO IS HELPING US TO INTEGRATE WITH OTHER DATA SOURCES OUT THERE IN THE WORLD THAT PROVIDES US SOMETHING WE CAN OFFER TO, FOR INSTANCE, THE COMMON FUND DATA ENVIRONMENT TO SAY LET'S SEE IF WE CAN CONNECT THIS DATA WITH YOUR DATA. SO IN THAT CASE, WE ARE HOUSING NOT NECESSARILY THE RAWEST OF RAW DATA, BUT DATA THAT PEOPLE CAN ACCESS AND DO WHAT WE WANT TO WITH. >> OF COURSE, MIKE, THOUGH, ONE OF THE MOTIVATIONS FOR DOING THAT WAS, IN FACT, TO CREATE A SINGLE POINT OF ACCESS FOR WHAT HAS BEEN A CONSTELLATION OF INDEPENDENT DIFFERENTLILY FORMATTED RESOURCES, RIGHT? >> YEP >> SO THERE IS A SYNTHETIC -- >> THAT'S CERTAINLY A VERY RICH DATA SORT. YOU'RE TYING THAT WITH THE PDB, IS THAT CORRECT? PROTEIN DATABASE? >> NOT THE ARRAY DATA, PAMELA. >> DO WE HAVE ANY OTHER QUESTIONS? >> I HAVE A QUESTION. THIS IS AXE ALEXANDRY. DO YOU KNOW -- YOU PROBABLY KNOW ANY STATISTICAL DATA ON YOUR USERS, HOW MANY PEOPLE ACCESS WHICH FUNCTIONALITY AND SO ON? I THINK THIS MAY BE USEFUL FOR -- JUST FOR THIS PROGRAM TO HAVE THAT INFORMATION. >> JUST OVER THE LAST YEAR, WE'VE BEGUN TO TRY TO BE MORE SOPHISTICATED AT UNDERSTANDING WHO OUR USERS ARE, SO THAT'S EVOLVING FOR US. WE DON'T, OF COURSE, COLLECT IDENTITIES, BUT WE CAN GET SOME GEOGRAPHICAL INFORMATION OF THE THINGS. >> OF COURSE, EXACTLY, AND MAYBE TO KNOW IF THESE ARE PEOPLE FROM BEYOND THE COMMUNITY WHERE YOU WERE ACTIVELY ADVERTISING IF SOME PEOPLE ARE ACCESS -- THAT WILL BE GREAT. THANKS. >> JUST THE RAW NUMBERS TO ME IN TERMS OF THE NUMBER OF USERS SAY TO ME THAT WE'RE REACHING BEYOND THE MEMBERSHIP OF THE SOCIETY FOR GLYCOBIOLOGY, AND THE MEMBERSHIP OF THE IGO, SO JUST THAT MASS -- WE'VE GOT TO BE GETTING IT OUT THERE SOMEHOW. >> THANKS. >> DO YOU KNOW WHERE -- I MEAN, IS IT ALL U.S. USE OR IS IT INTERNATIONAL? >> I DON'T HAVE THAT -- IT'S DEFINITELY INTERNATIONAL. I DON'T HAVE THE ACTUAL BREAKDOWN IN MY HEAD. SORRY, PAMELA. BUT WE HAVE THAT INFORMATION. >> THAT WOULD BE VERY HELPFUL, I THINK. ARE THERE ANY OTHER QUESTIONS? IF NOT, WE HAVE A BREAK COMING UP, SO WHY DOESN'T EVERYBODY TAKE A FEW MINUTES, GRAB YOUR CUP OF COFFEE, AND WE WILL BEGIN AGAIN AT -- LET'S TRY TO BE BACK BY 10:30. WE'LL SEE YOU ALL AT 10:30. >> WHY DON'T YOU GET STARTED. I KNOW EVERYBODY IS ITCHING TO HEAR WHAT YOU HAVE TO SAY. >> OKAY. THANKS. WE'RE ALL IN THE TOOLS GROUP. AND WE'VE BEEN WORKING ON ESSENTIALLY MASS SPEC-BASED SIMPLIFIED GLYCAN PROFILING WORKFLOWS THAT CAN BE ADAPTED TO ESSENTIALLY EVERYTHING. AND SO HERE'S MY SLIDE. MY SLIDES AREN'T ADVANCING. I DON'T KNOW HOW I DID THAT. BRIEF DISCLOSURES. DRS. DRAKE, MYSELF, DR. MEHTA AND DR. ANGEL, WE HAVE OWNERSHIP INTEREST IN TWO COMPANIES, ONE WHICH YOU'LL HEAR MORE ABOUT AND DR. MEHTA ALSO HAS INTEREST -- OWNERSHIP INTEREST AND ADVISORY BOARD IN GLYCO TEST DIAGNOSTICS. I'LL DO A QUICK OVERVIEW AND THEN GET INTO THE SUMMARY OF OUR AIMS AND THEN PROVIDE SOME -- SOME OUTREACH SUMMARY AND EFFORTS AND APPROACHES WE'VE BEEN DOING. THE ORIGINAL PREMISE OR ORIGINS OF THE METHOD CAME FROM OUR WORKFLOWS WE DEVELOPED FOR IMAGING N GLYCANS AND TISSUE USING IMAGING MASS SPECTROMETRY APPROACHES WHERE WE CAN SPRAY PANGASE ON TO THE TISH AND THEY'RE LINKED BACK WITH THEIR HISTOPA WILLING TO. WE'RE USUALLY DOING THIS ON FORMAL AND FIXED TISSUES WHICH IS GREAT BECAUSE THERE'S A LOT OF FORMALIN FIXED TISSUES IN MEDICAL SCHOOL. AND GENERALLY IT'S A HEAT MAP OF ALL THE LOCATIONS WHERE THE -- WAS DONE, SO IT'S AN INTENSITY HEAT MAP AND IT GETS CHANGED OVER TO COLOR PIXEL. AS I MENTIONED, OUR PREMISE WAS WHAT WE'VE NOTICED FROM THE TISSUE IMAGING, THESE WERE VERY REPRODUCIBLE AS FAR AS THE TYPES OF GLYCANS DETECTED, THE ABUNDANT ONES, THE LEAST ABUNDANT ONES. IT'S A VERY MATHEMATICAL APPROACH THAT YOU CAN USUALLY FIGURE OUT COMPOSITION QUITE EASILY. SO COULD WE ADAPT THAT TO DIAGNOSTIC APPLICATIONS, IN ESSENCE, A GLYCO-TYPIER, THE NAME IS BASED ON THE SUCCESS IN THE CLINICAL MICROBIOLOGY WORLD, WHERE NOW THERE'S ABOUT 6,000 LITTLE MASS SPECS DEPLOYED ACROSS THE WORLD THAT DO SPECTRAL BARCODE LIBRARIES, ID CLINICAL PATHOGENS IN THE MICRO LAB. SO WHY NOT GLYCANS? THAT'S ONE OF THE THEMES. THE POINT IS THESE AREN'T MICRO BIOLOGISTS WHO RUN THESE SO CAN WE DO THESE IN NON-GLYCOMIBG LABS. SO AGAIN AS MENTIONED, WE STARTED WITH THE IMAGING MS SIDE. WE'LL COME BACK TO SOME OF THIS IN A MINUTE, BUT WE'VE BEEN PRETTY SUCCESSFUL WITH DEVELOPING WORKFLOWS LOOKING AT TOTAL GLYCAN ON BIOFLUID, OORN WITH THAT ASSAY. URINE WITH THAT ASSAY. WE HAVE A LOT OF ANTIBODY ARRAYS, WE'LL COVER THAT, AND THEN AIM TWO WAS LOOKING AT IMMUNE CELL CAPTURE, AND THAT ONE IS THE MOST ONGOING AT THE MOMENT. INHERENT -- THIS ALSO IS TO BE ABLE TO LOOK AT INDIVIDUAL CELLS. SO THAT WAS AIM THREE. LARGELY DIRECTED BY DR. ANGEL. SO YOU CAN GET THE CELL GROWN ON A SLIDE. CAN WE DO THE GLYCAN IMAGING WORKFLOW ON IT. SO THE INITIAL PAPER WAS PUBLISHED IN 2019 AND IN THE LAST COUPLE MONTHS, WE HAVE A METHOD PAPER THAT DESCRIBES ALL THE DETAILS ON METHODOLOGY WORKFLOWS FOR DOING THIS. THIS WAS FROM THE ORIGINAL PAPER. IF YOU CAN GET THEM ON THE SLIDE, WE CAN GENERATE GLYCAN PROFILES. SHE ALSO ADAPTED AS PART OF THE ORIGINAL I DOG METHOD WHERE YOU USE A LABELED GLUTAMINE THAT WILL INCORPORATE A PLUS 1 INTO THE GROWING CELLS, AND I LIKE THIS SLIDE A LOT BECAUSE WE CAN DEMONSTRATE WE CAN INCORPORATE IT, BUT THEY'RE COLORED YELLOW BECAUSE THESE WERE THE ONES THAT INCORPORATED GLUTAMINE, THE BLUE ONES DO NOT, AND YOU'RE ESSENTIALLY LOOKING AT SINGLE CELL, SO WE'RE GETTING GOOD AT THAT KIND OF RESOLUTION, AND THEN LASTLY, OVER THE LAST I GUESS THROUGH PANDEMIC ACTUALLY, WE'VE BEEN BUSY WORKING WITH OTHER GROUPS. TWO INDEPENDENT PAPERS THAT INVOLVED DR. ANGEL AND SOME OF US HAVE BEEN PUBLISHED USING THIS METHOD, BESIDES THE ORIGINAL METHOD PAPERS. AND WE'VE GOT AT LEAST FOUR OUTSIDE LABS INCLUDING SOME OF THE GLYCOALLIANCE MEMBERS THAT ARE DOING DIFFERENT PROJECTS, SO THAT'S ALL MOVING FORWARD NICELY. ALSO DURING THE PANDEMIC, WE STARTED WORKING WITH RICHARD AND HEATHER STEET AT THE GREENWOOD EPIGENETICS CENTER, HAPPENS TO BE IN SOUTH CAROLINA, EVEN THOUGH IT'S STILL ABOUT FOUR HOURS AWAY, WORKING ON THE ZEBRAFISH MODELS, AND IT LOOKS VERY PROMISING. AGAIN, I DON'T WANT TO CALL THAT A SINGLE CELL, BUT THOSE ARE TINY. AND IT'S BEEN VERY PROMISING, AND THERE'S A PAPER SUBMITTED ALREADY AND MORE PLAN. SO THAT'S BEEN A GREAT USE OF THE TECHNOLOGY. IN AIM TWO, WE WANTED TO SEE IF WE COULD DO ESSENTIALLY THESE IMMUNE CELL SUBTYPES. THEY'RE NOT GROWING ON A SLIDE, WE HAVE ACCOMPLISHED DOING SOME MODEL CELL, T-CELL, B CELL LINES. THAT'S WORKED OUT. IT ISN'T AS ROBUST AS THE CELLS GROWN ON THE SLIDE. WE'RE WORKING ON THAT. ACTUALLY A LOT OF THESE STEPS, WE'RE LEARNING WHAT WE CAN FROM THE CULTURED CELL AIM THAT I MENTIONED WHICH IS AIM THREE, AND THEN THE ANTIBODY ARRAY AIMED -- FROM AIM ONE. PART OF THAT STUDY, I'M GOING TO DIVERT A LITTLE INTO THE TISSUE SIDE, BUT IT BRIX BRINGS IT BACK ON THE IMMUNE THEME. OF COURSE THE LEWIS ANTIGENS ARE INPARENT IN EVERYTHING ABOUT THE IMMUNE INTERACTION, CANCER, OF COURSE, SO EVEN THOUGH THE ANTIBODIES AREN'T THAT GREAT, THERE ARE SOME DECENT ONES YOU CAN USE, FFPE, IN PARTICULAR, WE'VE BEEN LOOKING AT SILO LEWIS A, LEWIS X AND LEWIS Y STAIN OF THE STAINING OF THE TISSUES. ACTUALLY DR. ANGEL DEVELOPED THIS FIRST FOR, I BELIEVE, P10, AND OTHER TUMOR MARKERS, BUT SO ESSENTIALLY WE CAN DO THE I HC, IMMUNOHISTOCHEMISTRY JUST LIKE YOU WOULD, STAIN IT WITH THE PEROXIDASE, GET THE IMAGE, AND THEN WE ACTUALLY TAKE THE COVER SLIP OFF AND JUST DO THE IMAGING WORKFLOW, SPRAY THE PANGASE AND THEN DO OUR GLYCAN IMAGING ON IT. THE ADVANTAGE OF THAT IS WITHIN THE SOFTWARE, YOU'RE AUTOMATICALLY COREGISTERRING YOUR DATA WITH THESE IHCs. SO IT'S QUITE ROBUST. THIS IS ACTUALLY LEWIS Y AND I WON'T -- BUT MY LAB IS LOOKING AT A LOT OF PROSTATE CANCER, SO AS WE'RE DOING SOME OF THIS, YOU CAN SEE THE BROWN COUNTER STAIN, THIS IS THE LEWIS Y DOWN AT THE BOTTOM, BUT AGAIN YOU DON'T NEED TO BE AN IMMUNOLOGIST TO FIGURE OUT WHERE IS THE IMMUNE INFILTRATE IN THIS TISSUE. THE COUNTER STAIN IN THIS WORKFLOW POPS THAT OUT REALLY NICELY AND ALLOWED US TO THEN START TARGETING, CAN WE SEE THE INDIVIDUAL GLYCOME WITH THIS? YES, OR WE WOULDN'T BE SHOWING IT. ACTUALLY WE CAN DO ALL TYPES OF PROFILING WITH ALL OUR TRICKS, BUT IN GREEN, THERE'S A GOOD HIGH MAN SIGNATURE, ESPECIALLY MAN 7 AND HIGHER, BUT ALSO WE CLEARLY SEE A THEME OF THESE BISECTING INFILTRATES. THREE OF THEM POP OUT. THIS ONE HERE, THE TRI DOWN HERE AND THEN THE TETRASEEM TO BE REALLY INDICATIVE OF THESE IMMUNE INFILTRATES IN THESE TYPES OF TISSUES. BACK TO THE IMMUNE PART, SO WHAT, WHAT ARE THESE CELLS LABELED UP? THIS IS AN EXAMPLE, IF WE DO THE OVERLAY, WE'RE NOT GETTING SINGLE CELL RESOLUTION AT THE TISSUE, SO YOU CAN KIND OF SEE IT LOOKS A LITTLE OUT OF FOCUS. BUT AGAIN, WE CAN DO THIS CO-LOCALIZATION QUITE NICELY. BUT WHAT IF YOU HAD A HYPERION MULTIPLEX ANTIBODY IMAGING PLATFORM TO REALLY LOOK AT THE SUBSETS. SO THIS IS JUST A SNAPSHOT OF AN ADJACENT SLICE OF THAT SAME TISSUE, AND IT POPS OUT QUITE NICELY, THE CD68, CD20s, AND CD3 STAINING. THERE ARE CD4s AND CD8s, BUT THEY WEREN'T AS -- THERE WEREN'T AS MANY ON THE PROFILE, BUT YOU GET THE IDEA WITH THIS KIND OF PANEL, THIS IS OUT OF 20 ABT BODIES USED BY OUR COLLEAGUE HERE AT MUSC. BUT YOU CAN GET THE IDEA, WE CAN START LINKING GLYCAN INFORMATION WITH THIS CELLULAR SUBSETS. SO WE'RE MAKING SOME PROGRESS ON THE IMMUNE SIDE. BUT I LOOK AT A LOT OF TISSUES, THIS IS GREAT, THIS IS EASY, LET'S GO TO TOWN ON THIS AND START MAPPING THE IMMUNE INFILTRATE ON ALL THESE TISSUES, WHENEVER IT'S PRESENT LIKE THIS. SO HERE WAS ANOTHER PROSTATE TUMOR. YOU CAN SEE THE STAINING. THIS ONE ACTUALLY -- THIS IS IMPORTANT, IT'S NEXT TO THE TUMOR. NOT SO MUCH TUMOR DOWN HERE IN THIS REGION. WELL, WE DID NOT GET AS ROBUST A PROFILE, BUT SOME OF THE USUAL SUSPECTS I MENTIONED IN THE PREVIOUS SLIDE FOR THAT OTHER TISSUE DID POP UP. YOU CAN KIND OF SEE T EVEN WITHOUT THE OVERLAY. NOTICE WHERE THE TUMOR IS. THERE'S NOT A LOT GOING ON THERE. I KNOW IT'S HARD TO SEE WITHOUT ZOOMING IN, WE STARTED SEEING THIS PATTERN. THERE IS A HINT OF, FOR INSTANCE, I BELIEVE IT'S RIGHT HERE, IN THIS MAN 9, BUT IT'S NOT THAT ROBUST. SO WHAT ABOUT A TISSUE WHERE ALL THE INFILTRATE IS ESSENTIALLY IN THE TUMOR OR SURROUNDED BY A TUMOR? AND THIS IS A LEWIS BISTAIN, YOUY STAIN, YOU CAN SEE THE TUMOR, IT'S ESSENTIALLY GONE, THOSE LITTLE DOTS AREN'T EVEN WHERE THE INFILTRATE IS, IT'S RIGHT ADJACENT TO THEM, AND WE CAN USE THIS SCALE OF LOOKING AT -- BACK AND FORTH AT THE LEWIS Y STAIN. IT'S HARD TO SEE BUT I'VE GOT THE ARROWS WHERE THE BLUE DOTS ARE HERE. SO ESSENTIALLY WE DON'T SEE -- THIS IS A TUMOR PROFILE ACTUALLY. SO THE IMPLICATION IS THE IMMUNE CELL INFILTRATE, IT'S NEAR THE TUMOR OR ADJACENT TO THE TUMOR, IT CLEARLY NOT AS ROBUST. SO IN THE IMMUNOTHERAPY WORLD, THIS HAS GOT TO MEAN SOMETHING, SO IT'S A NEAT PROJECT WE'RE MOVING FORWARD WITH THE PBMCs, YOU KNOW, FROM THESE TYPE OF PATIENTS. OUR OTHER AIM WAS TO DO ADAPT WHAT WE CALL THE GLYCOTYPER. THIS IS AN ANTIBODY ARRAY THAT WE'D PUBLISHED WHERE WE CAN PROFILE THE SUBTYPES OF IGG 1, 2, 3 AND 4, AS WELL AS THE A AND Ms. WE DIDN'T HAVE TO INVENT THE ANTIBODY ARRAY, WHICH IS GOOD, JUST HAD TO FIND THE RIGHT ANTIBODIES TO PUT ON IT. THAT'S BEEN THE BIGGEST CHALLENGE, NOT NECESSARILY GENERATING THE DATA. DOESN'T USE THAT MUCH SAMPLE. WE'VE GOT THIS PUBLISHED AS WELL AS THE CURRENT PROTOCOLS NOW AND IT'S STRAIGHTFORWARD. IT'S REALLY BECOMING A BIOINFORMATIC ISSUE, ONCE YOU GET INTO BIGGER NUMBERS AND YOU'RE ESSENTIALLY LOOKING AT 100 SAMPLES WITH 30 GLYCAN PEAKS, THAT'S JUST FOR THE IGG SUBSETS. THAT'S BEEN THE CHALLENGE. SO CAN YOU GET THIS BIOMARKER ALGORITHM BARCODE AND THAT'S ONGOING RIGHT NOW. WE'VE BEEN BUSY THIS SPRING BECAUSE ALL OF A SUDDEN OUR LITTLE COMPANY, GLYCOPATH LLC BECAME A GLYCOPATH INGE WITH INC WITH SERIOUS SEED FUNDING AND IMPLEMENTATION RESOURCES, QUICKLY MOVING TOWARDS THIS GLYCOTYPER AND GLYCOHC C-TYPIER APPROACH. C-TYPEER APPROACH. FOR COMPARISON, THAT'S A COMPUTER TOWER. IT'S NOT AS ROBUST AS THE MUCH LARGER MORE SENSITIVE INSTRUMENTS WE'RE WORKING ON, BUT IT WORKS. SO NOW WE'RE IN THE PROCESS -- ON THE COMPANY SIDE OF THE EQUATION, ADAPTING ALL THESE WORKFLOWS OVER INTO THIS SYSTEM TO SEE, AS YOU'D EXPECT, LIMITED DETECTION AND ALL OF THOSE OTHER PARAMETERS. SO THE IDEA IS WE CAN ADAPT THE SLIDE ARRAY TECHNOLOGY INTO THIS, AND I'M WRAPPING UP. SO WE HAD A COHORT OF 87 PATIENT SAMPLES WITH EARLY TO LATE FIBROSIS, AND YOU CAN CJIS LOOKING AT THE IGGs, WE'RE STARTING TO GET SOME VERY PROMISING RESULTS. AGAIN, THIS IS IN THE BIOINFORMATICS STAGE, BECAUSE IF WE CAN DO OTHER ANTIBODIES, WE START INCORPORATING DIFFERENT GLYCANS FROM ALPHA-1 ANTITRYPSIN, TRANS FAIRN, INTO THESE DIFFERENT ALGORITHMS, AND LOGISTIC REGRESSION IS THE GREEN, IF YOU LOOK AT THE ROCK CURVE IN THIS POPULATION, AND THEN A DEEP LEARNING SEEMS TO BE WAY TO GO, AND AGAIN, THIS IS ONGOING WORK. OUTREACH WISE, WE HAD WORKED WITH -- TO SET UP A CENTER OF EXCELLENCE FOR TRAINING. IN ALL THESE KIND OF METHODS. AND THEN THE PANDEMIC HIT, SO WE'RE KIND OF HITTING RECEIPT START BUTTON ON THAT SO WE'RE OPEN. WE'VE HL A FEW PEOPLE COME BY IN THE LAST COUPLE MONTHS ACTUALLY. SO ANYBODY IS WELCOME TO COME VISIT AND LEARN THESE METHODS. IT'S ALSO HAMPERED OUR DATABASE ANALYSIS WITH METABOSCAPE THAT WE'RE WORKING WITH KIOKO ALSO, AS WELL AS SOME IMAGING DATABASES THAT SHE AND I ARE WORKING ON TOGETHER TOO. AND MORE IMPORTANTLY TO THIS EFFORT, DR. MAZUMDER FROM GLYGEN, A SUPPLEMENT GRANT TO AN TATE ANNOTATE THAT IMMUNOGLOBULIN DATA AND THERE'S A PAPER SUBMITTED. VERY BRIEFLY, I LOVE THIS PAPER. >> YOU'LL HAVE TO WRAP IT UP RICK. >> GLYCOGEN IMAGING. WHO KNEW? ON THE MASS SPEC SIDE, IT'S 162 WHEN YOU SPRAY THE AMYLASE. AND WHEN WE START LOOKING AT TISSUES, IT LOOKS LIKE IT SUPPLIED THEM, THE TUMORS IN RED. LAST SLIDE, TWO SLIDES, SO OUTREACH EFFORTS, WE'VE GOT THIS GLYCOMICS CENTER FOR TRAINING, COLLABORATION, ALL THE DATABASE AND INSTRUMENTATION RESOURCES, WE'RE TRYING TO MOVE FORWARD COLLABORATIVELY AND INTERACTIVELY. SOME MULTI-LAB RING TRIALS FOR METHOD AND TECHNOLOGY, WE'VE GOT A GREAT PARTNERSHIP WITH FDA WITH SOME RESEARCH LABS ON THE COVID SIDE, AND GLYCOPATH IS THE ULTIMATE OUTREACH, AT LEAST FOR US TO GET THIS TO THE WORLD. I JUST LIKE GLYCAN CARTOONS. AND I WANT TO THANK A LOT OF PEOPLE FOR THEIR HELP. TA-DA! >> OKAY, THANK YOU, RICK. OKAY. WELL, WE'RE BASICALLY RIGHT ON TIME NOW, SO I'D HATE TO KIND OF USE UP THE LITTLE BIT OF SLACK THAT WE HAVE ON QUESTIONS. IF YOU HAVE ANY QUESTIONS, MAYBE YOU COULD SEND THEM TO DR. DRAKE VIA CHAT OR CONTACT HIM AFTER THE MEETING. I JUST WANT TO GIVE A LITTLE INTRODUCTION TO OUR NEXT TALK. NOT EVERYBODY THAT'S SPEAKING IS A PI IN OUR COMMON FUND PROGRAM. WE ALSO GAVE OUT A NUMBER OF SUPPLEMENTS TO PIs WHO CLAIMED NOT TO HAVE ANY EXPERTISE IN GLYCOSCIENCE FOR THE INTEREST OF TRYING TO ADOPT SOME OF THE TOOLS THAT WERE MADE IN THIS PROGRAM. SO WE'RE ALLOWING THOSE WHO WERE AWARDED THESE SUPPLEMENTS TO GIVE FIVE-MINUTE TALKS AT OUR SESSION HERE, AND WE'RE GOING TO HEAR THE FIRST ONE NOW BY DR. CARL ATKINSON. SO DR. ATKINSON, LET'S SEE, ARE YOU -- >> I'M HERE. >> READY? OKAY. GO AT IT THEN. >> ALL RIGHT. THANK YOU VERY MUCH. >> FIVE MINUTES. >> I WAS TOLD 8 SO I'LL DO MY BEST. SO I'M GOING TO TALK TO YOU A LITTLE ABOUT HOW WE'VE USED N-GLYCAN SIGNATURES TO LOOK AT PRIMARY GRAFT DYSFUNCTION IN COPD PATIENTS POST TRANSPLANT. NO DISCLOSURES OTHER THAN I'M SUPER NEW TO GLYCOSCIENCE SO BEAR WITH ME. JUST A LITTLE OVERVIEW OF LUNG TRANSPLANT FOR CONTEXT. IT'S EFFECTIVE THERAPY FOR END STAGE LUNG DISEASES, NOW RECENTLY COVID. WHILE IT EXTENDS SURVIVAL AND IMPROVES PATIENT OUTCOMES, IT'S ONE OF THE POOREST OF ALL ORGAN TRANSPLANTS OF AVERAGE SURVIVAL OF ABOUT FIVE YEARS. REALLY WHY IT HAS POOR SURVIVAL IS THAT THE LUNG SEEMS TO BE ECK QIS ECK QIS -- PRIMARY GRAFT DYSFUNCTION OCCURS IN THE FIRST 72 HOURS, CHARACTERIZED BY FLUID IN THE LUNG, IMMUNE ACTIVATION WHICH INHIBITS RESPIRATION. WHAT'S BEEN SHOWN IS THE SEVERITY OF WHICH CLEARLY LEADS TO THE EARLY ONSET OF CHRONIC LUNG ALLOGRAFT DYSFUNCTION. THERE'S NO BIOMARKERS AVAILABLE, 25 TO 35% OF PATIENTS DEVELOP PRIMARY GRAFT DYSFUNCTION. OVER THE LAST 18 MONTH, THIS IS THE NORMAL LUNG, THESE ARE FEATURES THAT OCCUR DURING PRIMARY GRAFT DYSFUNCTION AND ALSO DURING COVID AIDS, ACTIVATION OF THE VASCULATURE AND THE ALVEOLAR SPACE. AND ONE SYSTEM THAT SEEMS TO BE PARTICULARLY IMPORTANT, THIS IS THE COMPLEMENT SYSTEM, IMMUNE COMPONENT ACTIVATED BY ANTIBODY, THE ACTIVATION OF WHICH CAUSES INJURY TO EPITHELIAL CELLS, ENDOTHELIAL CELLS AND PRIMING OF IMMUNE RESPONSES. SO HOW DOES ALL THIS FIT IN TO GLYCOBIOLOGY? WELL, IN LUNG TRANSPLANTATION, A LOT OF OUR CHRONIC LUNG DISEASES ARE AUTOIMMUNE DISEASES, GIVING RISE TO LUNG REACTIVE AUTO ANTIBO ANTIBODIES THAT DRIVE DISEASE PROGRESSION PRE-TRANSPLANT AND WHAT WE MIGHT THINK IS SOME OF THESE COMMON AUTO ANTIBODIES CAN INCREASE SUSCEPTIBILITY TO PRIMARY GRAFT DYSFUNCTION. SO WE HYPOTHESIZE YOU DEVELOP KRONG LUNG DISEASE, DEVELOP ANTIBODIES, THEY BIND AND LEAD TO INCREASE IN PRIMARY GRAFT DYSFUNCTION. WE WERE LUCKY ENOUGH TO GET 16 PGD NEGATIVE AND 16 PGD POSITIVE. THE PGD NEGATIVE HAD NO SIGNS OF PGD. JUST A PATIENT SUMMARY TABLE, BASICALLY WE MATCHED THEM SO THERE WAS NO DIFFERENCE IN GENDER, AGE, BMI OR SMOKING HISTORY. WE ASKED THE QUESTION DO WE HAVE MORE OF THESE ANTIBODIES PRE AND POST IMPLANT, SO THIS IS SOME DATA LOOKING AT COMMON ELASTIN AUTO ANTIBODIES. WHAT YOU SEE, THERE'S JUST NO DIFFERENCE IN ELASTIN. WHEN WE LOOK AT COLLAGEN, THERE'S SIMILARLY NO DIFFERENCE. SO REALLY THE CONCLUSION FROM THIS WAS THAT I WAS COMPLETELY WRONG, AND THAT IT'S NOT REALLY THE EPITOPE THAT'S IMPORTANT, MAYBE THERE'S SOMETHING ELSE. AS YOU ALL KNOW -- OF THE ANTIBODY THAT CAN SEVERELY CHANGE THEIR EITHER PRO INFLAMMATORY NATURE OR ANTI-INFLAMMATORY NATURE. IN FACT, THERE'S A COUPLE OF PUBLICATIONS SHOWING IGG GLYCOSYLATION C COPD IS ASSOCIATED WITH SEVERITY. WAS THERE ANY DIFFERENCE PRE AND POST TRANS TRANSPLANT. SO WE DID A FULL SERUM SCREEN USING TECHNOLOGIES THAT DR. DRAKE AND DR. ANGEL HAVE DESIGNED. WE FOUND 120 DIFFERENT N-GLYCAN STRUCTURES. WE FOUND THERE WAS TWO DIFFERENT N-GLYCAN STRUCTURES ASSOCIATED PRETRANSPLANT AND TWO DIFFERENT POST TRANSPLANT, AND WHAT WE FOUND INTERESTING WAS, IS THAT THEY HAD THESE BISECTING DND AND SIALIC ACID RESIDUES COMMONLY SEEN IN IMMUNOGLOBULIN MOLECULES. ASKED THE QUESTION, ARE THERE ANY DIFFERENT? WE WERE INTERESTED IN LOOKING AT THE DIFFERENT SUBSTIEPS OF IGG, 1, 2 AND 3 AND TOTAL IG, AND WE COMPARED THIS PRE AND POST TRANSPLANT AND COMPARED THE 20 MOST ABUNDANT N-GLYCAN STRUCTURES FOUND ON THE IGG MOLECULES THAT WE SCREENED FOR. AND WHAT WE FOUND INTERESTINGLY, THERE WAS 11 DIFFERENT N GLYCANS SIGNIFICANTLY ELEVATED IN PGD HERE IN THE ORANGE VERSUS NON-PGD. EIGHT OF WHICH HAD THESE CHANGES WHICH WERE ASSOCIATED WITH A PRO INFLAMMATORY IGG 1 MOLECULE. WHEN WE LOOK POST TRANSPLANT FOR THE 24 HOUR SAMPLE, WE AGAIN SEE SEVEN DIFFERENT N-GLYCAN SIGNATURES THAT WERE SIGNIFICANTLY ELEVATED IN THE PGD VERSUS THE P TB. GD NEGATIVE AND SIX OF THOSE CHANGES WERE ALSO PRESENT PRE AND POST TRANSPLANT. AGAIN THEY HAVE THESE RESIDUES MORE ASSOCIATED WITH PREINFLAMMATORY STRUCTURE. THERE WERE NO DIFFERENCES IN THE IGG 1 AND 2 AND WE'RE STILL DEVELOPING THE 3 AND 4 DATA. CHANGES TO THE I IGG 1 MOLECULE N DPLIE CAN CHANGES -- THE FIRST PART OF THE CLASSICAL PATHWAY AND HAVE BEEN SHOWN IF YOU HAVE THESE GLYCOSYLATION CHANGES THAT YOU CAN GET AN INCREASED COMPLEMENT ACTIVITY BY THESE ANTIBODIES. SO IN CONCLUSION, COLLAGEN SCREENING IS NOT PREDICTIVE OF PGD SO I WAS VERY WRONG. HOWEVER, IN THIS SMALL SUBGROUP, N-GLYCAN SIGNATURE APPEARS TO BE A PROMISING BIOMARKER. THE LIMITATIONS, WE HAVE A SMALL SAMPLE SIZE, ABOUT 16 IN EACH GROUP. THE FACT THAT WE FOUND ANY SIGNIFICANT DIFFERENCE WITH SUCH A SMALL SAMPLE SIZE I THINK IS SOMEWHAT ENCOURAGING. OBVIOUSLY ANOTHER LIMITATION IS THAT WE RESTRICTED THIS TO C OSM PD. OPD. WE'RE CURRENTLY LOOKING AT THESE SAME SERUM SAMPLES AND TRYING TO CORRELATE IT WITH THESE N-GLYCAN SIGNATURES. WE HAD HAD HOPE TO INCREASE THE SAMPLE SIZE BUT OBVIOUSLY CHANGES IN COVID HAVE MADE THAT DIFFICULT. BUT THE ONE THING THAT WE'RE VERY INTERESTED IN MOVING FORWARD IS THAT THIS IS ALL SERUM ANALYSIS. AS WE KNOW FROM TRANSPLANT ANTIBODIES COMBINE IN THE GRAFT AND THEREFORE IT MAY NOT BE WHAT'S INCREASED IN THE SERUM SAMPLES ARE ASSOCIATED WITH PGD, IT MAY BE WHAT'S GONE MISSING IN SERUM SAMPLES, SO WE'RE INTERESTED IN DOING SAMPLES WITH LAVAGE AND SERUM. REALLY WITH THE GOAL TO FIND A BIOMARKER THAT MIGHT BE FAMILIAR WITH WHICH PATIENT ARE AT RISK BECAUSE THERE ARE A NUMBER OF THERAPEUTICS WE COULD APPLY TO THESE PATIENT GROUPS. SO IN CONCLUSION, I'D LIKE TO ACKNOWLEDGE A COUPLE MEMBERS OF MY LAB WHO HAVE BEEN LEARNING THIS TECHNOLOGY FROM DR. ANGEL AND DANIEL SCOTT AND DR. DRAKE. MY CLINICAL COLLABORATORS, JASON CHRISTIE AND ANDRES PELAEZ, AND BOONE PRENTICE, TO TRANSFER TECHNOLOGY TO PEOPLE TO KNOW WHO KNOW NOTHING ABOUT IT, BOONE PRENTICE IS WORKING WITH DR. DRAKE. THANK YOU VERY MUCH FOR YOUR ATTENTION. >> THANK YOU, DR. ATKINSON. ARE THERE ANY QUESTIONS FOR HIM? WE APPRECIATE THE FACT THAT YOU SHOW THE APPLICABILITY OF DR. DRAKE'S TOOLS FOR YOUR OWN RESEARCH. THIS IS REALLY WHAT THIS PROGRAM IS ABOUT. >> YEAH, IT'S BEEN A GREAT LEARNING EXPERIENCE FOR OUR WHOLE LAB. I THINK NOW THAT WE'VE OPENED PANDORA'S BOX, WE'RE VERY MUCH INTERESTED IN MOVING THIS FORWARD AND AS I SAID, LOOKING AT LIKE MATCH TISSUE LAVAGE AND SERUM SAMPLES SO WE CAN REALLY BUILD A MATRIX. >> DR. ATKINSON, DO YOU HAVE A MINUTE FOR ANOTHER QUESTION? I WAS JUST WONDERING ABOUT THE -- SORT OF THE MECHANICS OF ADOPTING THIS TECHNOLOGY. OBVIOUSLY I DIDN'T RUN OUT AND BUY A MASS SPECTROMETER AND SET UP A WHOLE PARALLEL SYSTEM FOR WHAT'S GOING ON IN RICHARD'S LAB, BUT I'M CURIOUS ABOUT HOW YOU FOUND YOUR WAY INTO DOING THE ANALYSES AND INTERPRETING THE DATA. >> SO I THINK THE FIRST WAY I FOUND THIS IS BECAUSE I HAD A COLLABORATOR ON PEGGY AND RICK'S FLOOR AT MUSC AND I USED TO WALK UP AND LOOK AT THEIR POSTERS, THEN WE GOT CHATTING. STO A COUPLE OF PEOPLE RSO A COUPLE PEOPL E IN MY LAB GOT TRAINED AND THEY LINKED ME WITH BOONE PRENTICE WHO -- I THINK THEY POSTDOC'D TOGETHER, SO HE HAS ALL THE EQUIPMENT AND IMAGING APPARATUS TO BE ABLE TO TRANSFER THIS TO UNIVERSITY OF FLORIDA AND I SEE THIS AS AN ONGOING COLLABORATION WITH THE GUYS AT MUSC AND NOW DOWN AT UF. >> CARL, IF YOU CAN INDULGE ME, ONE MORE QUESTION. >> VERY QUICK. >> HAVING PUBLIC EXPOSURE TO IT NOW, ONE OF THE THINGS THAT WE'VE TALKED ABOUT IN THE PAST WITH IMAGING MASS SPECTROMETRY IS PUTTING IT IN THE HANDS OF CLINICAL THAT POLL GISTS, IN PATHOLOGISTS , IN THAT LABORATORY SETTING. HOW DO YOU THINK THIS MIGHT FAIR IN TRYING TO MIGRATE IT INTO THE CLINICAL PATHOLOGY LAB? >> SO THAT'S MY BRACK GROUND, I ACTUALLY RAN A CLINICAL PATHOLOGY TRANSPLANT LABORATORY, AND I THINK IT GOES DOWN TO SPEED. SO FOR ANALYSIS OF ACUTE REJECTION, IT'S HOW QUICKLY CAN WE DO IT, SO THE LIQUID-BASED TECHNOLOGIES, WE CAN GET ANALYSIS QUITE QUICKLY. TISSUE-BASED, WE HAVE TO ACT QUITE QUICKLY IN THE CLINIC TO BE ABLE TO TREAT THESE PATIENTS, AS THEY'RE REJECTING, WE HAVE TO ACT VERY, VERY QUICKLY. SO I THINK IT WOULD COME DOWN TO SPEED FOR TISSUE-BASED ANALYSIS. >> THANK YOU VERY MUCH. >> OKAY, THANK YOU ALL. WE NEED TO MOVE RIGHT ON TO OUR NEXT SPEAKER, WHICH IS DR. ROBERT WOODS. OKAY, ROB, I'LL LET YOU TAKE IT FROM HERE. >> LET ME JUST UNMUTE MYSELF AND START THIS. ALL RIGHT. I HOPE EVERYBODY CAN HEAR ME. WHAT I WANT TO DO IS GIVE A BRIEF UPDATE ON SOME OF OUR MOLECULAR STRUCTURE TOOLS THAT ARE NOW COMING ONLINE AND EVOLVING. I'D LIKE TO THANK CATHERINE FOR THE GREAT INTRODUCTION THAT MADE ME CHUCKLE. AND ALSO OF COURSE TO THE NIH FOR MANY YEARS OF SUPPORT ON THESE PROJECTS. WHAT I'M GOING TO TALK ABOUT ARE TWO TOOLS, ONE FOR EXTRACTING STRUCTURES FROM THE PDB, WHICH WE CO-DEVELOPED WITH JASMINE YOUNG, AND ONE FOR MODELING GLYCANS WHICH HAS BEEN A LONG TERM PROJECT WITH SEVERAL MEMBERS OF THE GROUP. SO IF YOU GO TO DEV.GLYCAM.ORG, THIS IS OUR DEVELOPMENT SITE, SOON TO BECOME OUR MAIN SITE, YOU SEE TOOLS THAT ARE AIMED AT SORT OF NOVICE USERS. THEY'RE VERY SOPHISTICATED TOOLS BUT THEY TRY TO PRESENT THEMSELVES IN A WAY THAT NON-SPECIALISTS CAN SEE AND USE THEM. THE ONE I'M GOING TO TALK ABOUT FIRST IS SOMETHING CALLED GLY FINDER. AS OF THIS PAST MONTH, THERE ARE ABOUT 180,000 BIOLOGICAL MACROMOLECULAR STRUCTURES IN THE DATA BANK BUT NOBODY HAS AN IDEA REALLY HOW MANY OF THOSE STRUCTURES CONTAIN CARBOHYDRATES. SO THE VALUES HAVE VARIED FROM 10 TO 20%. WE NOW KNOW APPROXIMATELY 10% OF THE STRUCTURES CONTAIN CARBOHYDRATES. THIS DOES NOT INCLUDE NUCLEIC ACIDS, AS MUCH AS YOU MIGHT WANT TO THINK THAT DNA AND RNA ARE REALLY JUST CARBOHYDRATES. WHAT WE FOUND DURING THIS EXERCISE WAS THE PRESENCE OF A LOT OF INCONSISTENCIES IN 3D STRUCTURES. THE PDB HAS COMPLETED A FIRST PHASE OF REMEDIATION OF DATA AND WHAT THEY'VE DONE IS SOMETHING LIKE THIS, FOR EXAMPLE, IN THIS STRUCTURE, 1CAP, BECAUSE OF OUR TOOLS THEY WERE ABLE TO SAY THEY'VE INTRODUCED A CAVEAT IN THE PDB FILE WHICH SAYS THAT THIS PARTICULAR GLAK TOES RESIDUE HAS THE WRONG PLURALITY OF C1. THE OTHER THING WE'VE DONE IS CREATED A UNIFORMITY IN NAMING SO THAT THAT PREVIOUSLY. IF YOU GO TO DEV.GLYCAM.ORG/GSM.ORG/GF. IF YOU DO THAT, LU COME OUT WITH 53,000 HITS, AND IT REALLY FAST. I'LL TRY TO GIVE A QUICK DEMO. WHEN YOU LOOK AT THESE, WHAT YOU'RE GOING TO SEE, IT COMES OUT WITH THREE STRUCTURES. ONE OF THEM IS AN N-LINKED CORE GLYCAN, ONE OF THEM IS A LIGAND, WITH AN OH ON THE END, THEREFORE IT'S NOT ATTACHED TO ANYTHING. THEN YOU SEE THIS THING HERE, WHICH IS THE COUPLE WITH AN R GROUP ATTACHED TO A GLUTAMATE. THIS IS NOT A NORMAL SUE SUGAR. SO LET'S HAVE A LITTLE LOOK AT THAT. WHEN YOU DO, AHA, IT A COVALENT SUBSTREAM INTERMEDIATE. IT'S THE UNKNOWN R -- FOR US AND IT ATTACHED TO A GLUTAMATE. WHEN WE DO THIS, WE ALSO CALCULATE THE -- SOMETHING THAT THE PDB DOESN'T PROVIDE BUT IT GIVES YOU A SENSE OF THE ACCURACY OF THE COORDINATES. SO WHAT DID WE FIND OUT OF THIS? N LINK GLYCANS, WE FOUND 4.2% OF ALL THE PDB ENTRIES, FROM THIS YOU CAN WORK OUT THAT ON AVERAGE, THERE ARE 6N-LINKED GLYCANS PER GLYCOPRO TEEFNLT DOES THAT MATTER? I DON'T KNOW, BUT IT DIDN'T PLATTER BEFORE AND IT GIVES YOU A QUANTITATIVE NUMBER THAT YOU CAN CITE. I JUST WANT TO SHOW YOU THE PERFORMANCE BECAUSE IT'S PRETTY NICE. HERE WE ARE ON DEV.GLIECAM.ORG, CLICK ON PDB. YOU COULD ENTER JUST YOUR FAVORITE PDB ENTRY IF YOU HAD ONE YOU WANTED TO CHECK, BUT LET'S DO A SEARCH. LET'S SEARCH FOR EVERYTHING THAT CONTAINS XYLOSE, JUST FOR FUN, AS THEY'VE DONE. THERE YOU GO. 1,110 ENTRIES CONTAIN XYLOSE. THAT WAS PRETTY QUICK. IF WE LOOK AT IT, THIS GOES TO LIGHT MALL VIEWER AND YOU CAN SEE THE MOLECULE, XYLOSE IS A STAR, THAT'S WHY I CHOSE IT BECAUSE IT'S KIND OF FUN. ALL RIGHT. WHAT ELSE CAN WE DO? WELL, IF YOU CLICK ON THIS SIDE, IT WILL TAKE YOU RIGHT TO THE PDB ENTRY, WHERE YOU CAN DOWNLOAD ALL THE FILES. ALSO, IF YOU CLICK ON THE MORE BUTTON, IT WILL TELL YOU SOME INFORMATION. IT WILL TELL YOU THE AUTHORS, THE TITLE, YOU CAN CLICK ON THE PUBMED I.D. AND GET THE RESOLUTION AND SO FORTH. IF THERE WERE ANY ERRORS, THEY WOULD SHOW UP HERE. YOU CAN DOWNLOAD THE PDB COORDINATES OR JUST THE SUGAR COORDINATES, AND IT'S THE SUGAR COORDINATES SHOWN HERE, SO YOU CAN HAVE A LOOK AT THAT. SO THAT'S THE PDB SEARCH TOOL. LET'S LOOK AT ONE OTHER TOOL, MODELING OF 3D SHAPES OF SUGARS. WE'VE BEEN DOING THIS FOR A LONG TIME, BUT THERE'S A FEW DIFFERENCES NOW ON THE DEV SITE. THE DEV SITE, YOU CAN BUILD WITH OUR OLD SCHOOL TOOL WHICH IS MORE LIKE THE SEARCH INTERFACE FOR THE GLY FINDER AND MANY PEOPLE USE THAT, BUT WE WORKED OUT A VERY DIFFERENT WAY TO DO IT. IF YOU HAVEN'T SEEN THIS BEFORE, THIS MAKES IT EASY FOR GLYCOSCIENTISTS TO PICK OUT WHAT THE SUGAR IS, WE START BUILDING NOW FROM THE REDUCING TERMINUS. SO WE'RE GOING TO START AT THE ONE POSITION AND THEN GROW TO THE TWO POSITION. BUT OF COURSE LINKAGE NOMENCLATURE GOES THE OTHER WAY, 2-1 IS THE LINKAGE, SO HOW DOES THIS WORK? LET'S SAY WE CLICK ON GLUCOSE AND WE WANT TO HAVE IT LINKED TO GALACTOSE. WHAT'S THE LINKAGE? IT'S HERE. THESE ARE THE ALLOWED ONES, SO LET'S SAY IT'S A BETA 1-4. SO WE WANT TO MODEL THIS, WE CAN ADD BRANCHES, I'LL SHOW YOU THAT IF I HAVE TIME, AND THERE IT IS. THAT WAS PRETTY FAST. THIS IS A FULLY MINIMIZED STRUCTURE. YOU CAN VISUALIZE IT HERE, YOU CAN DOWNLOAD THE PDB STRUCTURE IF YOU WANT TO, YOU CAN DOWNLOAD IT IN TWO DIFFERENT SOLVENT MODELS OR YOU CAN DOWNLOAD ALL THE FILES IF YOU WANT TO DO A SIMULATION. BUT LET'S LOOK AT THIS, SUPPOSE YOU HAVEN'T BUILT THIS BEFORE. BECAUSE THAT WAS REALLY FAST. AND IT'S FAST BECAUSE ONCE A STRUCTURE IS BUILT, WE STORE IT IN A DATABASE SO THAT WHEN YOU RETRIEVE IT, IT'S AUTOMATICALLY THERE. IF YOU HAVEN'T BUILT IT BEFORE, IF IT'S A NEW STRUCTURE YOU HAVEN'T SEEN, LET'S SAY GLUCOSE, SAME THING, BETA 1-4, BUT LET'S PUT A BRANCH IN HERE OR SOMETHING UNUSUAL. SO ON THIS POINT, WE'RE GOING TO PUT A XYLOSE. I DON'T THINK WE'VE MADE THIS BEFORE. WE'LL CALL IT ALPHA-1-3. SOME TOTALLY RANDOM STRUCTURE. I'VE DONE THIS DELIBERATELY TO SHOW YOU THAT WHEN YOU FIRST MAKE SOMETHING, SEE, IT SAYS MINIMIZE, NO. THERE YOU GO. IT TOOK A LITTLE BIT OF TIME TO MINIMIZE, BUT NOW IT'S MINIMIZED, AND YOU CAN DOWNLOAD IT. IT'S NOW ALSO MINIMIZING THE SOLVENT TO SOLVATED FILES AND IT WILL STORE THOSE. SO FOR FUTURE REFERENCE, EVERY PERSON WHO WANTS TO BUILD THIS AGAIN WILL PULL UP ALMOST INSTANTLY ALL THE FILES THEY NEED. OKAY. SO THAT'S THAT. ANOTHER FEATURE OF THE CARBOHYDRATE BUILDER TOOL IS YOU CAN PASS THE STRING THROUGH A URL. WHAT DOES THAT MEAN? WELL, THAT MEANS SOMEBODY ELSE, SOME THIRD PARTY OR SOME OTHER USER OR YOU SITTING AT YOUR COMPUTER CAN TYPE IN THIS STRING IN YOUR SEARCH BAR. DEVGLYCAM.ORG/URL? SO THIS WOULD BE THE STRING YOU MIGHT HAVE BUILT THIS FROM SOMEWHERE ELSE OR GENERATED IT ELSEWISE, BUT IF YOU DO THIS, IF YOU CLICK ON THIS, IT TAKES YOU RIGHT TO OUR BUILD AREA AGAIN. NOW IN THIS CASE, THERE ARE ROTOMERS SO YOU CAN HAVE DIFFERENT -- THIS WOULD DEMONSTRATE SIX STRUCTURES. IF YOU WANTED TO GENERATE ONLY A SUBSET AND YOU KNEW EXACTLY WHAT THEY WERE, THAT'S WHAT YOU WOULD DO, GENERATE SELECTED STRUCTURES, AND IT'S GOING TO SUBMIT IT. AND THEN RUN IT. SO I WANT TO WRAP UP BY THANKING THE BLUE TEAM, WHICH ARE THE SCIENTISTS WORKING ON IT. DAVE MONTGOMERY, GLYFINDER, TOGETHER WITH DAN WENTWORTH, WHO'S ON THE GREEN TEAM, WHICH ARE THE PROGRAMMERS AND DEVELOPERS. DAN HAS BEEN INSTRUMENTAL IN PARTICULARLY THE WORK WITH OLIVER. IT'S REALLY A VERY INTERESTING COMBINATION OF CS PEOPLE AND SCIENTISTS WORKING TOGETHER. OF COURSE I WANT TO THANK THE NIH FOR ALL THE SUPPORT STARTING WAY BACK WITH A P41 THROUGH THE U01 SERIES AND NOW MOVING ON TO AN R24. SO I THINK THAT'S ALL I HAD TO SAY. >> THANK YOU VERY MUCH, ROB. YOU PUT US A LITTLE BIT AHEAD OF SCHEDULE SO WE HAVE TIME FOR A FEW QUESTIONS FOR ANYBODY. >> HEY, ROB. YOU KNOW I LOVE THIS STUFF AND I USE THIS STUFF. I'M GOING TO SHOW ONE YOUR STRUCTURES LATER. ONE ISSUE FOR THE -- WHAT DO I DO ABOUT SALVATION STAKE? DO I ALWAYS SOVVATE IT OR DO I NOT SOLVATE IT OR TO I COMPARE THE TWO? HOW DO I INTELLECTUALLY APPROACH THAT? >> SOLVATION ONLY MATTER FS YOU'RE GOING TO RUN MD, IT DOESN'T CHANGE THE SHAPE OF THE MINIMIZED STRUCTURE HART HARDLY AT ALL SO NEVER WORRY ABOUT IT UNLESS YOU'RE PLANNING TO RUN MD, AND THERE, IT'S IMPORTANT. >> THANK YOU. >> MUST BE CLEAR AS BEER. >> I GET TO SEE YOUR PROGRESS REPORTS, I THOUGHT IT MIGHT BE A GOOD IDEA IF YOU JUST INDICATE THE NUMBERS OF USERS THAT YOU HAVE COMING TO YOUR SITE. I THINK THAT'S VERY ENLIGHTENING. >> I COULD AND SHOULD HAVE PUT THIS INTO THE CONTEXT. MOST OF OUR USERS GO TO THE ORIGINAL SITE, GLYCAM.ORG AND NOT TO THE DEV SITE, WHICH ISN'T WIDELY ADVERTISED BECAUSE IT'S NOT AS STABLE AS THE ORIGINAL SITE. THE ORIGINAL SITE GETS OVER 100,000 USERS, 120,000 OR SO A YEAR IF I RECALL THE LAST YEAR'S FIGURES, BUT YEAH, IT IS WIDELY USED AND WHAT I PARTICULARLY LIKE AND A NUMBER OF PEOPLE HAVE MENTIONED THIS ALREADY IS THE INTEROPERABILITY WITH OTHER SITE, FOR INSTANCE, WITH GLYGEN OR WITH THE CFG WHERE YOU CAN GO BACK AND FORTH. PARTICULARLY IF YOU FIND SOMETHING ON ANOTHER SITE, YOU CAN MODEL IT THROUGH OUR SITE USING THE STRING PASSING. THAT GREATLY DISSEMINATE THE WORK SO IT'S NOT JUST A ONE PLACE. YOU CAN REACH IT FROM MULTIPLE PLACES. >> ANY OTHER LAST MINUTE QUESTIONS FOR DR. WOODS? >> PLEASE GO AHEAD AND TRY THESE TOOLS AND DROP US AN EMAIL WHEN YOU FIND A BUG OR IF YOU JUST WANT TO THANK US. THAT'S GREAT. >> GREAT. THANKS. MAYBE WE'LL TRY TO SNEAK AHEAD OF SCHEDULE A LITTLE BIT AND JOE, I GUESS YOU'RE NEXT, IF YOU WANT TO GET STARTED. >> ALL RIGHT. THANK YOU VERY MUCH, CARL. I WANT TO PRESENT AN UPDATE ON OUR SOFTWARE TOOLS FOR GLYCOMICS AND GLYCOPROTEOMICS, IN THIS CASE FOCUSING ON GLYCOPROTEOMICS. AND THESE ARE TOOLS THAT ARE AVAILABLE. THEY'RE PUBLISHED AND AVAILABLE. THESE INCLUDE GLYCOSOFT, GLYCODE NOVO, AND GAGFINDER AND GAGRANK WHICH ARE FOR GLIE GLYCOSITY AND GLYCANS. SO REALLY AIMED AT WHAT DO WE NEED TO DO TO FOLLOW CHANGES IN PROTEIN GLYCOSYLATION THAT OCCUR DURING BIOLOGICAL PRO PROCESSES, SO WE KNOW THAT GLYCOPROTEINS ARE HETEROGENEOUS THEY HAVE HETEROGENEITY AT INDIVIDUAL SITES AND THEN THEY HAVE THE ORAL HETEROGENEITY, THE MOLECULE. SO IT PUTS US IN A PLACE WHERE I IS DIFFERENT FROM WHAT WE HAD IN PROTEOMICS WHERE WE HAVE TO SAMPLE THE DISTRIBUTION OF GLYCANS AT EACH SITE IN ORDER TO REACH A QUANTITATIVE CONCLUSION. SO HOW DO WE DO THAT CONFIDENTLY AND HOW DO WE GET A CONFIDENT SOLID STATISTICAL COMPARISON. SO WE'LL TALK ABOUT APPROACHES FOR DOING THAT. THE STANDARD APPROACH WHICH WOULD BE USING DATA DEPENDENT ACQUISITION, JUST LIKE WE DO IN PROTEOMICS QUITE OFTEN, AND THEN COMPARING THE RESULTS USING DATA-INDEPENDENT ANALYSIS AND THEN TALKS ABOUT RESULTS USING A NEW INSTRUMENT. SO FOR INTERPRETING OUR GLYCOPEPTIDE TANDEM MASS SPECTRA, WE ARE REALLY TALKING ABOUT INTERPRETING, SAY, WE HAVE A SPECTRUM AND WE'RE GOING TO ASSIGN A GLYCOPEPTIDE STRUCTURE TO IT. IN OTHER WORDS, THIS IS NOT A SPECTRAL LIBRARY APPROACH. THIS IS INTERPRETING MASS SPECTRA. SO JUST KEEP THAT IN MIND AS WE GO THROUGH THE VARIOUS EXAMPLES. AND THE CLASSIC APPROACH IS DATA-DEPENDENT ACQUISITION, AND THAT MEANS THAT IF YOU YOU YOU HAVE -- THIS WOULD BE AN EXAMPLE OF A GLYCOPEPTIDE, AND IT IS GOT A DISTRIBUTION OF GLYCOFORMS AT THAT GLYCOPEPTIDE, AND IF WE USE DATA-DEPENDENT ACQUISITION, WE SEE THAT THE -- ESSENTIALLY WHAT WE'RE DOING IS SAMPLING THE TOP-MOST ABUNDANT GLYCOPEPTIDES. AT SOME POINT, THE INSTRUMENT WILL NOT BE ABLE TO SAMPLE THE LOWER ABUNDANCE ONES. SO IF THERE'S A LOT OF GLYCOPEPTIDES, A LOT OF GLYCOFORMS AT THAT GLYCO SITE, YOU'RE ONLY SAMPLING THE MOST ABUN ABUNDANT. SO THERE'S A FINITE AMOUNT OF INSTRUMENT SPEED WHICH IS AVAILABLE, AND SO THEREFORE, THE DEPTH OF SAMPLING AND ALSO THE REPRODUCIBILITY OF SAMPLING -- I WANT TO TALK ABOUT AN EXAMPLE THAT WE DID WORKING WITH HENRY WAN'S GROUP AT UNIVERSITY OF MISSOURI WHERE WE WERE COMPARING GLYCOSYLATION OF TWO VARIANTS OF HEMAGGLUTININ, ONE IS THE WILD TYPE, AND WE HAVE A TRIPLE MUTANT. THE THREE RED POSITIONS ARE THE MUTATED POSITIONS. YOU CAN SEE THAT THEY DON'T DISRUPT ANY OF THE SEQUONS SHOWN IN BOLD. SO THE QUESTION IS, HOW DO THE GLYCOSYLATION CHANGE BY THIS TRIPLE MUTATION THAT DOES NOT INTERRUPT ANY GLYCOSYLATION. WHAT WE DID WAS, CALCULATING SIMILARITY METRICS FROM THE DATA, WIRE ABLE TO IDENTIFY THAT THE GLYCANS AT POSITION 165 SHOWN IN THE GREEN BOX ARE DIFFERING IN THE EXTENT OF PROCESSING OF THE HIGH MANNOSE AND GLYCOFORMS. SO THAT IS THE POSITION, THE NVT RIGHT HERE. SO USING WHAT WE WOULD CALL BOTTOM-UP DATA DEPENDENT ACQUISITION, WE ARE IN FAVORABLE CASES ABLE TO ASSIGN AND QUANTIFY CHANGES IN GLYCOSYLATION. BUT THIS BECOMES MORE DIFFICULT AS THE GLYCOSYLATION CHANGES BECOME MORE SUBTLE AND AS THE GLYCOPROTEIN COMPLEXITY INCREASES. IN OTHER WORDS, IS YOUR GLYCOPROTEIN PURE, IF IT'S NOT PURE, IF YOU'RE TRYING TO DO SOMETHING FROM A COMPLEX MIXTURE, THERE'S MORE STRUCTURAL POSSIBILITIES THERE, ALSO HOW MANY GLYCOCYTES ARE PRESENT, AS IT GETS COMPLEX IT GETS TO BE MORE CHALLENGING. SO DATA INDEPENDENT ACQUISITION, WHICH YOU MIGHT BE FAMILIAR WITH, IS A WAY OF SAYING, WE'RE GOING TO SAMPLE EVERYTHING THAT COMES OFF THE LC COLUMN, WE'RE GOING TO DO THAT BY STEPPING A WINDOW, A MASS WINDOW ALL WAY ACROSS THE MASS RANGE, AND SO THEREFORE EVERYTHING GETS SAMPLED. ALL THE PRECURSOR -- GET SAMPLED BUT IT TAKES SOME AMOUNT OF TIME TO STEP THROUGH THE MASS RANGE, AND AS A RESULT OF THIS SCANNING WINDOW PROCESS, WE LOSE A LITTLE BIT OF SENSITIVITY, BUT IT'S VERY, VERY COMPLETE. SO IF YOU LOOK IN THE PROTEOMICS DOMAIN, THE INSTRUMENTS ARE BEING DESIGNED TO TAKE ADVANTAGE OF DATA INDEPENDENT ACQUISITION BY BEING DEVELOPED WITH BRIGHTER SOURCES IN ORDER TO REALLY MAXIMIZE THE POWER OF THAT APPROACH. FOR GLYCOPROTEOMICS, HOW DO WE INTERPRET THE DATA? SO WHAT WE DO IS, WE HAVE TO -- THIS IS WHERE WE'VE BEEN MODIFYING GLYRESOFT OVER THE PAST YEAR OR SO, THE CONVOLUTION STEP HAS TO INCLUDE SOMETHING WHICH IS KNOWN AS A PEARSON CORRELATION, KNOWN AS DIA UMPIRE, SO WE DID SOMETHING SIMILAR. ESSENTIALLY WHAT WE'RE TRYING TO DO IS GENERATE SUE TOE-TANDEM MASS SPECTRA, WE'RE TRYING TO INTERPRET MASS SPECTRA BASED ON A PEPTIDE-CENTRIC APPROACH AND THEN WE HAVE TO COME UP WITH A MODIFIED SCORING MODEL. THIS IS ESSENTIALLY WHERE HE WITH WE ARE, WHERE WE'VE ADAPTED GLYCRESOFT. SO THAT DOES REINQUIRE A BIT OF AN ADJUSTMENT IN TERMS OF HOW WE'RE DESIGNING THE SOFTWARE. SO THE THING THAT WE HAVE TO DO GOING FORWARD IS TO IMPROVE THE SCORING BY ADDING RETENTION TIME MODELING WHEN WE COME TO INSTRUMENTS THAT PRODUCE THE HIGH MOBILITY SEPARATIONS. ALL OF THIS WILL BE NECESSARY AND AT THE END TO ADJUST THE METRICS TO MAKE THEM MORE RELIABLE. SO LET'S LOOK AT AN EXAMPLE. THIS IS A COMPARISON OF SARS COV2 SPIKE PROTEIN COMPARING IT USING DDA VERSUS DIA AND AS YOU SNOW, THAT SARS COV-2 SPIKE PROTEIN IS VERY HIGHLY GLYCOSYLATED, BUT FLONT LESS, GROUPS AROUND THE WORLD HAVE BEEN ABLE TO ASSIGN GLYCOSYLATION USING DDA APPROACHES BUT OUR APPROACH WAS TO COMPARE STANDARD DDA VERSUS DATA-INDEPENDENT ANALYSIS. SO IN RED, YOU SEE THE BAR PLOTS OF THE VARIOUS GLYCOCYTES, FOR GIVING GLYCOCYTE THE GLYCOFORMS THAT ARE PRESENT, AND GREEN IS DIA AND RED IS DDA. THE VIOLIN PLOTS, THE WIDTH OF THE VIOLIN PLOTS ARE SHOWING YOU HOW MANY ASSIGNMENTS THERE ARE FOR COMPLETE -- THESE ARE DATA RUN IN TRIPLICATE, SO IF WE HAD THREE ASSIGNMENTS, IT SHOWS UP AT THE TOP REGION OF THE VIOLIN PLOT, TWO OUT OF THREE SHOWS UP IN THE MIDDLE, AND THEN ONE OUT OF THREE IS AT THE BOTTOM. SO BECAUSE DATA INDEPENDENT ANALYSIS REQUIRES MORE TIME, IT ESSENTIALLY IS LOSING A LITTLE BIT OF SENSITIVITY, BUT WE FIND THAT AS LONG AS WE GET SUFFICIENT NUMBER OF ASSIGNMENTS AND A LOT OF THE SPECTRA ARE FULLY ASSIGNED, WE GET BETTER PERFORMANCE USING DATA INDEPENDENT ANALYSIS. AND THAT'S WHAT WE SEE HERE. WE'RE ABLE TO SAMPLE A LOT MORE GLYCOPEPTIDES INCLUDING THOSE AT LOWER ABUNDANCE. LOOKING AT DIFFERENT DPLIE COSITES FOR GLYCOCYTES FOR DIFFERENT PROTEINS, THIS PATTERN IS BORNE OUT. WE START TO SEE MORE SUBTLETY WHEN WE LOOK AT THE LOWER ABUNDANCE GLYCOCYTES, THE ONES THAT PRODUCE HIGHER ABUNDANCES, MEANING THE DATA INDEPENDENT ANALYSIS -- ACQUISITION UNDERPERFORMS IN THOSE CASES SIMPLY BECAUSE THE SCORING FAILS DUE TO LOW EYE ON ABUNDANCE. ION ABUNDANCE. SO THIS IS NOT SURPRISING. ESSENTIALLY THIS REFLECTS WHAT HAS BEEN DRIVING PROTEOMICS IN TERMS OF NEEDING BRIGHTER SOURCES AND FASTER SCANNING TO FAVOR DATA-INDEPENDENT ANALYSIS. SO WE GOT INTERESTED IN USING THE MOBILITY DIMENSION AND ARE WORKING WITH WATERS CORPORATION ON THEIR SICKLY ION MOBILITY INSTRUMENT. IN THIS IS AN INSTRUMENT THAT TAKES A DIFFERENT APPROACH TO DATA-INDEPENDENT ACQUISITION. WHAT I MEAN BY THAT IS THAT IT ESSENTIALLY IS SAMPLING ALL OF THE GLYCOPEPTIDES AT ONCE. THERE IS ABSOLUTELY NO PRECURSOR ION SELECTION. IT TAKES THE ENTIRE MASS RANGE AND SENDS IT THROUGH THE INSTRUMENT AT ONCE. SO OBVIOUSLY IF THERE'S NO STEPPING THROUGH THE MASS RANGES, IT'S GOING TO IMPROVE SAMPLING RATE AND SENSITIVITY, BUT WE HAVE TO COMPENSATE FOR THE LOSS OF PRECURSOR ION SELECTIVITY. SO IN THEORY, THIS IS GOING TO BE A VERY SENSITIVE WAY OF SAMPLING GLYCOPEPTIDES BUT WE HAVE TO CHANGE THE WAY WE ARE DOING OUR SOFTWARE ANALYSIS. SO HERE'S THE INSTRUMENT, IT IS A KUTOFF INSTRUMENT AND IT HAS A DONUT IN THE MIDDLE WHICH IS WHERE THE IONS PASS THROUGH A CYCLIC ION MOBILITY REGION ABOUT A METER LONG. YOU CAN SEND IT THROUGH ONE OR MORE TIMES BUT IN THE DATA I'LL SHOW YOU HERE, IT'S ALWAYS JUST GOING THROUGH ONCE, AND THEN IT GOES INTO THE MASS ANALYZER. SO WE'RE COMPARING THE MASS SPECTRA OF THE IONS AFTER THEY'VE GONE THROUGH THE CYCLIC CELL, AND THEN REQUIRING A TANDEM MASS SPEC, LOW ENERGY, HIGH ENERGY, LOW ENERGY, HIGH ENERGY, JUST ALTER NATEING. THIS IS CALLED AN HDMS TO THE E EXPERIMENT. SO ESSENTIALLY WHAT WE DO IS, WE NEED TO INTERPRET THE TANDEM MASS SPECTRA AND THE MASS SPECTRA IN TERMS OF GLYCOPEPTIDES, AND COMING UP TO CONCLUSIONS BASED ON A SPECTRUM-CENTRIC APPROACH. AND SO IF YOU LOOK AT THE SEPARATION OF, SAY, A TBLIE GLYCOPEPTIDE FROM MAN 9 ALL THE WAY TO MACHINE 4, YOU GET A NICE SEPARATION IN DRIFT SPACE AND DRIFT TIME, AND THEN WHAT WE'RE DOING IS WE'RE ACQUIRING MS AND TANDEM MS ACROSS ALL THESE PEAKS. SO WE'RE GETTING HUNDREDS AND HUNDREDS ACROSS THESE PEAKS. >> DO YOU THINK YOU CAN WRAP UP IN ABOUT TWO MINUTES? >> SURE. AND SO THIS HAS BEEN USING THE APPROACH THAT WE DEVELOPED WHERE WE WERE DOING ESSENTIALLY DI UMPIRE LIKE EXTRACTION OF MASS SPEC SPECTRA AND IMPROVING OUR SCORING MODELS. WHAT WE HAVE ALSO FOR SARS SPIKE PROTEIN, COMPARING BEFORE WE ADDED THE DIA UMPIRE STEP IN RED AND THEN AFTER IN GREEN, SO WE'RE SEEING AN IMPROVEMENT IN OUR ABILITY TO SCORE GLYCOPEPTIDES FROM THIS VERY COMPLEX GLYCOPROTEIN. SO AT THIS POINT, WHAT WE HAVE TO DO IS START ADDING IN MORE RETENTION TIME AND ARRIVAL TIME MODELING, BUT WE VIEW THIS AS REALLY THE FUTURE IN TERMS OF THE ABILITY TO SAMPLE CONFIDENTLY AND COMPLETELY GLYCOPROTEIN GLYCOSYLATION AND HOW IT'S CHANGING OVER TIME. SO IN SUMMARY, DATA INDEPENDENT ACQUISITION ACHIEVES MORE COMPLETE COVERAGE THAN DOES DATA DEPENDENT AND THEN WE SEE THE ION MOBILITY HDMS TO THE E TO BE THE OPTIMAL APPROACH FOR DEEP CONFIDENT GLYCOPEPTIDE PROFILING AND ARE ESSENTIALLY EXTENDING AND DEVELOPING GLYCRESOFT SO IT WILL HANDLE THAT DATA. OKAY, I ACKNOWLEDGE MY COLLABORATORS, ESPECIALLY HENRY WAN OF MISSOURI STATE UNIVERSITY, AND ROB WOODS AT UNIVERSITY OF GEORGIA. AND ACKNOWLEDGE THE COMMON FUND FOR FUNDING, AND THANK YOU VERY MUCH. >> OKAY, THANK YOU, JOE. IS THERE ANY ONE QUICK QUESTION OUT THERE IN THE AUDIENCE? >> I HAVE A QUICK QUESTION. SO I'M CURIOUS HOW YOU ARE THINKING ABOUT SEPARATING OUT DIFFERENT ISOMERS, GLYCOISOMERS, USING DIA. >> I'M NOT THINKING ABOUT SEPARATING GLYCOISOMERS. I'M TRYING TO FIGURE OUT HOW TO ASSIGN SINGLY GLYCOSYLATED PEPTIDES. I DON'T THINK THERE IS AN AVENUE FOR SAYING IN YOU HAD IF YOU HAD A GLYCAN EYE OWE MER, I DON'T THINK WE'RE GOING TO GET AT IT IN THIS APPROACH. >> EVEN WITH DEEP LEARNING FOR EXAMPLE WITH RETENTION TIME MODELING, BEING ABLE TO KNOW WHICH PEAK IS REPRESENTED OF THE SPECIFIC ISOMER BASED ON WHERE IT IS RELATIVE TO OTHER PEPTIDES? THIS SORT OF WORK HAS BEEN DONE WITH PHOSPHOPEPTIDES, FOR EXAMPLE, WITH POSITIONAL ISOMERS. >> YEAH, I SEE RETENTION TIME MODELING AS REALLY GOOD FOR IMPROVING SCORING OF GLYCOPEPTIDES, AND ELIMINATING FALSE POSITIVES, BUT I AM NOT CONFIDENT IN THE ABILITY TO SEPARATE POSITIONAL EYE ISOMERS YET. I'D HAVE TO REALLY CONVINCE MYSELF OF THAT AND I HAVEN'T YET. >> OKAY. THANKS. >> THANK YOU. WE'LL HAVE TO MOVE ON. SO OUR NEXT TALK IS GOING TO BE IN A SIMILAR VEIN. THIS IS FROM THE LAB OF -- GOLDMAN AND I BELIEVE MILOSLAV SAN KA WILL DA WILL BE PRESENTING. >> SPECIFICALLY HIGH SENSITIVITY GLYCOPEPTIDE DIA AND THE GPD TRANSITION LIBRARY. DURING THE LAST FEW DECADES, THE NUMBER OF PROTEOMICS PUBLICATIONS SIGNIFICANTLY INCREASE EVERY YEAR. SIMILAR TRENDS, YOU CAN SEE IN CASE OF -- >> MILO, WE CAN'T SEE YOUR SLIDES. >> REALLY? I'M SORRY. SORRY. I FORGOT TO SHARE MY SCREEN. HOW ABOUT NOW? >> YEP. THANKS. >> I'M SORRY. SO AGAIN, I WILL TALK ABOUT HIGH SENSITIVITY GLYCOPEPTIDE INDEPENDENT ANALYSIS AND GPTWIKI TRANSITION LIBRARY. SO DURING THE LAST FEW DECADES, A NUMBER OF THE PROTEOMICS PUBLICATIONS SIGNIFICANTLY INCREASED AND THE SAME TREND IS IN DIA GLYCOPROTEOMICS INTRODUCED IN THE PROTEOMICS APPLICATIONS. AND WITH THE GLYCO -- DIA, WE ARE JUST BEGINNING. SO THERE ARE SOME HERE AND JUST A FEW APPLICATIONS. THE ADVANTAGE OF INDEPENDENT ANALYSIS, A FEW ADVANTAGES. ONE IS HIGHER SENSITIVITY, YOU CAN SEE IT HERE, OVER THE DDA, AND -- ALSO THE POSSIBILITY OF RETROSPECTIVE DATA SO YOU CAN BASICALLY -- OF TARGET -- IDENTIFY -- SO VERY IMPORTANT PART OF DATA INDEPENDENT METHODOLOGIES ARE SPECTRA OR TRANSITION LIBRARIES. IN THE CASE OF PROTEOMICS ARE CURRENTLY AVAILABLE FOR MANY -- CELL LINES OR TISSUE OR LIPID BIOPSIES. ALSO IN THE LAST GLYCOPROTEOMICS SPECIAL ISSUE MCP, THERE WERE TWO PAPERS FOCUSED ON THE DATA INDEPENDENT ANALYSIS. ONE WAS ON THE -- AND GLYCOSYLATION -- SO HERE YOU CAN SEE OTHER CONTRIBUTION TO INDEPENDENT -- SO THE FIRST GROUP WHO INTRODUCED DATA INDEPENDENT ANALYSIS PROTEOMICS, AND WE ALREADY PUBLISHED SEVERAL PAPERS. AND SOME OF THEM ARE ON THE WAY. YOU CAN SEE HERE THE SELECTIVITY OF THE TRANSITION OF IN THIS CASE OUR TRANSITION OF -- GLYCOFORM IN UNFRACTIONATED HUMAN -- OR WE PUBLISH WITH COLLABORATION WITH PROFESSOR MEHTA THE STUDY OF -- [INAUDIBLE] AND WHY IS THAT INDEPENDENT GLYCOPROTEOMICS MORE -- ON THE BOTTOM, YOU CAN SEE -- GLYCOPEPTIDE WITH -- FRAGMENTATION SPECTRA, AND YOU CAN SEE THE HIGHEST INTENSIVE POPULATION -- OF SPECIFIC -- IN CASE OF COMPLEX GLYCANS AND MULTIBRANCH GLYCANS, YOU WILL GET MUCH LOWER SENSITIVITY OR INTENSITY OF SPECIFIC IONS WHICH COULD BE USED FOR DIA ANALYSIS OR BUILDING THE TRANSITION -- SO FOR EXAMPLE, THE PUBLISHED PAPER IN MCP WAS BASED ON THE ANALYSIS OF -- [INAUDIBLE] SO COULD BE THE CHANGED CONDITION, SO YOU HAVE DIFFERENT APPROACH -- CHANGE CONDITION OF THE -- SO IN MILD FRAGMENTATION, YOU BASICALLY WILL GET HIGH INTENSITY -- AND LOW INTENSITY -- MAJOR FRAGMENTATION IS A LOSS OF -- IN SOME SMALL -- FRAGMENTATION. IN HIGH COLLISION ENERGY, YOU BASICALLY FRAGMENT WHOLE GLYCAN GLYCAN -- IONS, AND SOME VERY SMALL SELECTIVE BUT LOW INTENSIVE PEPTIDE -- AND USUALLY YOU'LL END UP WITH SLIGHTLY HIGHER INTENSIVE PEPTIDE WITH THE -- SO HERE IS OUR WORKFLOW OVER TRANSITION LIBRARY. WE START WITH THE GLYCOPROTEIN DIGEST. -- TO BASICALLY NORMALIZE THE RETENTION TIME, SIMILAR TO PROTEOMICS APPROACH. AND WE ACQUIRE SAMPLES WITH OR WITHOUT -- AND FIRST SCAN IS FOCUSED ON THE IDENTIFICATION, WHICH IS BASED ON THE ETHCD. AND IN CASE OF PRESENCE OF OXONIUM IONS, THE RECORD ALSO THE LOW COLLISION THAT SERVE AS A SOURCE FOR LIBRARY BUILD. FOR THIS TWO STEP, BAYICALLY BASE BUILDING FROM FOUNDATIONAL PROTEOMICS FOR THE IDENTIFICATION AND DIA TRANSITIONS ARE FROM THE SAME FRAGMENTATION. AS I MENTIONED, TRANSITION LIBRARY COULD BE USED FOR MRM AND -- SO WE HAVE DEVELOPED A HIGH SENSITIVE METHODOLOGY USING LIMITED FRAGMENTATION. HERE YOU CAN SEE THE PUBLISHED WORKFLOW OF MRM ANALYSIS, LOW COLLISION MRM ANALYSIS OR PRM ANALYSIS OF LOW ABUNDANT PLASMA GLYCOPROTEIN IN LIVER DISEASE PROGRESSION -- CARCINOMA, AND -- AND YOU CAN SEE THAT WE ACTUALLY ANALYZE SEPARATELY -- CHANGES. FOR OUR OTHER COLLABORATIVE PROJECT, ONE FROM UNIVERSITY OF MARYLAND FOCUSED ON THE HIGH -- RATE AND SENSITIVE QUANTIFICATION OF IGG DPLIE COFORMS IN UNFRACTIONATED HUMAN SERUM, USING INTER-AND INTRASCAN NORMALIZATION. SO WE RECORDED DATA, OPTIMIZED THE WORKFLOWS, AND NATHAN EDWARDS CREATED GPTWIKI WHICH SERVES AS A TRANSITION LIBRARY AND DATA AND METHODOLOGY. THIS IS THE MAIN PAGE, AS YOU CAN SEE, WE HAVE SO FAR INCLUDED TRANSITION LIBRARY FROM HUMAN SERUM AND -- WE ALSO INCLUDED SOME IMPORTANT TARGETS, FOR EXAMPLE, SARS COV-2 GLYCOPROTEIN OR HUMAN ACD. AND HIS EXAMPLE OF ONE GLYCOPEPTIDE, YOU CAN SEE THE DESCRIPTION INCLUDED METHODOLOGY AND SOME PREPARATION AND SOME ADDITIONAL INFORMATION. HERE YOU CAN SEE THEY CALL IT SPECTRA USED FOR IDENTIFICATION. SO THAT IS A POPULATION OF THE SPECIFIC IONS USED FOR IDENTIFICATION OR SOME ADDITIONAL SPECIFIC IONS WHICH BASICALLY COULD SERVE AS A CONFIRMATION OF IN THIS CASE -- WHICH IS HERE AND HERE, OR ADDITIONAL IN THIS CASE IS CHARGE LOSS TYPICAL FOR FRAGMENTATION. EACH COULD SERVE AS A CONFIRMATION OF MONO ISOTOPE IONS -- ASSIGNMENT [INAUDIBLE] AND HERE WE ALSO HAVE INCLUDED IN THE GPTWIKI THE ACCEPTED -- WE HAVE TESTED OPEN SOURCE SOFTWARE FOR GLYCOPEPTIDE DIA. SO USE OF -- IDENTIFICATION DATA TO CREATE TRANSITION LIBRARY GLYCOPEPTIDE DIA. EACH COULD BE USED AND WE TESTED THE SIMILARITY OF THE FRAGMENTATION SPECTRA BETWEEN THE -- BASED INSTRUMENT AND -- SPECIFICALLY 6600 TRIPLE TOF. AND WE CONFIRMED THAT WE ARE ABLE TO USE ONE LIBRARY PERUSE ON THE ORBITRAP FOR ANALYSIS BY TRIPLETOF. SO YOU CAN SEE EXAMPLE OF THE TRANSITION LIBRARY, AS I MENTIONED THE MAJOR FRAGMENTATION IS -- AND SOME OF THE SPECIFIC FRAGMENTS AS I MENTIONED ARE LABELED -- STOPS. SO GPTWIKI CURRENTLY CON CONTAINS SAMPLES OF DATA FROM TRIPLETOF AND HERE YOU CAN SEE [INAUDIBLE]. SO NATHAN EDWARDS IMPLEMENTED MANY QUALITY PROGRAM PARAMETERS, FOR EXAMPLE, -- MODELING AND GLYCOPEPTIDES FRAGMENTATION SPECTRA QUALITY -- AND WE ARE CURRENTLY WORKING ON THE EXPANSION OF THE LIBRARY. SO IN LESS THAN TWO YEARS, WE HAVE PUBLISHED SEVERAL PAPERS OF COMPREHENSIVE GLYCOSYLATION CHARACTERIZATION OF -- IMPORTANT TARGETS SUCH AS, FOR EXAMPLE HERE, PDL1 AND PD-1 TARGETS FOR CANCER. EACH WILL BE IN THE TRANSITION LIBRARIES. AND ALSO WE ARE WORKING FOR THE IMPLEMENTATION OF THE NEWEST TECHNOLOGIES LIKE ION MOBILITY -- [INAUDIBLE] WHICH WILL INCLUDE -- ION MOBILITY. >> DR. SANDA, CAN YOU WRAP UP IN TWO MINUTES? >> YES. AND THE LAST PART IS VALIDATION STUDY, SO WE CHOOSE AN INTERLABORATORY TEST. SO WE ARE WORKING FROM THE TWO INTERLABORATORY STUDY, WHICH ONE WILL BE FOCUSED ON THE TESTING OF INTERLABORATORY REPRODUCIBILITY OF DIA METHODOLOGY. WITH ALL INFORMATION PROVIDED BY GPTWIKI. AND THE SECOND WILL BE THE COMPARISON OF ALL METHODOLOGIES FOR -- ANALYSIS OF GLYCOPEPTIDE. THAT'S IT. THANK YOU. >> OKAY, THANK YOU. I GUESS WE HAVE TIME FOR A QUESTION. CAN I ASK IF YOUR LIBRARY THAT YOU'VE BEEN POSTING IN THAT WIKI SITE, HAVE OTHER PEOPLE BEEN USING THAT YET? >> THAT ACTUALLY IS MORE QUESTION FOR DR. EDWARDS. I DON'T KNOW HOW MANY VISITORS. I DON'T KNOW CURRENTLY ANYONE WHO PUBLISHED INDEPENDENT ANALYSIS USING OTHER LIBRARIES. >> OKAY. LET'S MOVE ON TO THE NEXT TALK. AND AGAIN, THIS TALK IS BY A SUPPLEMENT AWARDEE, DR. CHRISTOPHER ANDERTON. DR. ANDERTON, ARE YOU -- OKAY, YOU'RE SHARING YOUR SCREEN, SO YOU CAN GO AHEAD AND GET STARTED. >> ALL RIGHT. THANK YOU FOR THE INTRODUCTION. I'M A SPATIAL GLYCOMICS SCIENTIST AT THE NORTHWEST NATIONAL LAB. THE WORK I'M PRESENTING ON IS BASED ON ADMINISTRATIVE SUPPLEMENT GRANT WE WERE AWARDED THIS YEAR IN CONJUNCTION WITH OUR WORK WITHIN THE KIDNEY PRECISION MEDICINE PROGRAM. OUR GROUP CONSISTS OF FOLKS AT UT HEALTH SAN ANTONIO, PACIFIC NORTHWEST NATIONAL LAB AND THE EUROPEAN MOLECULAR BIOLOGY LAB. WE ARE A TISSUE INTERROGATION SITE FOCUSED OND SPATIAL ON SPATIAL METABOLOMICS. I HAVE NO DISCLOSURES. A LITTLE BIT ABOUT THE KPMP. THIS IS A LARGE CONSORTIUM EFFORT THAT CONSISTS OF TISSUE OOO HE RECRUITMENT SITES, LONGITUDINALLY ACROSS DEMOGRAPHICS AND TISSUE INTERROGATION SITES THAT HAVE THEIR OWN EXPERTISE IN SPECIALIZED TECHNOLOGIES FOR ANALYZING THE KIDNEY BIOPSIES GENERATED FROM THE RECRUITMENT SITES AND THEN WE HAVE A CENTRAL HUB THAT COWARDS NAIS THESE COORDINATES TH ESE. THE AIM OF THE KPMP IS ORGANIZING AND INTEGRATING THIS MULTIPLE -- TO CREATE A KIDNEY TISSUE ATLAS AND HOPES WILL BE ABLE TO FIND CRITICAL CELLS AND PATHWAYS AND TARGETS FOR NOVEL THERAPIES RELATED TO ACUTE KIDNEY INJURY OR CHRONIC KIDNEY DISEASE. SO AS NOSED, WE ARE A TISSUE INTERROGATION SITE FOCUSED OND SPATIAL METABOLOMICS, AND THE PRIMARY TECHNOLOGY WE USE IS MATRIX-ASSISTED LASER DESORPTION/EYE ONIZATION OR MALADY, I'M NOT MALDI, I WILL NOTE THAT THIS TECHNOLOGY PERMITS US TO VISUALIZE WITHIN KIDNEY TISSUE AND WE CAN ALIGN THIS WITH DATA FROM HISTOCHEMICAL IMAGING OR OTHER IMAGING MODALITIES, AND THIS HELPS US LOCALIZE BIOCHEMICAL PROCESSES WITHIN DIFFERENT MICRO ANATOMICAL REGIONS OR EVEN CELL TYPES WITHIN THE KIDNEY. ONE OF THE BIG TOOLS WE USE FOR PROCESSING AND ANNOTATING THIS BIG MASS SPEC IMAGING DATA WE'RE GETTING IS META SPACE. THIS WAS DEVELOPED -- HE HAS BEEN FURTHER EXPANDING THE UTILITY OF THIS AS PART OF OUR TISSUE INTERROGATION SITE. ESSENTIALLY THINK OF META IT IS SPACE, IT IS A CLOUD OPEN METABOLITE ENGISH AND KNOWLEDGE BASE SO IT'S LIKE MASS SPEC IMAGING GOOGLE, IF YOU WILL, AND IT FOLLOWS THE PRINCIPLES OF FAIR DATA. SO OVER THE LAST FEW YEARS OR COUPLE YEARS, OUR TISSUE INTERROGATION HAS LOOKED AT EXPANDING FROM LOOKING AT THE MA TAB LOAM TO BEING LOOING AT THE GLYCOME. WE'RE HOPING TO MAKE THE TECHNOLOGY UTILIZED FOR ANALYZING KIDNEY BIOPSIES LATER THIS SUMMER. AS PART OF THIS EFFORT, WHAT WE DID IS CREATED THIS CUSTOMIZED N-GLYCAN DATABASE, N CLIE N-GLYCAN DB, AND WE USE SOME OF THE TOOLS THAT WERE DEVELOPED AS PART OF THIS EFFORT TO CREATE THIS DATABASE AND THIS TOOL IS NOW AVAILABLE FOR USERS TO USE WORLDWIDE. THIS GIVES YOU A LITTLE BIT OF AN IDEA OF WHAT THIS INTERFACE LOOKS LIKE, SO IF ONCE YOU'VE UPLOADED YOUR DATA, YOUR SPATIAL N IMLIE COMIX DATA TO META SPACE, YOU CAN CLICK ON ALL THE TYPES OF DIFFERENT -- YOU CAN SEE ONCE YOU CLICK ON ONE OF THESE ANNOTATIONS, THE ION DISTRIBUTION WITHIN YOUR SAMPLE. AND ALSO THE TYPES OF POSSIBLE N-GLYCANS THAT ARE WITHIN -- MOLECULAR FORMULA, AND ALSO YOU CAN REGISTER AND OVERLAY THE DATA WITH AN OPTICAL IMAGE OF YOUR SAMPLE. SO OUR MAIN EFFORT WITHIN THIS FUNDING WAS -- AND IN ADAPTING DR. SUNG'S OXIDATIVE RELEASE OF NATURAL GLYCANS FOR MASS SPEC IMAGING. HAS THE POTENTIAL TO EXPAND OUR SPATIAL GLYCOMICS CAPABILITY BEYOND JUST N GLYCANS. SO AS WE STARTED ASKING OURSELVES A FEW QUESTIONS IN GETTING THIS TO WORK. FIRST OFF, DOES ADDING AN OXIDATIVE AGENT TO OUR SAMPLES -- DOES THAT AFFECT OUR TISSUE MORPHOLOGY, AND WHAT IS THE OPTIMAL AMOUNT THAT WE CAN SPRAY ON TO OUR TISSUE? MOREOVER, WHAT IS THE BEST TYPE OF TISSUE TO USE, AND PRESERVATION METHODS? SO FOR EXAMPLE, WE USE PRESH PRO 16 TISSUE FOR SPATIAL METABOLOMICS BUT -- AND FINALLY, AFTER WE'VE OPTIMIZED METHOD FOR ADAPTING THE ORNG TO MALDI WORKFLOW, DO WE SEE MOLECULAR DIFFERENCES AND ANYTHING WE CAN RELATE BACK TO SOME ANATOMICAL MICRO COMPARTMENT OR SOMETHING WITHIN OUR TISSUE? SO FIRST UNDERSTANDING HOW ADDING AN OXIDATIVE AGENT TO OUR TISSUE AFFECTS THAT, WE FIRST FOLLOWED THE PROCEDURE DR. SUNG REPORTED ON USING 1% SODIUM CHLORIDE AND SPRAYED THAT DOWN. ONE OF THE ISSUES WE SAW WITH THAT IS WE DO SEE IT DOES AFFECT SOME OF THE TISSUE MORPHOLOGY AND WE SEE SOME THESE LARGE SALT CRYSTALS THAT END UP FORMING ON THE TISSUE. THIS WILL DEFINITELY AFFECT OUR MALDI AND CREATE ION SUPPRESSION. BUT ONCE WE LOOKED AT REDUCING THE AMOUNT OF OXIDATIVE AGENT WE WERE SPRAYING, WE DID ACTUALLY SEE THE OPTIMAL AMOUNT WAS ABOUT 10 TIMES LESS WHEN WE'RE DOING THIS ON TISSUE SAMPLE DIGESTION, SO WE WERE ABLE, LOOKING AT THIS GLYCOPROTEIN STANDARD A MINIMAL AMOUNT OF OXIDATIVE AGENT, WE WERE ABLE TO SEE THAT THESE DIFFERENT GLYCAN PEAKS. COMPARING BETWEEN TISSUE PRESERVATION TYPES, WE FOUND THAT FRESH FROZEN TISSUE WORKED MUCH BETTER THAN FFPE. WE DIDN'T SEE ANY CHARACTERISTIC MASS SPEC PEAKS FROM THE FFPE, BUT WE DID SEE IT FROM THE FRESH FROZEN. SO THIS COULD BE POTENTIALLY PRETTY PROMISING FOR OUR TISSUE INTERROGATION SITE BECAUSE WE'RE ALREADY USING FRESH FROZEN TISSUE FOR SPATIAL METABOLOMICS, WE COULD END UP USING THE SAME TISSUE SAMPLE AND GET -- OR TISSUE SECTION RATHER AND GET TISSUE METABOLOMICS FOLLOWED BY SPATIAL GEICO MIX FOLLOWING THIS METHOD. ONE OF THE ISSUES WE RAN INTO IS WE DIDN'T REALLY KNOW HOW TO AN TATE ANNOTATE THIS DATA BECAUSE THIS REACTION IS PRETTY MESSY AND THERE'S NUMEROUS DIFFERENT OXIDATION REACTIONS THAT COMPLICATE THE ANNOTATION, BUT WE'VE BEEN TALKING TO DR. SONG AND WE HAVE SOME IDEAS ON HOW TO AN ANNOTATE THESE SPECIES THAT WE'RE SEEING. SO THIS IS THE OPTIMIZED WORKFLOW WE'RE USING ON THE LEFT HERE, AND HERE WHAT WE'VE BEEN ABLE TO SEE IS WE CAN SEE SOME CHARACTERISTIC SPECIES AND HETEROGENEOUS DISTRIBUTIONS THAT DO INDICATE THERE PROBABLY IS SOME KIND OF BIOCHEMISTRY WE'RE SEEING IN THESE DIFFERENT COMPARTMENTS WITHIN THE KIDNEY TISSUE ON THE LEFT. ALL THESE ISSUES ARE OF UNTREATED TISSUE AND ON THE RIGHT, ALL THESE -- TRYING SOME STANDARD STUFF OUT JUST TO SEE -- TO VALIDATE WHAT WE'RE LOOKING AT. SO IN SUMMARY OVER THIS LAST YEAR, WE HAVE SPATIAL N N GLYCOMICS TECHNOLOGY THAT WE SHOULD HAVE IT READY FOR BIOPSIES BY THE END OF THIS SUMMER AND WE DO THINK THE ORMG METHOD IS ADAPTABLE FOR MALDI-MSI, AND TO AN TATE -- TYPES OF GLYCANS THAT WE WOULD BE ABLE TO MEASURE AND WHAT THE MOLECULAR FORMULAS ARE. SO I'D LIKE TO THANK THE FOLKS ON OUR TIS, WHOSE EFFORTS HAVE MADE ALL THE WORK I SHOWED TODAY POSSIBLE, AND ALSO DR. ZHANG, DR. DRAKE FOR THEIR HELP WITH GLYCOMICS KIDNEY TISSUE. THANK YOU. >> OKAY, THANK YOU. VERY INTERESTING. ESPECIALLY EXCITING TO SEE HOW A SUPPLEMENT AWARDEE CAN ACTUALLY HELP OUT OUR PIs AS WELL, KIND OF EXPAND THE RESEARCH. ANY QUESTIONS FOR DR. ANDERTON? I GUESS IT'S VERY CONVENIENT FOR YOU THAT IT TURNS OUT THAT THIS IS MORE APPLICABLE TO FRESH FROZEN RATHER THAN FFPE. DO YOU HAVE ANY IDEA WHY FIXATION KIND OF MESSES UP THIS REACTION? >> THAT'S A GREAT QUESTION. I DON'T, AND I'D HAVE TO THINK A LITTLE BIT MORE ABOUT THAT. I'D HAVE TO GET BACK TO YOU ON THAT ONE. >> I HAVE A QUICK QUESTION. >> ALL RIGHT. EXPWR HOW DO YOU DEAL >> HOW DO YOU DEAL WITH DIFFUSION ISSUES? I IMAGINE WITH SMALL MOLECULES IT'S MUCH EASIER TO DIFFUSE THINGS OUT WHILE YOU'RE PROCESSING. >> YES, THINGS ARE GOING TO DIFFUSE WITH ANY OF THE MALDI APPROACHES WHEN YOU'RE SPRAYING MATRIX -- IN THIS CASE, YOU'RE SPRAYING AN OX DATED AGENT. THE DROPLETS ARE A VERY FINE MIST AND THE MOLECULE EXTRACTION THAT HAPPENS IN THE CASE OF THE REACTION WITH THE OXIDATIVE AGENT, IT'S VERY LOCALIZE TODAY A LEVEL THAT IT'S BELOW WHAT OUR LATERAL RESOLUTION IS. SO IT'S GOING TO BE SO SMALL, IT'S UNRESOLVABLE BY CURRENT METHODS. >> OKAY, WE'LL NEED TO MOVE ON TO THE NEXT TALK. THANK YOU VERY MUCH, DR. ANDERTON. OKAY. DR. LINGJUN LI, IT LOOKS LIKE YOU'RE SHARING YOUR SCREEN, SO GO AHEAD. >> CAN YOU SEE ME AND HEAR ME? >> YES, WE CAN. COMING IN LOUD AND CLEAR. >> GREAT. GOOD MORNING, EVERYONE. IT'S A GREAT PLEASURE TO SHARE WITH YOU SOME OF OUR PROGRESS ON THE DEVELOPMENT AND APPLICATION OF CHEMICAL TAGS FOR QUANTITATIVE GLYCOMICS AND GLYCOPROTEOMICS. I HAVE NO CONFLICT OF INTEREST TO DISCLOSE. PERHAPS FOR THIS AUDIENCE IT'S NOT SO IMPORTANT TO STRESS GLYCOSYLATION COULD REALLY CHANGE THE STRUCTURE AND ALSO FUNCTION OF THE PROTEINS AND SO AS A RESULT, THE ABERRANT EXPRESSION OF GLYCOSYLATION HAS BEEN IMPLICATED IN NUMEROUS DISEASES, RANGING FROM CANCER, CARDIOVASCULAR DISEASE AND NEUROLOGICAL DISORDERS. SO QUANTIFICATION OF GLYCANS AND GLYCOPEPTIDES ARE HIGHLY IMPORTANT. SO IN THIS RECENT REVIEW THAT WE SUMMARIZE A GREAT DEAL OF DIFFERENT STRATEGIES FOR GLYCAN AND GLYCOPEPTIDE QUANTIFICATION, AND THIS CAN OCCUR AT MULTIPLE LEVELS, SO EITHER WAS LABELED PRE OR INCORPORATING, FOR EXAMPLE, METABOLIC LABELING OR THE MS1 STAGE WITH ISOTOPIC LABELING, ALSO MULTIPLEX LABELING QUANTIFICATION AT THE MS2 STAGE, WE JUST HEARD SEVERAL GREAT TALKS ON THE STRATEGY OF USING EITHER DATA-DEPENDENT OR DATA-INDEPENDENT ACQUISITION, SO DESPITE THESE ARRAY OF ISOTOPIC LABELING STRATEGIES, THERE ARE STILL LIMITATIONS FOR EACH INDIVIDUAL APPROACHES. FOR EXAMPLE, METABOLIC LABELING CAN BE VERY ACCURATE BUT IT ALSO COULD BE VERY EXPENSIVE. ALSO OFTENTIMES THAT REQUIRES EITHER DOING THE CELL CULTURE, METABOLIC INCORPORATION, AND ALSO THE MS1 BASED QUANTIFICATION IN GENERAL WOULD ONLY ALLOW YOU TO DO EITHER DUPLEX OR TRIPLEX AND THIS COULD ALSO INDUCE SOME OF THESE ISOTOPE OVERLAPPING ISSUE. SO TO SOLVE SOME OF THESE ISSUE, THERE IS THIS DEVELOPMENT OF MS2 BASE QUAN TIFICATION IN ONE OF THE MORE SUCCESSFUL AMINOXYTMT IS THE COMMERCIAL APPROACH BUT THERE'S STILL SOME LIMITATIONS. SO JUST A QUICK SLIDE TO SHOW THE LABELING QUANTIFICATION STRATEGY SHOWN HERE. THIS IS THE 6 PLEX YOU CAN SEE THE REPORTER ON AND THE BALANCER BASICALLY ADDING TOGETHER AS IDENTICAL MASS. SO THIS ALLOWS US TO SIMULTANEOUSLY QUANTIFY FOR EXAMPLE 6 PLEX SAMPLES AND AT THE MS1 LEVEL, THESE GLYCAN SPECIES EACH SHOW AS A SINGLE PEAK SO THERE'S NO COMPLEXITY HOWEVER THE SECOND STAGE WOULD RESPOND TO GENERATE -- FOR RELATIVE QUANTIFICATION. SO SHOWING HERE IS THE 6 PLEX TMP LABELING REACTIONS WHERE IT CAN INSTALL DIFFERENT ISOTOPES. HERE WE HAVE THE REPORTER, THE MASS BALANCER AND THIS IS AN ALDEHYDE AND KETONE-REACTIVE GROUP. SO THIS TAG ACTUALLY HAS ACTUALLY A PRETTY GOOD SUCCESS BUT THERE ARE SOME LIMITATIONS, IN PARTICULAR WHEN LABELING MORE COMPLEX N-GLYCANS. AND THIS WOULD REQUIRE US OFTENTIMES TO DO TWO SEPARATE -- COLLISIONAL ASSOCIATION, ALLOWS US TO IDENTIFY THESE GLYCANS BUT THE LOW REPORTER ION YIELD WOULD ACTUALLY COMPLICATE THE QUANTIFICATION. SO IN ORDER TO BOOST UP THE REPORTER ION, WE NEED TO ACTUALLY PERFORM THIS HIGH COLLISIONAL ENERGY TO GENERATE THESE MORE ENHANCED REPORTER ION BUT HERE YOU DON'T SEE MUCH BACKGROUND FRAGMENTS. SO WE'VE BEEN DEVELOPING A DESIGN SET OF THESE ISOBARIC MULTIPLEX REAGENT, WE TERM IT SUGAR. SO THIS ACTUALLY SHOWS THE VERY SIMPLE THREE-STEP SYNTHCIRCLES BASICALLY WE STARTED OUT WITH LEUCINE DIE METHYLATION TO BASICALLY INSTALL THE GLYCINE AS A BALANCER AND IN THE -- REACTING TO FORM THIS HYDROCIDE -- FROM THE SUGAR, BY INSTALLING DIFFERENT STABLE ISOTOPES, WE CAN ACTUALLY HERE CREATE A FOURPLEX -- WHERE WE CAN GENERATE THE REPORTER ION RIGHT HERE DIFFERENT FROM ONE ANOTHER IN MS2. SO BASED ON THIS IDEA, WE HAVE DESIGNED WORKLOAD TO LOOK AT, FOR EXAMPLE, SO TO LOOK AT A GLYCOPROTEIN AFTER RELEASING THESE GLYCANS WITH PNGASEF. IN MS1 FOR THE FULL MS SCAN, THERE WILL SHOW EACH OF THESE SPECIES AND THEN FRAGMENTATION AT MS2 FOR RELATIVE -- SO THE FIRST WE WANT TO CARE IS LABELING EFFICIENCY. SO WE WANT TO COMPARE THE NATIVE AND ALSO THE AMINO XYTMT. WE STILL HAVE ABOUT 50% LABELED 100% LABELING EFFICIENCY. ANOTHER VERY IMPORTANT FACTOR TO LOOK AT IS THE FRAG MEN TAIG. FRAGMENTATION. SO HERE WE'RE COMPARING TWO DIFFERENT CONDITIONS, HCD25 AND 30. IN BOTH CONDITIONS, THE SUGAR TAG ACTUALLY PRODUCED MORE ABUNDANT REPORTER ION AND ALSO GENERATE MORE INTENSE BACKGROUND FRAGMENTS. SIMILARLY WE LOOK AT THE GLYCANS -- AGAIN WE CAN SEE THAT THE TMT HAS THE MINIMUM REPORTER ION SUGAR ACTUALLY PRODUCED A LOT MORE STRONG INTENSE ION SO THAT ALLOWS US TO QUANTIFY LOWER ABUNDANT SPECIES. SO THEN WE APPLY THIS TO LOOK AT THESE COMPLEX GLYCAN MIXTURES LABELED WITH ONE TO ONE TO FIVE TO 10 IN THIS, WE CAN SEE WE HAVE A PRETTY GOOD QUANTIFICATION ACURA HE ZOO AND SHOWING ON THE BOTTOM OF TWO REPRESENTATIVE SPECIES AND SHOWS PRETTY GOOD QUANTIFICATION ACCURACY. SO AFTER PROOCH OF PROOF OF PRINCIPLE, WE WANT TO MOVE ON TO SOMETHING MORE COMPLEX. SERUM SAMPLES COLLECTED FROM CHILDREN WHO RECEIVE CHEMOTHERAPY FROM THE B CELL LEUKEMIA, SO TEAM UP WITH DR. AMEDO AT MEDICAL SCHOOL OF WISCONSIN. SO HERE LEUKEMIA ALL IS PROBABLY ONE OF THE MOST PREDOMINANT CHILDHOOD CANCERS, SO THEY HAVE TREATED THESE -- THE KIDS WITH THESE CHEMOTHERAPY AND WE WANT TO LOOK AT THE GLYCAN LEVEL CHANGES IN THE BLOOD PRIOR AND ONE, THREE AND SIX MONTHS AFTER CHEMOTHERAPY. USING OUR 4 PLEX TAGS, SUGAR TAG. HERE AS YOU CAN SEE FROM THIS BAR GRAPH, ALL TOGETHER, WE WERE ABLE TO QUANTIFY 145 N GLYCANS, 68 OF THESE N GLYCANS ARE COMMONLY SHARED IN ALL THREE PATIENTS AND ALL OF THESE ACTUALLY SHOW IN PARTICULAR -- SHOWS DOWN REGULATION AFTER THE CHEMOTHERAPY TREATMENT. SO NEXT WE WANT TO FURTHER IMPROVE OR INCREASE -- THERE ARE SEVERAL -- WHY WE WANT TO INCREASE THE MULTIPLEXING. ONE IS THAT IT CAN HELP TO REDUCE THE SAMPLE AMOUNT FOR ANY ONE GIVEN SAMPLE, ALSO REDUCE THE SAMPLE PREPARATION ANALYSIS TIME, ALLOWS US MORE READILY TO LOOK AT LARGER NUMBER OF BIOLOGICAL CONDITIONS OR REPLICATES OR CLINICAL SPECIMENS. THIS CAN ALSO IMPROVE THE LC-MS DATA OVERLAP. IN ORDER TO DO THIS, WE TURN TO THIS MASS DEFECT PHENOMENON. FOR EXAMPLE HERE, IF WE SWAP WITH CARBON 13 THIS WOULD INTRODUCE A 2.9 MDA DIFFERENCE DUE TO THIS BINDING DIFFERENCE, SO IF WE DO THIS FOR OUR 4 PLEX WE CAN ACTUALLY CREATE A 6 PLEX VERSION -- A 12 PLEX VERSION OF THESE SHOE TBAR TAGS WHERE WE CAN ACTUALLY USE HIGH RESOLVING INSTRUMENTATION EVEN SOME OF THE HIGH END -- SYSTEM TO RESOLVE THEM IN THIS CHANNEL AT 30,000 RESOLVING POWER. SO WITH THIS IDEA, WE HAVE DESIGNED AND SYNTHESIZED THESE SUGAR TAGS THAT ALLOWS US TO BASICALLY TRIPLE THE THROUGH PUT OF OUR QUANTIFICATION STRATEGY WITH OUR SUGAR TAGS. NEXT I WANT TO TALK A LITTLE BIT ABOUT OUR EFFORT BESIDES LOOKING AT FREELY RELEASED GLYCANS OR GLYCOMIBG TO ALSO LOOK AT INTACT GLYCOPEPTIDE BECAUSE THERE ARE A NUMBER OF DISEASE CONDITIONS DREBTLY RELATED TO THE GLYCOFORM CHANGES WHERE WE LOOK AT THE SEEN RAL CEREBRAL SPINAL FLUID, WE EXTRACT THE PROTEINS, ENRICH THESE GLYCOPEPTIDES AFTER HYBRID -- TO GET SITE-SPECIFIC CHARACTERIZATION WE THEN USE OUR CHEMICAL TAGS TO DO QUANTIFICATION. TO TARGET THE PRIMARY -- OF THE PEPTIDE. SO HERE THIS IS SHOWING PROOF OF PRINCIPLE WITH THE DUPLEX 115 AND 117 DILUTE TAG WHERE WE CAN DIFFERENTIALLY LABEL TWO SAMPLES MI THEM IN MS1, AND MS2 ALLOWS US TO QUANTIFY THEM. IN THREE DIFFERENT CATEGORIES, THE CONTROL GROUP, MILD COGNITIVE IMPAIRMENT AND ADVANCED DEMENTIA. SO USING OUR ANALYTICAL WORKFLOW WITH THE 12 PLEX TAGS WE WERE ABLE TO QUANTIFY MORE THAN 1500N GLYCOPEPTIDES AT THE SPECIFIC LEVEL THAT MAP TO 107N IMLIE N GLYCOPROTEINS FROM THE CEREBRAL SPINAL SAMPLES. SO WE CAN USE THIS APPROACH, HERE I'M HIGHLIGHTING THREE DIFFERENT GLYCOPROTEINS, L1-LIKE PROTEIN WHERE ENCODE FOUR DIFFERENT GLYCOSITES, AND COMPLEMENT IS A NEUROINFLAMMATORY PROTEIN HAS A SINGLE GLYCOSITE BUT IT HAS 22 DIFFERENT GLYCOFORMS AND CLUSTERING IS A REALLY INTERESTING ONE BECAUSE THAT'S THE THIRD MOST -- GENE IN ALZHEIMER'S DISEASE, THIS ENCODES 449 AMINO ACID RESIDUES THAT HAVE FOUR DIFFERENT GLYCOSITES. SOME ARE MINIMUM -- HETEROGENEITY AND AT 374, THIS ONE ACTUALLY ENCODES 56 DIFFERENT GLYCOFORMS THAT IS REALLY INTERESTING TO INTERROGATE. SO WE HAVE ALSO LOOKED AT A MORE GLOBAL -- DISREGULATED IN GLYCOPEPTIDES USING A VOLCANO PLOT. AS WE CAN SEE, THERE ARE 69 OF THEERS N GLYCOPEPTIDES THESE MILD COGNITIVE CONDITION AND 32 SHOWS DYSREGULATION AT 80 STAGE. SO USING GENE ONTOLOGY ANALYSIS WE CAN ACTUALLY REVIEW SOME OF THE TOP-RANKED BIOLOGICAL PROCESSES RANGING FROM PLATELET DEGRANULATION TO NEGATIVE REGULATION OF THE ENDOPEPTIDASE ACTIVITY, ALL LEAD TO DEGENERATION IN ALZHEIMER'S DISEASE. WE THEN TOOK THIS 19 OF THE N GLYCOPEPTIDES SHOWING CONSISTENT CHANGING TRENDS IN THE MCI AT THE SAME LOCATION, WE WANT TO ASK THE QUESTION WHETHER THESE CHANGES ARE OCCURRING AT THE TBLIE COST GLYCOSYLATION SITE OR ACTUALLY AT THE PROTEIN. SO TO DO THAT, WE HAVE PERFORMED IN PARALLEL THE QUANTITATIVE PROTEOMIC ANALYSIS, SO IN THIS ONE EXAMPLE AS CAN YOU SEE PROTEIN D, THERE IS NOT STATISTICAL CHANGE AT THE PROTEIN LEVEL, HOWEVER, AT THIS TWO GEICO SIDE N65 -- WE OBSERVE NINE OF THESE GLYCOPEPTIDE, ACTUALLY EIGHT OF THESE SHOW UPREGULATION IN BOTH THE MCIN80 AND THIS IS OCC PAYING THE BETA STRAND AND N98 IS ACTUALLY SHOWING DOWN REGULATION AND THIS OCCUPIED THE COIL JUNCTION CONNECTING THE TWO BETA STRANDS THAT SUGGESTS THESE STRUCTURAL TRANSLATION. ANOTHER EXAMPLE IS TO USE THIS APPROACH THAT ALLOWS US TO LOOK AT A DISEASE STAGE SPECIFIC N GLYCOPEPTIDE CHANGES, AGAIN, WE'RE HIGHLIGHTING A FEW EXAMPLES, FOR EXAMPLE, THE PROSTAGLANDIN ISOMERASE, IT THE N51, THERE WAS ACTUALLY THREE OF THESE GLYCOPEPTIDE SHOWDOWN REGULATION AT 78, AT MCI, THESE TWO GLYCOPEPTIDES SHOW UPREGULATION AND RECEIPT MAINING ALL SHOWDOWN REGULATION. SAME THING FOR THE ION TRANSPORT PROTEIN AT 630, AGAIN, WE HAVE AT THE -- SOME OF THESE OTHER GLYCOPEPTIDES SHOWDOWN REGULATION AT AD STAGE. SO TO KIND OF SUMMARIZE WHAT I'VE TALKED TO YOU TODAY, WE HAVE DEVELOPED A SET OF THESE NOVEL GENERIC GLYCAN LABELING REAGENT THAT CAN BE ACTUALLY BROADLY USED BY THE GLYCOMICS COMMUNITY FOR HIGH THROUGHPUT MASS SPEC QUANTIFICATION AND ALSO STRUCTURAL CHARACTERIZATION OF GLYCANS. I DIDN'T HAVE TIME TO TALK ABOUT OUR EFFORT IN ALSO COUPLING THESE FOR A VARIETY OF DIFFERENT SEPARATION PLATFORMS, AND ALSO HILIC SEPARATION AND WE'RE IN THE PROCESS OF TRYING TO FURTHER VALIDATE SOME OF THESE TOOLS SO JUST A FEW QUICK HIGHLIGHTS OF SOME OF THE COLLABORATION PROJECTS AND ALSO PUBLICATIONS THAT HIGHLIGHTED HERE IN THE INTEREST OF TIME I'M NOT GOING TO NAME THEM, BUT HERE YOU CAN SEE SOME OF THE HIGHLIGHT OF THE APPROACHES, AND ALSO THESE PUBLICATION FOR THE MS1 BASED DIPYRO TAGS AND ENHANCED SEPARATION OF SOME OF THESE GLYCAN ISOMERS, LOOKING AT BREAST CANCER CELLS, AND JUST A QUICK ONE SUMMARY SLIDE ABOUT OUR DISSEMINATION AND OUTREACH EFFORTS, SO THIS IS OUR YEAR THREE OF THE PROJECT, SO WE TOGETHER PRODUCED 20 PEER REVIEWED PUBLICATIONS, THERE ARE SEVERAL STILL IN PROCESS, EVE ALSO PARTICIPATED IN THE MCP GLYCOPROTEOMICS SPECIAL ISSUE. WE'RE ALSO PRESENTED SEVERAL -- AT SEVERAL OF THE CONFERENCES, ALSO ACTUALLY ORGANIZING A GLYCOPROTEOMICS AND -- AT THE PACIFICHEM AT THE END OF THIS YEAR, HOPEFULLY SOME OF YOU CAN ATTEND, AND ALSO WE ARE COLLABORATEING WITH A NUMBER OF RESEARCH LABS BOTH ON AND OFF CAMPUS TO FURTHER PROMOTE OUR TECHNOLOGY AND EXPAND THE APPLICATION. SO WITH THAT, I'D LIKE TO THANK THE STUDENTS AND ALSO IN PARTICULAR, THE U01 GEICO01 GLYCOSCIENCE COMMON FUND AND THANK ALL OF YOU FOR YOUR ATTENTION. THANK YOU. >> OKAY. WE'RE STARTING TO RUN A LITTLE BIT SHORT ON TIME HERE, SO IF ANYBODY HAS QUESTIONS FOR DR. LI, MAYBE YOU CAN SEND A PRIVATE CHAT TO HER, AND I'D LIKE TO MOVE ON TO THE NEXT TALK. ACTUALLY THE NEXT TWO PRESENTATIONS WILL BE BY SUPPLEMENT AWARDEES. SO THE FIRST ONE WILL BE COMING FROM THE LAB OF DR. RICHA SAXENA. I BELIEVE DR. GILL WILL BE PRESENT PRESENTING. PLEASE GO AHEAD. >> THANK YOU FOR THE OPPORTUNITY TO PRESENT OUR WORK. I WILL BE TALKING ABOUT OUR WORK TO UNDERSTAND HUMAN GENETIC LOCUS IN THE BEGINNING, AND THIS IS SUPPORTED BY A SUPPLEMENT THROUGH DR. RICHA SAXENA. I HAVE NO CONFLICTS OF INTEREST TO DISCLOSE. SO THE CONTEXT IN WHICH WE WORK IS THE UK BIOBANK, WHICH IS A COHORT OF ABOUT HALF A MILLION PEOPLE RECRUITED IN THE UK ON WHICH BOTH OBJECTIVE AND SUBJECTIVE MEASURES OF HEALTH HAVE BEEN COLLECTED AS WELL AS THEIR ELECTRONIC MEDICAL RECORDS, THEY HAVE BEEN GENOTYPE AND SEQUENCED. WE ARE PARTICULARLY INTERESTED IN STUDYING SLEEP IN THIS COHORT, AND SO WE USE THE SLEEP QUESTIONS AS WELL AS THE GENOTYPES TO GET CORRELATIONS AND END UP WITH THESE SORTS OF MAN OF -- WHAT THEY DON'T TELL US IS THE CAUSAL GENE THAT UNDERLIES A CERTAIN LOCUS, THE DIRECTIONALITY OF EFFECT, AND ULTIMATELY THE BIOLOGICAL MECHANISMS. SO TO ADDRESS THESE QUESTIONS, WE HAVE BEEN COLLABORATING FOR SEVERAL YEARS NOW, THREE LABS, THE LAB OF DR. JAMES WALKER AT MGH, DR. RICHA SAXENA AND THE LAB OF DR. STUART SCHREIBER AT THE BROAD. ANOTHER COMMON FUND GRANTEE, DR. CHRISTINA WOO AT HARVARD. OUR STORY BEGINS WITH A GENETIC VARIANT ON CHROMOSOME 12 THAT WE KISS DISCOVERED TO BE ASSOCIATED WITH CHRONOTYPE AND EASE OF WAKING UP. >> CHROMOSOME IS IT NUMBER 1 A GENE CALLED ALG10 AND ALG10B ASSOCIATED WITH SLEEP TRADES IN BLUE AND CARDIOVASCULAR TRAITS IN RED AND A FEW OTHER TRAITS. SO WHEN WE LOOKED AT WHAT ALG10 IT'S THE END ENZYME IN THE OLIGOSACCHARIDE PATHWAY. SO SHOWN HERE ARE THE OTHER ENZYMES IN THE PATHWAY AND INTERESTING THING ABOUT ALG10 WAS UNLIKE THE OTHER ENZYMES IN THE PATHWAY THERE IS NO KNOWN CVG FOR ALG10. TO UNDERSTAND ALG10 MAY BE DOING AND WE USED A TRICK WHICH ALLOWS US TO KNOCK DOWN THE OVER EXPRESSED GENES AND TISSUES OF INTEREST. USING THAT SYSTEM WE GENERATED WITH A KNOCK DOWN OF THE ALG 10 GENE AND FLIES ONLY HAVE ONE UNLIKE HUMANS WHERE I SHOWED YOU THERE IS AN ALG 10 AND THE ALG 10B. TO STUDY THE SLEEP IN THE FRUIT FLY WE HOUSE THEM IN THESE INDIVIDUAL TUBES WITH AN INFRARED BEAM RUNNING TO THE MIDDLE AND EVERY TIME THE FLY WALKS THROUGH IT BREAKS THE BEAM AND WE ARE ABLE TO ASSESS THIS BECAUSE OF THEIR BEHAVIOR. WHEN WE PUT THE FLIES IN THE TUBES WE IMMEDIATELY NOTICED USING TWO RNA LIGHTS A WEAK AND STRONG KNOCKDOWN AND THESE FLIES HAD FRAGMENTED SLEEP. WHAT THAT MEANS IS INSTEAD OF THE FLY GOING TO SLEEP AND WAKE UP IT WOULD SLEEP, WAKE UP, SLEEP, WAKE UP, SLEEP, ULTIMATELY IS A MEASURE OF POOR SLEEP. AS A RESULT THE FLIES SLEEP MORE DURING THE DAYTIME. THIS PARALLELS OUR FINDINGS IN HUMANS WHERE WE DISCOVERED THIS AS A SLEEP LOCUS. WHAT WAS UNEXPECTED TO US WAS THE FLIES ALSO HAD SEIZURES. SO IN THE PROCESS OF HANDLING THEM WE SAW WHEN WE BANGED THE WALL THEY HAD SEIZURES AND TOOK LONGER TO RECOVER. A CLASSIC MODEL OF EPILEPSY. A PHENOTYPE BY PERFORMING ELECTROPHYSIOLOGICAL RECORDINGS ON THE FLIES. HAVING STUDIED THESE IN HUMANS AN FLIES WE WONDERED IF WE COULD EXPAND THEM AND HAD A ZEBRAFISH MUTANT USING CRISPR AND SAW THE FISH HAVE THE SAME FRAGMENTED SLEEP. WE ALSO MADE USE OF AN EXISTING LOSS OF FUNCTION OF ALG 10 AND NOTICED CONSISTENT WITH OUR FRUIT FLY AND ZEBRAFISH FINDING THEY SLEEP LONGER. WE ASKED WHETHER WE COULD TAKE IT STEP AND SEE IF THE FUNCTION OF ALG 10 MAY COME TO THE FLAG AND THE MUTANT FLIES TAKE LONGER TO GERMINATE AND CAN RESCUE IT BY OVEREXPRESSING THE GENE. THE FUNCTION OF THE ALG 10 IS CONSERVED ALL THE WAY FROM ANIMALS. THERE ARE TWO ALG 10 AND ALG 10B AND ALMOST 96% IDENTICAL AND WHEN YOU LOOK AT THE NUMBER OF COPIES OF ALG 10 AND ALG 10 LIKE GENES SPECIFICALLY IN PRIMATES THERE IS A DOUBLING OF THE NUMBER TIMING CORRESPONDS TO AROUND WHEN THIS TELLS US IS THAT IN HUMANS BECAUSE THERE ARE TWO VERY SIMILAR ALG 10 AND ALG 10B GENES HIGHLY IDENTICAL IN THEIR SEQUENCE THIS IS PROBABLY THE REASON WHY WE DO NOT HAVE AN ALG 10CBG OR BCBG. TO UNDERSTAND THE FUNCTION IN HUMANS WE NEED ANYONE WHO LOST BOTH COPIES OF ALG 10 AND ALG 10B AND SHOWED IT IN AN EPILEPSY COHORT AND WAS DIAGNOSED WITH EPILEPSY AND THIS IS THE ONLY PERSON WE KNOW OF WITH THIS GENOTYPE. SHE HAS A MUTATION IN ALG 10 AND IN THE OTHER AS SHOWN HERE USING OUR FRUIT FLY MODELS WE CAN OVEREXPRESS AND RESCUE THE SEIZU SEIZURE PHENOTYPES. WE'RE VERIFYING THERE'S A LOSS OF FUNCTION. OUR FLIES HAVE PREDICTED THIS PERSON MIGHT HAVE DISRUPTED SLEEP. SO WHEN WE PERFORMED THE ASSESSMENTS AND UNLIKE A HEALTHY PERSON WHO DURING REM SLEEP UNDER GOES -- BECOMES IMMOBILE THIS PERSON'S MUSCLES AS SHOWN ON THE ELECTRODE HER MUSCLES DISPLAY UNCONTROLLED MOVEMENT SO SHE DOES HAVE A SLEEP DISORDER AND WITH HAS A LOT OF ALG 10 AS WELL AS THE FACT THE PATHWAY IS LINEAR AND WONDERED WHAT WOULD BE THE EFFECT OF KNOCKING DOWN OTHER COMPONENTS OF THE PATHWAY AND TO SUMMARIZE A LOT OF OUR WORK WE NOTICED WHEN WE KNOCK OUT ENZYME ARE EARLY IN THE PATHWAY WE SEE THE STRONGER PHENOTYPE WHICH IS SEIZURES WHEN WE KNOCKOUT ENZYMES LATER IN THE PATHWAY WE SEE FRAGMENTS SLEEP. TO UNCOVER WHAT THE PROTEIN TARGETS MIGHT BE OF ALG 10 THAT UNDER LIE THESE NEUROLOGICAL TRAITS WE COLLABORATED WITH THE LAB OF DR. WU AND USED OUR FRUIT FLIES WHERE WE EITHER HAD CONTROL FLIES OR TWO STRAINS WITH ALG 10 KNOCK DOWN AND USED GLYCOPEPTIDE ENRICHMENT AND DID GLYCOPROTEOMICS AND WE ANALYZED THE DATA AT THE LEVEL OF THE GLY GLYCOPROTEIN AS WELL AS THE GLYCOSITE AND LOOKED AT PROTEIN WHICH IS ARE DOWN REGULATED AND GLYCOSYLATED AND SOME ARE PROTEINS WE RECOGNIZED AS BEING IMPORTANT FOR SLEEP SUCH AS A RECEPTOR AND CHINASE RECEPTORS AND FIRSTLY WE NOTICED THERE WASN'T A MAJOR CHANGE IN THE PROTEOME BUT WHEN WE LOOK AT PROTEINS THAT CAN BE DETECTED AT THE PROTEOME THERE'S A POSITIVE SUCH THAT IF WE HAVE LOWER GLYCOSYLATION WE HAVE LOWER AMOUNTS OF THE PROTEIN. THIS IS USEFUL AS GENETICISTS BECAUSE WE CAN RECAPITULATE THIS AND KNOCK DOWN THE PROTEIN AND ASK IF IT'S IMPORTANT FOR THE PHENOTYPE WE'RE STUDYING. WE DID THAT AND THE FIRST THING WE UNCOVERED WAS A PROTEIN UNTIL THEN WAS A PROTEIN OF UNKNOWN FUNCTION SIMPLY CALLED CG2915 WHICH IS A PROTEASE AND NOTICED WHEN WE KNOCK IT DOWN WE GET THE SAME SEIZURE PHENOTYPES IN THE FRUIT FLIES. AN ADDITION WE HAD A HOST OF OTHER ENZYMES WHICH WE PREDICTED TO BE ALG 10 TARGETS TO BE FUNCTIONALLY IMPORTANT FOR SLEEP. SO TO SUMMARIZE OUR WORK WE THINK OF GLYCOSYLATE AS HEALTHY AND WHAT WE COVERED BY STARTING WITH COMMON VARIANTS PRESENT IN THE HEALTHY POPULATION IS THERE MAY BE INTERMEDIATE SUCH THAT WHEN WE HAVE A MORE SEVERE DISRUPTION OF THE PATHWAY WE END UP WITH SEVERE TRAITS SUCH AS EPILEPSY AND CONGENITAL DISORDER OF GLYCOSYLATION. BUT AS WE SAW WITH A MINOR DISRUPTION IN THE VARIANTS WE ENDED UP WITH SLEEP ABNORMALITIES WHICH MECHANISTICALLY MAY MEAN THE SAME PROTEIN BEING GLYCOSYLATED TO A LESSER EXTENT OR THE NUMBER OF PROTEINS GLYCOSYLATED. WITH THAT I HAVE IN THE INTEREST OF TIME I WON'T GO THROUGH THE ACKNOWLEDGEMENTS. THIS WORK HAS BEEN A VERY BIG COLLABORATION AND THANKF FUFUL NIH AND GLYCOSCIENCE PROGRAM AND IF YOU HAVE QUESTIONS YOU CAN E-MAIL THEM TO ME. >> THANK YOU VERY MUCH. WE'RE RUNNING BEHIND AND NEED ONE MORE TALK IN BEFORE WE BREAK OUT. NEXT TALK IS ANOTHER FROM OUR SUPPLEMENT AWARDEES FROM THE LAB OF DR. JOHN BLENIS AND BELIEVE DR. SCHAFFER WILL PRESENT. >> I'M A POST-DOC AT CORNELL AND WOULD LIKE TO THANK THE ORGANIZERS FOR GIVING THE ME OPPORTUNITY TO DISCUSS MY WORK ON DISCUSSING THE N ACETYL D GLUCOSAMI GLUCOSAMINE. WITH THE DOWN STREAM DERIVATIVES VIA THE SYNTHETIC PATHWAY THE SUGARS CAN BE USED BY THE CELL THROUGH THE PHOSPHORYLATION OF SPECIALIZED KINASES AND NAGK PHOSPHORYLATES ALLOWING THE RE-UTILIZATION AND DESPITE THE IMPORTANCE OF THE PATHWAY AND MODELLING IN CANCER AND CANCER PROGRESSION LITTLE IS KNOWN ABOUT THE ROLE OF THE SALVAGED PATHWAYS TO THE PROCESSES. AS AN INITIAL ATTEMPT TO UNDERSTAND WHEN AND WHERE NAGK AND GULK ARE ARE RELEVANT A KNOCKED DOWN THE KINASES AND FOUND KNOCK DOWN OF NAGK AND LESS GUL 2 SHOWS THE CELL LINES AND HERE'S THE BREAST EPITHELIAL CELL AND THE TRIPLE NEGATIVE BREAST CANCER CELL. WE ALSO OBSERVED BUT I'M NOT GOING TO DISCUSS THE LOSS OF NAGK CAN HAVE EFFECTS OF THE ENT PROCESS AND PHENOTYPES ASSOCIATED WITH CANCER PROGRESSION. STRIKINGLY WE DIDN'T OBSERVE CONSISTENT EFFECT WHEN NAGK WAS KNOCKED DOWN ON ALL CELL LINES. SHOWN ON THE RIGHT ARE TWO CELL LINES WHERE WE SEE VERY LITTLE TO NO AFFECT ON PROLIFERATION AND I OBSERVED THE SAME THING WITH A KNOCKOUT OF THE NAGK AND A GRADUATE STUDENT HAS ALSO BEEN WORKING ON NAGK AND WITH CRISPR KNOCKOUTS SHE OBSERVED SIMILAR PHENOTYPES AND NON-PHENOTYPES IN PROLIFERATION IN THE CELLS BUT WHEN SHE KNOCKED OUT NAGK IN THE CELLS AND GREW THEM AS UNIGRAPHS THERE WAS A REMARK REDUCTION IN THE ABILITY OF THE CELLS TO GROW SUGGESTING THE ROLFE NAGK MAY BE MORE PRONOUNCED IN VIVO THAN IN 2-D CULTURE. SO TO BEGIN TO GET AN IDEA OF WHY LOSS OF NAGK MAY BE AFFECTING THE PROLIFERATIVE CAPACITY OF THE CELLS, MY INITIAL HYPOTHESIS WAS THE ULTIMATE LEVELS WOULD BEING COMPROMISED WHEN THE SALVAGED PORTION WAS GONE BUT IN METABOLOMICS ANALYSIS IN COLLABORATION I DIDN'T OBSERVE ANY DEFECTS IN THE ULTIMATE LEVELS WHEN NAGK WAS KNOCKED DOWN AND MADE A SIMILAR OBSERVATION FOR THE PATHWAY AS WELL. HOWEVER, WHEN I LOOKED AT OTHER ASPECTS OF THIS METABOLOMICS DATA I FOUND WHILE KNOCK DOWN OF NAGK INCREASED THE AMOUNT OF GLUCOSE IN THE CELLS WHETHER INCREASING THE INCREASING THE GLUCOSE UPTAKE OR ONCE TAKEN UP WHEN I LOOKED AT THE BRANCH POINT THERE'S A REDUCTION IN THE LEVELS I OBSERVED SUGGESTING WHEN THE CELL HAS LOST THE ABILITY TO SELL VAG IT IS SHUNTING RESOURCES AWAY FROM GLYCOLYSI GLYCOLYSIS. LYTIC AND THERE'S A REDUCTION AS A RESULT OF KNOCK DOWN OF NAGK. THE CELL IS WILLING TO COMPROMISE FOR THE SAKE OF INCREASING THE AMOUNT OF EDP AND THE GRADUATE STUDENT FOUND A TYPE OF GLUCOSE WAS ADMINISTERED TO CELLS WITH AND WITHOUT CRISPR KNOCKOUT OF NAGK THERE'S AN INCREASE IN THE AMOUNT OF GLUCOSE INCORPORATED IN THE SUGGESTING DE NOVO SYNTHESIS AND SUGGESTING IT'S NOT AFFECTING CERTAIN ASPECTS. SIMILAR TO OUR FINDINGS SHE FOUND THE OVERALL LEVELS OF GLCNAC BUT AND THERE'S AN AMOUNT THE CELLS ARE ABLE TO GENERATE. AND WE THINK IT REPRESENTS A STRESSED INDUCED PHOSPHORYLATION EVENT AS PROTEIN AS THE PHOSPHORYLATION IS KNOWN TO RETARD THEM AND WHEN I REMOVED ONE OF THE INHIBITORS I CAN REMOVE OUR ABILITY TO OBSERVE THIS MOBILITY SHIFT. SO THIS INITIAL OBSERVATION HAS BEEN PUBLISHED AS PART OF A COLLABORATION WITH SIDNEY ON THE PAPER AND DOES A GREAT JOB OF DESCRIBING THE FLEXIBILITY IN THE PATHWAY IN DE NOVO VERSUS SALVAGE USE FOR THE GENERATION OF GLCNAC AND WE'RE CONTINUING TO WORK WITH THE LAB ON DESCRIBING THE FUNCTION OF THE PHOSPHORYLATION FURTHER. IN ADDITION OF LOOKING AT THE MOBILITY SHIFT IN RESPONSE TO LOW GLUTAMINE IT ALSO OCCURS IN RESPONSE TO LOW GLUCOSE AND MULTIPLE CELL LINES AND A RESPONSE TO HYPOXIA SUGGESTING IT MAY BE A GENERAL STRESS RESPONSE THE CELL IS UNDERGOING. SO JUST TO BRIEFLY SUMMARIZE WHAT I'VE BEEN ABLE TO TELL YOU WE FOUND THE CELL OF NAGK INHIBITS PROLIFERATION IN SOME BUT MINIMAL AFFECTS ON GLCNAC WHEN THE CELLS ARE GROWN UNDER NORMAL GROWTH CONDITIONS BUT THE LEVEL OF GLYCOLYTIC REDUCE THE AMOUNT AND IT'S LIKELY PHOSPHORYLATED IN RESPONSE TO STRESSES. IN THE FUTURE SOME QUESTIONS WE'RE HOPING TO ADDRESS ARE WHAT ASPECTS NAGK LOSS ARE DRIVING THE INCREASE IN PROLIFERATION AND TUMOR GROWTH. ONE HYPOTHESIS IS THAT THE AND THERE IS IN THE TUMOR UNDER WHAT WE OBSERVE UNDER LOW CONDITIONS THAT INCREASES THE NEED FOR GLCNAC SALVAGED AND THE OTHER HYPOTHESIS IS WITHOUT NAGK THE CELL HAS TO SHUNT RESOURCES FROM BIO ENERGETICS TO GLCNAC DE NOVO AND ALSO POSSIBLE AND WE HAVE SOME EVIDENCE THAT NAGK IS PARTICIPATING IN NON-CANONICAL FUNCTIONS. THERE'S SOME LITERATURE THAT SUGGESTS NAGK PLAYS INDEPENDENT ROLES IN SCAFFOLDING. NOW WE'RE TRYING TO WORK OUT WHAT ARE THE RELATIVE CONTRIBUTIONS OF NAGK AND WHY IT'S RESULT INNING SUCH A PRONOUNCED EFFECT ON PROLIFERATION IN TUMOR GROWTH. THE REASON WE'RE A PART OF THE COMMON SCIENCE PROGRAM IS TO USE ISO TAG IN COLLABORATION TO UNDERSTAND THE CONSEQUENCES OF LOSS OF NAGK ON GLYCAN SYNTHESIS AND STRUCTURE. AS WE WORK ON NAGK MORE WE REALIZE THE ROLE IS NOT ADDS STRAIGHTFORWARD IN THE CELL AS WE INITIALLY THOUGHT AND SO WE'RE WORKING TO DEFINE THE BEST CIRCUMSTANCES UNDER WHICH TO LOOK AT IT'S EFFECTS ON GLYCAN IN THE STRUCTURE AND FINALLY WE'RE VERY INTERESTED IN HOW THE PHOSPHORYLATION EVENT MAY CHANGE THE SALVAGE CAPACITY AND HOW IT COULD BE REGULATED AND WHAT ARE THE CONSEQUENCES ON TUMOR GROWTH. AND WITH THAT I WOULD LIKE TO THANK OUR LABORATORY. WE'RE A SIGNALLING LABORATORY AND NOT A GLYCOLABORATORY THOUGH IT'S BEEN FUN FOR ME AS WELL AS MANY COLLABORATORS AND WE HAVE THE COMMON FOUND GLYCOSCIENCE PROGRAM AND MY POST-DOCTORAL TRAINING FUNDING AND WITH THAT I'LL TAKE ANY QUESTIONS. >> THANK YOU, DR. SCHAFFER. SO YOU'RE THE LAST TALK BEFORE LUNCH. WE'LL SEE IF ANYBODY HAS ANY QUESTIONS FOR YOU. >> LAURA HAS ONE IN THE CHAT. >> DOES PHOSPHORYLATION CHANGE CELL TURNOVER OR ACTIVITY? >> SO WE DON'T KNOW THE ANSWER TO THAT YET. WE DO THINK IT CHANGE THE ACTIVITY GIVEN WE CAN FIT WHEN THE CELLS ARE UNDER THE NUTRIENT LIMITED CONDITIONS WHERE WE OBSERVE THE MOBILITY SHIFT. WE DO EXPECT THAT THAT IS PROBABLY GOING TO ENHANCE THE SALVAGE CAPACITY OF NAGK. AND WE'RE GOING TO WORK WITH THE WILEAN LAB AND WE HAVE SAMPLES FROM MASS SPECT NOW AND. WE ISN'T YET LOOKED AT LOCALIZATION OR TURNOVER. IN GENERAL WHEN THE CELLS UNDER GO ENERGY STRESS THERE'S A LITTLE BIT LESS NAGK SPECIFIC TO NAGK BECAUSE THEY DON'T HAVE THE RESOURCES TO SYNTHESIZE NEW PROTEINS BUT THOSE ARE STILL OPEN QUESTIONS. >> I THINK LOCALIZATION WOULD BE ESPECIALLY COOL TO LOOK AT ESPECIALLY IF YOU THINK IT HAS OTHER SCAFFOLDING FUNCTIONS YOU MAY BE ABLE TO DETECT THAT. >> THAT'S A VERY GOOD POINT. THANK YOU. >> OKAY. THANK YOU VERY MUCH. PAM OR AMANDA DO EITHER ONE OF YOU WANT TO TAKE OVER FROM HERE FOR THE LAST FEW ANNOUNCEMENTS BEFORE BREAK. >> YEAH, THIS ISN'T REALLY A BREAK. IT'S A WORKING LUNCH. WE'LL GIVE YOU A COUPLE MINUTES TO REFRESH YOUR COFFEE AND GRAB YOUR SANDWICH AND WE'RE INVITING FOLKS HERE WHO ARE IN COMPANIES THAT ARE INTERESTED IN GLYCOSCIENCE TO JOIN US AROUND THE TABLE AND LET'S START THIS AT 1:45. GIVE EVERYBODY A CHANCE TO STRETCH THEIR LEGS, GRAB A CUP OF COFFEE AND COME BACK AND SIT DOWN AND OUR COMPANIES ARE GOING TO INTRODUCE THEMSELVES AND TELL US ABOUT WHAT GLYCOTOOLS AND REAGENTS AND WHAT'S COMING UP AND INTERESTED IN MAKING OR COLLABORATING ON AND THEN WE'LL ASK THEM ABOUT WHAT ADDITIONAL TOOLS AND RE-AGENTS THEY SEE AS BEING IMPORTANT TO COME TO MARKET. THIS WILL BE A CHANCE FOR YOU TO CHAT WITH THEM ABOUT YOUR NEEDS AND FOR THEM TO GIVE YOU SOME FEEDBACK. SO 12:45 AND WE'LL START WITH DR. MORGUE JAB -- MORGAN WHEN WE GET BACK. -- STABILITY OF THE RECOMBINANT PRODUCT AND FOR AN APPLICATION YOU PREFER TO NOT HAVE IT AS A GFP FUSION. WE ARE ON THE WEB AS GLYCOEXPRESSTECH.com. YOU CAN GET ACCESS TO ANY CONTACT INFORMATION ABOUT THE COMPANY THERE AND WE'RE IN THE PROCESS OF LEVERAGING THOSE ENZYME FOR A VARIETY OF SECOND-GENERATION PRODUCTS IN CHEMO ENZYMATIC SYNTHESIS OF GLYCAN STRUCTURES AND HOPEFULLY HOLE TO WILL COME ALONG IN THE FUTURE. WE'RE EXPANDING THE CATALOG SIGNIFICANTLY OVER THE NEXT COUPLE OF MONTHS BECAUSE WE WERE RE-ENGINEERING EVERYTHING BUT HAVE AN EXPANDED CATALOG OF ENZYMES WE'LL BE ABLE TO PRODUCE AND PROVIDE TO THE COMMUNITY AT LARGE. >> THANK YOU. MIKE, WOULD YOU LIKE TO INTRODUCE YOURSELF AND YOUR COMPANY? >> THANK YOU FOR INVITING US. WE STARTED IN 2002 AND WE'RE THE DISTRIBUTOR FOR LIBNER FROM THE U.K. SO WE HAVE A LARGE AMOUNT OF GLYCAN STANDARDS. THE LABELLING KITS AND GLYCOPROTEIN STANDARDS AND I THINK OUR FOCUS HAS REALLY CHANGED IN THE LAST FIVE YEARS WHERE THE INTERNATIONAL ASPECT HAS BEEN THE BIGGEST CHANGE WE'VE SEEN. WE'RE PROBABLY DOING ONE-THIRD PERHAPS MORE OF THE BUSINESS NOW OVERSEAS BOTH IN ASIA AND EUROPE. THAT'S BEEN ONE OF THE BIG CHANGES WE'VE SEEN. I'M NOT SURE IF THAT'S SOMETHING JUST WITH US PEOPLE FINDING US OR THAT PART OF THE MARKET HAS GROWN GREATLY. I SUSPECT THAT. >> DO YOU HAVE AN E-MAIL OR WEBSITE? >> YES. QA-BIO.com AND E-MAIL IS MGIBSON G-I-B-S-O-N LIKE THE GUITAR AT QA.BIO.com. >> THANK YOU AND WE'LL ASK QUESTIONS BUT WANT TO GET THROUGH THE INTRODUCTIONS. MOHAMED, DO YOU WANT TO INTRODUCE YOUR COMPANY? YOU'RE STILL MUTED. WE CAN'T HEAR YOU. WE'LL GO TO THE NEXT PERSON WHILE YOU WORK THAT OUT. ALEX WOULD I LIKE TO INTRODUCE YOUR COMPANY? >> CAN YOU HEAR ME OKAY? >> YES. >> I'M A PRODUCT SPECIALIST WE'RE A U.K. BASED COMPANY SO IT'S MORE OF A WORKING DINNER FOR ME. WE HAVE AROUND 30 YEARS OF EXPERIENCE IN PROVIDING SMALL MOLECULES TO RESEARCHERS. THERE'S ABOUT 5,000 SMALL MOLECULES AS A BROAD RANGE OF RESEARCH AREAS IN THE PORTFOLIO. WE HAVE A WEB ESTABLISHED FRAMEWORK TO DISSEMINATE THESE TOOLS IN A QUICK AND EFFICIENT MANNER. WE'RE PART OF A LARGER CORPORATE ENTITY. THIS ENCOMPASSES BRANDS SUCH AS R&D SYSTEMS, AND GLUTAMATE AND CROSSING MANY AREAS OF SCIENCE. I AM NO EXPERT IN GLYCOSITES MAKING ME STAND OUT TO EVERYONE AND OUR INTEREST IS RELATIVELY NEW AND IN PART HAS BEEN SPURRED BY THE PUBLICATIONS BY THE GLYCOSCIENCE COMMON FUND PROGRAM. WE HAVE LIVE CELLS AND THANK YOU FOR THE INVITE AND I LOOK FORWARD TO LEARNING MORE IN THE PROGRAM AND ASSIST TO COMMERCIALIZE SOME OF THESE TOOLS. THERE SEEMS TO BE A REPEATING THEME I'VE HEARD CATHERINE DOESN'T WANT HARD WORK TO GO TO THE FREEZER AND I'LL USE THE EXPRESSION OF PUSHING THE DOOR ON THE TOOLS AND THE MODEL OF COMMERCIALIZING IS THE WORK WITH THE INVENTERS AND WE CAN ALSO GIVE BACK TO THEM AND THERE'S LARGER CORPORATE ENTITY WE CAN HARNESS THE NORM HAVE WEBINARS AND SYMPOSIUM AND GIVING SPEAKERS A CHANCE TO PRESENT RESEARCH TOOLS AND I DON'T WANT IT TO SEEM LIKE A SHAMELESS PLUG BUT RELEVANT TO THE MEETING NEXT WEEK WE WILL PRESENT OUR WORK ON THE DISCOVERY OF CHIMERAS AND I THOUGHT I'D MENTION THAT. MAYBE ON MY INTRO TO MENTION THE PROCESS OF COMMERCIALIZATION. IF INVENTORS CAME TO SEE THE TOOLS MADE COMMERCIALLY AVAILABLE WE CAN STILL GIVE FULL ACKNOWLEDGEMENT NOT JUST TO THE PAPERWORK BUT TO THE GROUP AND IT'S OF BENEFIT AND MIGHT BE USING IN HIGHLIGHTING COMMERCIAL VALUE. I'LL CONCLUDE THERE AND MY DETAILS ARE ALEX MALONEY AT TOCRIS.com. >> WONDERFUL, THANK YOU. NEXT, ANDREW, DO YOU WANT TO INTRODUCE YOURSELF? >> SURE. THANK YOU FOR THE INVITE. MY NAME IS ANDREW LEE. ONE OF THE CO-FOUNDERS FOR IMCS STANDS FOR A LONG CEINTEGRATED PRODUCT AND WE HAVE EXPRESSIONS AND THE OTHER IS THE MICROCHROMATE OGRAPHY AND RIGHT NOW WE ARE REACHING OUT TO COLLABORATORS OR OTHERS WHO MAY BE INTERESTED IN THAT TECHNOLOGY. SO WE'RE GOING THROUGH THE ENZYMES AND EXPRESSING THAT IN LARGER SCALE. I BELIEVE HER EXPRESSIONS WERE A RANGE OF 20 MILLIGRAMS TO MAYBE 100 MILLIGRAMS PER LITER. WE SCALE THOSE UP 200 FOLD GRAMS PER LITER PRODUCTIONS AND WE ARE A CERTIFIED COMPANY. WE OVERSEE ABOUT 45 PEOPLE AND WE'D LIKE TO HEAR FROM PEOPLE INTERESTED IN THE QUITS TO TEST THE ENZYME BEING PUSHED OUT AS PART OF THE CONSORTIUM FOR THE GROUP THE WEBSITE IS WWW.IMCS.TMCS.COM AND MY E-MAIL ADDRESS AND CONTACT INFORMATION IS ON THE WEBSITE AS WELL. I LOOK FORWARD TO HEARING FROM YOU. THE ENZYMES ARE BETA TESTING AND FREE OF CHARGE. >> I THINK EVERYBODY LOVE FREE OF CHARGE. >> I ASSUME SO. >> ERICA, WOULD YOU LIKE TO TELL US ABOUT YOURSELF AND VECTOR. >> LEAVE YOUR VIDEOS ON. >> SO MY NAME IS ERA LEONARD AND A WORK FOR VECTOR LABORATORIES IN THE VECTOR AND QC DEPARTMENTS. WE WERE AROUND SINCE 1976 AND THE COMPANY WAS STARTED MAKING LECTINS AND WE HAVE A VERY BROAD PORTFOLIO OF PURIFIED LECTINS AS WELL AS LECTIN CONJUGATES AND OPTIMIZED NOT ONLY THE MANUFACTURING PROCESS FOR THOSE LECTINS BUT THE CONSISTENCY LOT TO LOT TO RELIABLY PURCHASE THEM FROM VECTOR AND KNOW YOU'LL HAVE A PRODUCT THAT GIVES CONSISTENT RESULTS. WE GIVE THEM IN SEVERAL CONJUGATES WITH BIOTIN AND DIFFERENT TAGS AND THE GREAT THINK ABOUT VECTOR IS BEING A SMALL COMPANY WE'RE ABLE TO PIVOT AND CAN WORK WITH RESEARCHERS TO HELP YOU DEVELOP TOOLS THAT YOU NEED IN YOUR LABORATORY AND YOU CAN REACH OUT TO OUR TECH SUPPORT TO HELP DEVELOP ANY ASSAYS OR RE-AGENTS YOU NEED AND CAN WORK WITH YOU. WE'RE NOW LOOKING AT THE NEW TECHNOLOGIES AND GS IS HOT IN THE MARKETPLACE AND LOOKING TO SEE HOW WE CAN UTILIZE OUR TOOL TO EMPOWER THAT AREA OF RESEARCH. ALSO WE'RE LOOKING INTO RECOMBINANT PRODUCTS TO IMPROVE THE AFFINITIES FOR THE GLYCAN TARGETS BUT HOW THEY COMPARE WITH NATURAL LECTINS. REACH ME AT E LEONARD L-E-O-N-A-R-D AT VECTOR LABS.com AND REACH OUT TO OUR TECH SUPPORT IF YOU HAVE QUESTION OUR LECTIN PRODUCT. WE'VE BEEN MANUFACTURING THEM OVER 45 YEARS AND MASTERED THAT MANUFACTURING PROCESS AS WELL AS THE QC. I WILL LET LANCE SPEAK TO THE PRODUCT MANAGEMENT AREA OF OUR PORTFOLIO. >> THANKS, ERICA. ASKER -- AS ERICA SAID I'M PART VECTOR LABORATORIES AND INTERESTED IN THE LECTINS AND ALL PLANT BASED OVER 40 YEARS AND ALTHOUGH PROBABLY PEOPLE RECOGNIZE VECTOR MOST FOR IMMUNOHISIMMUNO HISTOFLUORESCENCE AND WE'VE BEEN AROUND AND A ROLE IS TO LOOK HOLISTICALLY AT HOW WE SORT OF ENGENDER MORE OF THIS THE PEOPLE AND MORE COMMON AREAS OF ONCOLOGY AND AUTOIMMUNE DISEASE AND THOSE TYPES OF MAINSTREAM SCIENTIFIC RESEARCH ENDEAVORS AND PULL THEM IN THE GLYCOBIOLOGY SPACE. AS EVERYONE HERE IS KEENLY AWARE IT'S VERY RICH IN TERMS OF OPPORTUNITIES TO FURTHER RESEARCH. HEARING OF ALL THE TECHNOLOGIES PEOPLE ARE USING NOW TO ADVANCE THEIR RESEARCH ARE ALL TOOLS THAT ARE INTERESTING AND POTENTIALLY WE CAN PULL THOSE IN THE INNOVATION SPACE. WE'RE EVEN LOOKING MORE AT THE ENGENDERING MORE PEOPLE IN THE MAINSTREAM RESEARCH AREAS AS SOME OF THE PEOPLE ALLUDED TO THEY DON'T THINK OF THEMSELVES AS GLYCOBIOLOGISTS AND THEY'RE KEEN IN PULLING IN SO THERE'LL BE GREATER PARTICIPATION AND MORE PEOPLE LIKE YOURSELVES WILL BEIZING THE TOOLS YOU'RE USING. THAT'S OF PARTICULAR INTEREST. THANK YOU FOR THE CHANCE TO SPEAK. >> WONDERFUL. >> LORI, WOULD YOU LIKE TO INTRODUCE YOURSELF? >> SURE. THANK YOU FOR INVITING US ALL TO THE ROUND TABLE. I'M LORI YANG CO-FOUNDER AND CEO OF LECTENZ BIO AND COMMERCIALIZING NOVEL RE-AGENTS AND TOOLS AND FOR GLYCO-SANSS -- GLYCO-SANS AND IT'S ENGINEERED WITH SPECIFICITY FOR A CARBOHYDRATE SUBSTRATE AND USING THE ENZYME IT'S ACTIVATED AND INFINITY FOR THE SUBSTRATE. THE TOOL IS A MULTI-PLEX FEED BASED ASSAY FOR FLOW CYTOMETRY THAT GENERATES A SIMPLIFIED GLYCOPROFILE. IT'S ESSENTIALLY AN ELECTROMICRORAY AND CATHERINE NICELY INTRODUCED THE TOOL I'LL SPEAK MORE ABOUT LATER ON TODAY. AND WE HAVE PRODUCTS SUCH AS GLYCOENZYMES. SO IN TERMS OF THE GLYCOMARKET I CAN SPEAK FOR OUR COMPANY. CURRENTLY WE HAVE LECTIN ARE SPECIFIC FOR AN ACID AND HAVE NEW REAGENTS COMING SOON AN ALPHA 2 SPECIFIC GLYCAN AND PROTEASE AND AS FAR THE NEEDS IN THE GLYCOMARKET, PEOPLE HAVE ASKED HAVE WE DEVELOPING OTHER LECTINS AND INDEED WE ARE. CHECK OUT OUR WEBSITE AT WWW.LECTINS.com OR CONTACT ME BY E-MAIL AT L-Y-A-N-G AT LECTENZ.com. >> THANK YOU. DAVE SMITH. >> I'M DAVID SMITH. I'M WITH NAT GLYCAN. WE STARTED UP SEVERAL YEARS AGO. SPIR SUPPORT AND WE APPRECIATE THAT AS THAT'S THE ONLY THING KEEPING US GOING RIGHT NOW. WE HAVE BEEN FOCUSSING ON USING THE VERY SIMPLE PROCESS THAT WAS DEVELOPED AT EMORY AND SIMPLE BLEACH AND WE TREAT NATURAL PRODUCTS WITH BLEACH AND GENERATE FROM THAT FREE GLYCANS. WE'LL, THERE'S O GLYCANS OR GLYCOLIPID DERIVED GLYC JAB. WE CAN PURIFY THEM THROUGH LARGE-SCALE PURIFICATION. FROM THESE WE GENERATE LARGE QUANTITIES OF NATURAL GLYCANS AND HOW WE NAMED OURSELVES NAT GLYCAN AT N-A-T-G-L-Y-C-A-N.com AND WE ARE ABLE TO PROVIDE MILLIGRAM QUANTITIES OF MEN 9 THROUGH MEN 5 GLYCAN. THESE ARE FLUORESCENT DERIVATIVE AND CAN PROVIDE THEM AS FREE GLYCANS AND THE PURITY OF THEM BECAUSE WE OBTAIN IN LARGE QUANTITIES ARE IN THE 90% TO 95% RANGE. WE'RE BEGINNING TO LOOK AT INTERESTING BYPRODUCTS AS WE LEARN TO CONTROL THE REACTION. ONE OF THIS MOST INTERESTING PRODUCTS IS THE MONOGLUCONATE GLYCANS NOW AVAILABLE IN SUBSTRATE QUANTITIES FOR USING THE REVERSE SYNTHESIS WITH THE ENDO GLYCOSIDASES AND SELLING THOSE FOR PEOPLE MAKING GLYCOPEPTIDE PROTEIN. WE'VE MOVING ON TO O GLYCANS. HAVEN'T DONE MUCH WITH GLYCOLIPID DERIVED GLYCANS AND WE'VE ALSO DEVELOPED A HUMAN MILK GLYCOSACCHARIDE PROTEIN AND IT'S BEEN VERY SUCCESSFUL IN THE PAST. WE'RE ALSO MOVING TOWARDS PROVIDING THESE MATERIALS. THOSE INTERESTED IN TRYING TO DEVELOP LARGE SCALE QUANTITIES OF GLYCANS LET US KNOW AND WE'RE WORK ON THE COMPLEX TYPE GLYCANS AND THE FOCUS WILL BE ON THOSE THAT ARE FOUND ON HUMAN IGG. SO IF YOU HAVE INTEREST IN THOSE COMPOUNDS, PLEASE LET US KNOW AND WE'D LOVE TO WORK WITH YOU ANY WAY WE CAN. >> THANK YOU. MOHAMED, WERE YOU ABLE TO GET YOUR VIDEO? >> HELLO >> THERE YOU ARE. >> I CAME BACK AND FORTH. >> THANK YOU. OUR COMPANY IS ALMOST A DECADE AND ALWAYS BEEN FUNDED WITH THE SPIR GRANTS AND WE ARE VERY FORTUNATE AND THANKFUL FOR THAT. I'M A CHEMIST AND IT'S FOCUSSED MOSTLY ON SUGARS AND I OVERSEE THE CHEMICAL SIDE AND WE ALSO HAVE OTHER FELLOWS WHO DO THE ENZYMATIC PART AND WE'RE TAKING CONCENTRATION OF OUR PORTFOLIO WE HAVE A VERY DIVERSE PORTFOLIO. WE DO OUR PRODUCT WHICH WE COLLABORATED WITH AND PRODUCED LIKE 300 GLYCAN AREA. IT'S PART OF THE IMPORTANT GLYCANS ALREADY THERE AND APART FROM THAT WE CAN ALSO STUDY THE AREA FOR CUSTOM SYNTHESIS AND RECENTLY IT'S FOCUSSED ON THE LARGE SCALE SYNTHESIS AND WE MAKE SCALES OF THE NUCLEO TIDES AND 100 MILLIGRAM SCALES OF THE METABOLOMICS PROBES OF THE BIOTINS AND CAN MAKE MANY OF THOSE AREAS AND ALL OF THEM ARE AVAILABLE ON THE GLYCOSANS WEBSITE AND REACH OUT TO US OR REACH OUT TO ME PERSONALLY. APART FROM THE PRODUCTS LISTED ON THE WEBSITE WE CAN SYNTHESIS THE OLIGOSACCHARIDES TO THE GRAM SCALE SO REACH OUT TO US IF YOU HAVE NEED FOR THE GLYCAN RNA OR THE SUGAR NUCLEOTIDES OR THE OLIGOSACCHARIDE MIX. THANK YOU. >> SO WE HAVE A BROAD SPECTRUM OF RE-AGENTS AND TOOLS BEING PROVIDED. WHAT ABOUT THE MARKET? WE ALWAYS HEAR THE GLYCOMARKET IS RELATIVELY SMALL AND HERE YOU'RE TALKING TO PEOPLE THAT ARE TOOL MAKERS AND ACADEMICS BUT IS THE MARKET OPENING UP? ARE COMPANIES SEEKING YOUR BUSINESS? PHARMA SEEK YOUR BUSINESS? WHERE DO YOU SEE THE MARKET -- WHAT I'M SAYING IS WHERE DO YOU SEE THE MARKET IN GLYCOSCIENCE AS GROWING? MICHAEL I'LL START WITH YOU. YOU'RE ON MUTE. >> OUR FOCUS HAS BEEN ON GLYCOAND WE NEEDED GOOD PRODUCTS AND IF YOU GET IN ONE COMPANY IT'S THE SAME PERSON BUYING OVER AND OVER FOR BIO MANUFACTURING. WHEN YOU GET IN THE RESEARCH WORLD THE DIFFICULTY IS -- I CAME FROM THE DNA SEQUENCE AND PROTEIN SEQUENCE IING AND THEY HAD SO MANY MORE CUSTOMERS PER UNIVERSITY OR BUSINESS UNIT THE MARKET WAS LARGER. THE MARKET IS SMALLER HERE BUT IF YOU CAN FIND PRODUCT ARE NEEDED OVER AND OVER THAT MAKES UP FOR IT. THAT'S BEEN OUR FOCUS AND KEPT US IN THE BUSINESS. IT IS REALITY. WHERE DO I SEE GROWTH? ENGINEERING OF GLYCANS. GLYCOTRANSFERASES. IF WE CAN GET A GOOD SET THOSE WOULD BE INTERESTING BUT THEY CAN TELL IN VOLUME. IF THEY GOT IN THE MANUFACTURING COMMUNITY THAT IS DEFINITELY A BIG MARKET. AS FAR AS THE ANALYSIS TECHNIQUES, FOR OUR CUSTOMER BASE IT'S PRETTY STABILIZED WITH THE MASS SPECT AND I DON'T SEE THEM LOOKING FOR A LOT OF NEW THINGS IN THAT REALM. THEN AGAIN I'M NOT LOOKING IN THE GLYC OMICS OR HIGH THROUGHPUT TYPE OF LAB. THAT'S MY SPIN ON WHERE THE MARKET IS NOW AND WHERE I THINK IT MIGHT BE GOING. >> INTERESTING. I SAW THE FOLKS PRODUCING ENZYMES STARTING TO SMILE AND SHAKE THEIR HEADS THERE. MADE A LOT OF THEM HAPPY. WHERE DO YOU SEE IT'S GOING? >> I'M NEW TO THIS WORLD SO WOULD HAVE LITTLE INFORMATION. IT'S A CHICKEN AND THE EGG DILEMMA AND IN THE DIAGNOSTIC WORLD THEY'RE NOT CHEAP FREE AGE ENS BECAUSE THEY HAVE TO RUN MILLIONS OF ASSAYS. IF THEY'RE PAYING A DOLLAR PER MILLIGRAM OR MER MICROGRAM OF ENZYME IT'S TOO COSTLY TO RUN THE ASSAYS. OF COURSE YOU NEED MASS PRODUCTION TO GROW THOSE COSTS. I THINK WE'RE SCALING UP THE ENZYME PRODUCTION SO THE REAGENTS ARE AVAILABLE IF A TURNING POINT DOES OCCUR. IF THEY'RE USING A PARTICULAR ENZYME THERE NEEDS TO BE A COMPANY CAPABLE OF MAKING THESE IN A RELIABLE, CONSISTENT MANNER. THAT'S ONE PROGRAM THAT NEEDS TO BE SET UP AND THE GLYCANS OR GLYCOLIPIDS THAT POTENTIALLY HAVE A THERAPEUTIC APPLICATION AND WILL BE AT LARGER SCALE AND THE MODEL I SEE IS TO THE BIO LABS WITH THE RESTRICTION ENZYME ENZYMES HAVE YOU SMALL PIECES OF THE TRANSFERASES OR A VARIETY OF ENZYME ARE KIND OF THERE BUT DOESN'T NEED TO BE PRODUCED IN LARGE SCALE. THOSE ARE GOING TO BE YOUR ENZYME ENZYM ENZYMES THAT'S PRETTY LIMITED. >> ALEX, YOU'RE COMING AT THIS FROM THE CHEMICAL END? >> I'M NO EXPERT IN GLYCOSCIENCE BUT I THINK THERE'S NO ARGUING INFECTIOUS DISEASE IS GOING TO BE AN INCREASED AREA OF RESEARCH AND A WAY IT STUDY PATHOGENS IN THE SPACE IS GOING TO BE IMPORTANT. THIS IS OUR FIRST INTRODUCTION TO THE SPACE. I KNOW THE MODALITY IS THE TACKS AND FANTASTIC WORK WAS DEMONSTRATED TO HARNESS TISSUE SPECIFIC RECEPTORS FOR PROTEIN DEGRADATION AND FANTASTIC APPLICATIONS. >> IF THEY ENDED UP IN CLINICAL TRIALS WOULD THAT GIVE YOU A STEP UP IN THE MARKET? >> THERE'S BEEN A HUGE NUMBER AND SEVERAL COMPANIES NOW ARE MOVING TO CLINICAL TRIALS AND START WITH THE RESEARCH TOOLS THERE'S INCREASED NEED IN THE RESEARCH SPACE. >> ERICA OR LANCE, WHAT ABOUT VECTOR? >> I THINK WE'RE TRYING TO UNDERSTAND HOW TO BEST APPROACH OUR TOOL DEVELOPMENT BECAUSE THERE'S SUCH A VARIATION IN HOW PEOPLE ARE ANSWERING THESE QUESTIONS WHETHER IT BE IN THE IMMUNOLOGY WORLD OR IN CANCER RESEARCH THERE'S MANY GROUPS THAT DON'T KNOW THAT MUCH ABOUT GLYCOMICS AND NEED TO INVEST IN EASY TO USE AND EASY TO UNDERSTAND TOOLS SO THEY CAN BEGIN THE GLYCORESEARCH AND THEN WE ALSO NEED TO EMPOWER THOSE DEEPLY INVOLVED IN THE GLYCOMICS AND GLYCOPROTEIN RESEARCH AND AS THE BEEN OUR STRUGGLE, HOW DO YOU PRODUCE THE MOST USEFUL TOOL FOR THE COMPLEXITY THAT'S OUT THERE AND THE GLYCONAIVE ARE TRYING TO UNDERSTAND HOW IT IMPACT THE QUESTION AND EMPOWER THOSE WHO ARE REALLY DELVING DEEP INTO GLYC OMICS. >> THIS IS IMPORTANT AND WHY ONE OF THE REASONS WHY THE COMMON FUND SET UP THIS SECOND PROGRAM IN THE SPIR TTIR LINE. THERE'S A DEFINITE NEED FOR TOOLS IN A VARIETY OF TOOLS FOR THE GLYCOCOMMUNITY BUT THIS PROGRAM SPECIFICALLY WANTED TO INTEGRATE GLYCOTO THE BROADER SCIENTIFIC COMMUNITIES AND I THINK IN TERMS OF BEING ABLE TO OPEN UP THE MARKET AND GET NON-GLYCOSCIENTISTS INVOLVED IN USING SIMPLE EASY TO USE TOOLS, THIS IS REALLY IMPORTANT IF WE'RE GOING TO MOVE THE FIELD FORWARD HEARING WHAT YOU LEARN IN TERMS OF MARKET SIZE AND WHERE THINGS ARE GOING AND ONE OF THE REASONS WHY WE'RE HAVING THE ROUND TABLE NOW. DOES ANYBODY SEE A NEED? DO YOUR COMPANIES GET ASKED FOR TOOLS YOU DON'T ALREADY HAVE OR TO MAKE THINGS THAT YOU DON'T ALREADY HAVE ON THE SHELF REPEATEDLY? IS THERE NEEDS BUBBLING UP AND ARE THEY FROM GLYCOSCIENTIST OR OTHER COMPANIES OR FROM PHARMA? ANYBODY HAVE AN ANSWER? >> FOR VECTOR IT'S ALL OF THE ABOVE. WE HAVE A CUSTOM OEM SECTOR WHERE WE WORK WITH OTHER COMPANIES OR RESEARCH GROUPS TO DEVELOP A SPECIFIC REAGENT OR A PROGRAM THEY'RE WORKING ON. IT'S BEEN QUITE VARIABLE AND WHAT'S REALLY INTERESTING IS IT'S OPING UP OUR EYES TO WHERE GLYC OMICS IS HEADING AS FAR AS FOR DIAGNOSTICS OR FOR TRACKING DISEASE PROGRESSIONS OR CONTAMINATION IN WATER WE HAD NOT THOUGHT OF USING OUR PRODUCTS FOR BUT RELY ON ALL OF YOU TO COME TO US TO SAY, HEY, HAVE YOU EVER THOUGHT OF USING THIS LECTIN IN THIS MANNER OR THIS TYPE OF DETECTION? WE THAT'D EXPERTISE AND CONJUGATION IN VARIOUS LECTINS AND WE'VE BEEN ABLE TO WORK WITH RESEARCH GROUPS AND COMPANIES TO HOPEFULLY PUSH THAT RESEARCH FORWARD. >> SCOTT MILLER? >> THIS IS BRIEF. WE HAD EXPERIENCE A FEW YEARS AGO MAKING I THINK SOME NEAT CHEMISTRY. A BUNCH OF PHOSPHO LIPIDS AND WE WERE OVERWHELM ED FOR A MILLIGRAM OF THIS OR THAT AND WERE HAPPY TO COMPLY AND WE WERE MAKING HUNDREDS OF MILLIGRAMS AND PERHAPS BECAUSE OF MY OWN LIMITATIONS FOR PERSON AT OR SOMETHING I NEVER EVEN OCCURRED TO ME TO START A COMPANY. I THINK THE GENERAL PRINCIPLE OF HIGH QUALITY SPECIALIZED MANUFACTURE SMALL QUANTITY DEMAND IS SOMETHING OTHER SECTOR OR INDUSTRIES HAVE CONFRONTED. MAYBE INDUSTRIAL PANELISTS HAVE DONE IT AND WONDER IF THERE'S VALUE IN REACHING OUT TO ECONOMISTS OR MINE THE LITERATURE. IT'S NOT FIRST ORDER SUPPLY/ DEMAND ECONOMICS 101 SORT OF STUFF BUT WAY OUT OF MY EXPERTISE AND WONDERING OUT LOUD. I EXPECT OTHER SECTORS CONFRONTED SIMILAR THINGS SO MAYBE THERE'S OTHER MODELS. >> HAS ANYBODY SEEN WHITE PAPERS OR PURCHASED THE WHITE PAPERS FOR MARKET IN GLYCOSCIENCE OR GLYC OMICS? IT SEEMED LIKE THE MARKET WAS GROWING AND ENZYMES IS ONE PLACE THEY HAD A REAL INTEREST IN SEEING EXPANSION AND ALSO IN LABELLING TOOLS. IT WOULD SEEM ALL OF ARE YOU IN AREAS THAT ARE RELATIVELY HOT RIGHT NOW. HAVE YOU SEEN AN INCREASE IN YOUR MARKET WITHIN YOUR OWN COMPANIES? >> >> WHEN COVID HIT WE SAW A GOOD INCREASE. WE'VE GONE THROUGH A COUPLE WAYS. IN THE 2000s THERE WERE COMPANIES WE WERE WORKING WITH AND OCCURRING THE NEXT 10 YEARS ALL THE SMALL COMPANIES JUST DROPPED OUT. THEY WERE STILL WORKING BUT THEY WEREN'T LOOKING AT THE GLYCANS AS MUCH ANYMORE. COVID CAME IN AND ALL THOSE PEOPLE CAME BACK. WE STARTED SEEING SCIENTIST WE HADN'T HEARD FROM FROM 10 YEARS AGO AND WERE WORKING ON OTHER ASPECTS NOW THEIR BOSSES REMEMBER GLYCAN IMPORTANT SO PROJECT STARTED FUNNEL BACK IN AND THEY WEREN'T JUST COVID PROJECTS. IT'S JUST THE OVERALL UNDERSTANDING THAT GLYCAN IMPORTANT WAS BACK AND A GENERAL OVER ALL INCREASE AND LOOKS TO BE A TREND THE NEXT THREE YEARS. >> THAT'S GOOD TO KNOW. SO THE PANDEMIC WAS A GREAT DETRIMENT IN SOME WAYS BUT -- >> BITTER SWEET. YOU DON'T WANT TO CHEER FOR IT. >> BUT NEEDS WERE THERE. IT PUT THE SPOTLIGHT BACK ON GLYCANS FOR SURE. THERE WAS A COMPUTER VIDEO SOMEBODY DID THAT SHOWED THE DIFFERENT GLYCANS ON THE SPIKE PROTEIN WIGGLING AROUND AND PEOPLE SAW THAT AND WERE LIKE, OH, MY, THESE ARE IMPORTANT. >> THEY USED OUR TOOLS TO PUT THEM -- IT WAS ON THE NEWS ON ONE OF THE MAJOR NEWS CHANNELS. >> WELL, WE HAVE A COUPLE MINUTES LEFT. DOES ANYBODY HAVE SOMEBODY THEY WOULD LIKE TO SAY OR CONTRIBUTE WE HAVEN'T ALREADY COVERED? >> THIS IS DAVE. >> GO AHEAD. >> I WANTED TO MENTION WHAT WE'RE SEEING IS PROTEIN FUNCTION SEEMED TO BE ASSOCIATED WITH GLYCAN INTERACTIONS AND SOME OF THE MOST SUCCESSFUL COMPANIES WE HAVE ARE THOSE THAT FOCUSSED ON DRUGGABLE INTERACTIONS WHILE OUR COMPANY IS PRIMARILY FOCUSSED ON MAKING GLYCANS WE SEE USING THIS APPROACH TO LOOK AT NATURALLY OCCURRI OCCURRING GLYCANS AND WE CAN FOCUS ON SPECIFIC GLYCOMS. ONE WE WANT TO FUNCTION IS THE HUMAN IGG GLYCOME AND HUMAN MILK AND BUILD LIBRARIES AND SCREEN THEM WITH A HIGH THROUGHPUT PROCESS AND WE'VE BEEN WORK ON THAT IT GENERATES PROGRAMS THAT ARE DISCOVERY PLATFORM. WE'D LIKE TO FOCUS ON WORK WITH PHARMACEUTICAL COMPANIES THAT HAVE AN INTEREST IN POTENTIALLY DRUGGABLE PROTEIN GLYCAN INTERACTIONS THAT MAY NOT HAVE BEEN DISCOVERED BEFORE. THEY MAY BE OUT THERE AND OUR APPROACH WHERE WE USE LARGE QUANTITIES OF NATURAL PRODUCT, THE IDEA IS WE WOULD GENERATE THE COMMON GLYCAN EVERYBODY SEES BUT PROBABLY A LOT WE HAVEN'T SEEN AND DON'T THE KNOW THE STRUCTURES AND THEN PURIFY AND CAN GET THEM PURE AND NOT KNOW THEIR STRUCTURE. THESE CAN BE SCREENED RAPIDLY FOR SPECIFIC INTERACTIONS AND THEN WE HAVE BASICALLY IN A SENSE A COMBINATORIAL LIBRARY. >> >> I WANT TO THANK YOU. THIS IS BEING RECORD AND IF YOUR COMPANY ISN'T LISTED WE'D LOVE TO LIST IT ON THE COMMON FUND WEBSITE THAT PROVIDE TOOLS AND TECHNOLOGIES TO THE GLYCOCOMMUNITY THAT'S A ONE-STOP REFERENCE LIBRARY OF COMPANIES. WE'RE HAPPY TO PUT YOU UP THERE. AND PLEASE CONTINUE TO DO GREAT THINGS. WE NEED MORE GLYCANS AND AFFINITY AND RELABELLING RE-AGENTS. HOPE TO CONTINUE SEEING YOU AT OUR MEETINGS. THANK YOU VERY MUCH. I'LL TURN IT BACK TO CARL. >> THANK YOU, PAM. WE'LL START AGAIN WITH MORE PRESENTATIONS ON OUR TOOLS THAT HAVE BE HAVE BEEN DEVELOPED. IS ADAM LINSTEDT THERE. >> I'M HERE. >> OVER TO YOU. >> GETTING BACK I'LL TALK ABOUT OUR FOCUS ON THE ENZYMES CALLED THE GALNAC. GEF GEF INTERESTING THING ABOUT THE FAMILY OF THESE ENZYME WE HAVE 20 DIFFERENT GENE PRODUCT CARRY OUT THE SAME REACTION ON SPECIFIC SUBSTRATES BUT THERE'S A LOT OF OVERLAP AND THIS LEADS TO SOME INTERESTING BIOLOGICAL QUESTION HOW THE NETWORK OF GLYCOSYLATION TAKES PLACE. THESE ENZYMES ARE MODIFYING PROTEINS THAT CONTRIBUTE AND PLAY KEY ROLES IN MANY DISEASES AND OFTEN IT'S ACTUALLY THE GLYCOSYLATION THAT PLAYS AN IMPORTANT FUNCTION AND WE JUST HEARD SOMETHING ABOUT THE ROLE OF GLYCANS IN VIRAL INFECTION BUT THERE ARE MANY OTHER SCENARIOS. YET, FOR THESE ENZYMES WHICH ARE INITIATING O GLYCOSYLATION. THEY'RE DETERMINING WHETHER A PROTEIN GETS O GLYCOSYLATED OR NOT IN THE GOLGI AND AT WHAT SITES THERE'S NO DRUG LIKE COMPOUNDS AVAILABLE TARGETING THESE. TO ADDRESS SOME OF THIS STUFF WE HAVE A SET OF TOOLS THAT ARE FLUORESCENT BIOSENSORS THAT DETECT THE ACTIVITY OF THE ENZYMES IB A SPECIFIC WAY AND -- IN A SPECIFIC WAY AND CAN BE USED TO EITHER OUT RIGHT MEASURE THE ACTIVITY OR CAN BE USED TO LOOK FOR DRUGS. ONE SET OF OUR BIO SENSORS ARE DESIGNED SO IF THERE'S A DRUG THAT INHIBITS THE ENZYME BEING TARGET THE FLUORESCENCE WILL INCREASE. I'LL SHOW AN EXAMPLE OF THAT. YOU'RE LOOKING AT A BUNCH OF CELLS GROWING IN TISSUE CULTURE AND THE BIO SENSOR IS A GENETICALLY ENCODED BIO SENSOR AND WE HAVE A GFP TAG AND YOU CAN SEE THE PROTEIN ACCUMULATES ON THE CELL SURFACE OF THE CELL. BUT IF THE CELLS ARE EXPRESSING THE ISOSIME AND I BELIEVE THIS IS FOR THE T2 ISO SIME AND THIS GLYCOSYLATES THAT BLOCKS A CLEAVAGE SITE. THEN THIS PERSISTS AND DOESN'T ALLOW THE FLUORESCENT ACTIVATING PROTEIN TO BIND TIE DYE. IF THE INHIBITOR COMES ALONG AND BLOCKS THE ENZYME IN THIS CASE T2, THEN THE CLEAVAGE WILL TAKE PLACE AND THE BLOCKING DOMAIN WILL DIFFUSE AND THE FAT WILL DIMERIZE AND BIND THE MG DYE AND ACTIVATE THE FLUORESCENCE. A HIGH SIGNAL TO NOISE BIOSENSOR AND IN NORMAL CONDITIONS WHEN THE ISOZYME IS EXPRESSED THERE'S LITTLE SIGNAL BUT IF WE MUTATE THE SITE THERE'S AN EXPRESSION AND IF WE EXPRESS THE SENSOR IN CELLS WHERE WE'VE GENETICALLY KNOCKED OUT THE T2 ISOZYME THAT ALSO ACTIVATES THE SENSOR. SO THE SENSOR'S A GOOD DETECTER OF ACTIVITY IN THAT CASE OF THE T2 ISOZYME AND WE HAVE A NUMBER OF THESE TO GO AFTER SPECIFIC ACTIVITY MEASUREMENTS FOR EACH ONE. I'M JUST GOING FINISH REAL QUICK WITH EXAMPLES OF HOW WE USED THE SENSORS. THE LAST ONE IS NOT US. IT'S A DIFFERENT LAB AND I'M USING THAT AS AN EXAMPLE OF VALIDATION. IN THE FIRST EXAMPLE WE USED T2 AND T3 SENSORS WHERE DRUGS ARE ADDED AT A 10 MICROMOLAR CONCENTRATION AND SCREENED FOR THEIR ABILITY TO ACTIVATE ONE OR BOTH SENSORS. IF THEY ACTIVATE ONLY ONE THERE'S A GOOD CHANCE THAT IS A VERY SPECIFIC AFFECT AND IN FACT WE WERE ABLE TO OBTAIN AN INHIBITOR OF THE ISOZYME AND CARRIED THAT INHIBITOR THROUGH OUR SECONDARY SCREEN ANALYSIS AND THE MOST IMPORTANT STEP OF THAT SECONDARY SCREENING IS TO MAKE SURE THE INHIBITOR WORKS ON THE PURIFIED ENZYME AND CHARACTERIZE HOW IT WORKS AND ALL THAT STUFF. WE ALSO CARE ABOUT THE FIZZOLOGICAL AFFECTS AND -- PHYSIOLOGICAL AFFECTS CAN DO AND AGAINST THE ENZYME IS SELECTIVE AGAINST THE T3 ISOZYME. WE'VE DONE LOTS OF WORK CHARACTERIZING THAT PARTICULAR COMPOUND. CAN SEE IT DOWN HERE. INTERESTINGLY, THIS IS ABLE TO WORK IN CELLS AND EVEN IN MICE IN CONTROLLING KNOWN LEVELS OF A KNOWN T3 TARGET IN OTHER WORDS A SUBSTRATE AND THAT IS SGF23 AND I WON'T SAY MORE ABOUT THAT RIGHT NOW BUT WE ALSO FOUND THE INHIBITOR IS QUITE POTENT IN BLOCKING INVASION OF A BREAST CANCER CELL LINE IN FACT MANY AND THE RESULTS ARE REMARKABLE. THERE'S TWO DIFFERENT TIME POINTS IN THE PROGRESSION OF INVASIVENESS OF THE BREAST CANCER CELL LINE IN A CULTURE ASSAY BUT IN THE PRESENCE OF THE INHIBITOR, THERE'S REALLY VERY LOW IF ANY DETECTED INVASIVENESS OF THE CELLS LEFT. IT'S REALLY QUITE A POTENT AFFECT AND WE'VE CONFIRMED IT HAS TO DO WITH INHIBITING THE T3 ISOZYME BECAUSE WE CAN KNOCK OUT THE ENZYME AND GET THE SAME BLOCK OF INVASIVENESS OF THE BREAST CANCER CELLS. WE'VE GONE THROUGH ALL THE STEPS AND MY NEXT EXAMPLE IS WHAT WE'RE CURRENT LYING ON WHICH HAS TO DO WITH VERY SIMILAR SCREEN BUT HERE WE'RE SCREENING AGAINST T1 AND T2 SIMULTANEOUSLY. WE'RE LOOKING FOR WILL INHIBITERS SELECTIVE AGAINST T1 AND THIS IS EARLY STAGES BUT I'M SHOWING ONE WHERE WE GOT ONE PROMISING HIT. YOU CAN SEE THIS IS WHAT IT SHOWS UP LIKE IN OUR SCREEN AND HERE'S THE COMPOUND. AND WHEN WE ADD THIS TO CELLS AND LOOK AT A PHYSIOLOGICAL SUBSTRATE OF THE PROTEIN WE CAN SEE IT INHIBITS THE GLYCOSYLATION OF THE SUBSTRATE. ONE OF THE REASONS WE TALKED ABOUT LAST YEAR ONE REASON WE'RE SO INTERESTED IN INHIBITERS AGAINST T1 IS BECAUSE WE'VE DONE A BUNCH OF EXPERIMENTS IN LOOKING AT THE GLYCOSYLATION OF THE EBOLA PROTEIN AND AND WE'VE SEEN MUTATION OF THE AND WE'VE SHOWN MUTATION OF THE SITES BLOCKS THE TOXICITY EFFECT OF THE EBOLA GP PROTEIN. WE HAVEN'T YET DONE THE ULTIMATE TEST WHERE WE TAKE OUR NEW T1 CANDIDATE CANDIDATES AND TEST FOR GLYCOSYLATION AND IT'S TOXIC EFFECTS. IT'S AN EXPERIMENT WE HOPE TO DO VERY ZOON. AND THE LAST IS EXPERIMENTS CARRIED OUT BY A GROUP STUDYI G GLYCOSYLATION AND INTERESTED HOW THE ENZYMES INTERACT WITH EACH OTHER AND THE SPECIFIC QUESTION ADDRESSED BY JOHN IN THE EXPERIMENTS WAS IF YOU CHANGE THE EXPRESSION LEVEL THE ACTIVITY OF JUST ONE SINGLE ISOZYME WILL THE AFFECT AND ACTIVITY LEVEL ONLY AFFECT THE T2 SPECIFIC SUBSTRATES OR WILL THE ENZYME AT HIGHER LEVELS SAY START TO GLYCOSYLATE TARGETS IT DOESN'T NORMALLY GLYCOSYLATE AND THEN CHANGE THE PROTEOME IN A SIGNIFICANT WAY. TO CARRY OUT THIS SET OF EXPERIMENTS THE APPROACH WAS TO USE OUR BIO SENSORS IN CELL LINES WHERE WE KNOCKED OUT THE PARTICULAR ISOZYME AND EXPRESSED THE CORRESPONDING BIOSENSOR AND INDUCIBLY EXPRESS THE ENZYME. HERE YOU SEE AN EXAMPLE WHERE T2 IS INDUCIBLY EXPRESSED BY ADDING DIFFERENT CONCENTRATIONS OF DOXO DOXOCYCLINE AND THE ACTIVITY GOES UP BECAUSE THE RATIO IS GOING DOWN. THAT CONFIRMS THE DESIRED EFFECT ON ENZYME ACTIVITY IN THE CELLS. THEY DID THIS ON T2 AND T11 AND THE RESULT COMPLICATED TO PRESENT MOST O GLYCAN NOT CHANGED BY THE OVER EXPRESSION AND THIS IS SHOWN ON THE HISTOGRAMS ON THE RIGHT SIDE WHERE THERE'S CLUSTERING AROUND ZERO WHERE THERE'S NO CHANGE. YOU DO SEE INDIVIDUAL DISCREET TARGETS AND SHOWING INCREASED GLYCOSYLATION AS THE ENZYME RAMPS UP. THE TAKE HOME IS THE ISOZYMES ARE SELECTIVELY REGULATING TARGETS AND NOT CROSSING OVER TO AFFECT THE PROTEOME AT LARGE. I'LL TAKE YOU WITH THE TAKE HOMES IN DEVELOPING SENSORS FOR THE FAMILY OF THE GALNAC AND WE HAD SOME SUCCESS AGAINST T3 AND SOME SUCCESS AGAINST T1. THE SENSORS CAN BE USED IN ALL SORTS OF STUDIES THAT LOOK AT THE REGULATION OF GALNAC Ts AND THEY'RE ESPECIALLY YOU'VES BECAUSE THEY MEASURE ACTIVITY NOT JUST EXPRESSION LEVEL AND DO SO IN LIVING CELLS. I'D LIKE TO THANK EVERYBODY ON THE SLIDE. THE RESULTS COULD NOT HAVE BEEN CARRIED OUT WITHOUT THEIR HELP. OF COURSE THE NIH COMMON FUND. >> THANK YOU. ANY QUESTIONS ON THE VERY INTERESTING WORK? VERY GOOD. >> TO MAKE SURE IT SEEMED LIKE YOU WERE ABLE TOE FIND INHIBITERS FROM JUST T1 AND T3 AND YOU HAD T11. >> THEY'RE NOT INHIBITERS. THOUGH WE HAVE DONE SCREENS AND CANDIDATE COMPOUNDS BUT WAS ON THE SLIDE WERE THE SENSOR. WE HAVE SENSORS THAT ARE SPECIFIC AND CONFIRMED, VALIDATED OR A NUMBER OF THE DIFFERENT ISOZYME. NOT ALL OF THEM YET. AS FAR AS COMPOUNDS THAT BLOCK, I'D REALLY ONLY GO TO BAT WITH THE T3 COMPOUND BUT THE T1 COMPOUNDS ARE LOOKING VERY PROMISING. >> OKAY. >> I GOES YOU'RE ALL SET UP TO START SCREENING FOR SOME OF THE OTHER ISOZYMES THEN? >> WE HAVE SCREENED AGAINST T2 BECAUSE IT'S PART OF OUR STRATEGY AND HAVE COMPOUNDS THAT LOOK LIKE THEY HAVE ACTIVITY AGAINST T2. SOME DROPPED OUT BECAUSE IT'S MORE OF A MORE PAN ACTIVITY THAN ISOZYME SPECIFIC ACTIVITY. THERE'S A LOT OF WORK THERE IN GOING THROUGH THE SECONDARY SCREENING PROCESS. >> ANYBODY ELSE HAVE A LAST MINUTE QUESTION. IF NOT WE'LL MOVE ON TO THE NEXT TALK. >> I WILL START. MY PROJECT IS TO DEVELOP GENETIC AND ENCODED BIOSENSORS FOR MONITORING UDP SUGARS AND I DON'T HAVE CONFLICT OF INTEREST. MY PROJECT IS BASED ON USING FLUORESCENT PROTEINS. THIS IS AN EXAMPLE WITH THE GFP. WE USE IT AS THE SCAFFOLD TO BRIDGE AND BIND TO DETECT THE MOLECULE. IT'S A PROOF OF PROTEIN TO LOOK AT WILD TYPE GFP AND WE HAVE THE PROCESS WITH DEHYDRATION AND OXIDATION TO FORM THE SYSTEM. IT'S RESPONSIBLE FOR THE FLUORESCENCE AND SAME IN THE MIDDLE OF THE PROTEIN AND CONVERT THIS INTO AN ACTIVE BIO SENSOR AND A COMMON STRATEGY IS TO USE A GENETICALLY FUSED LINE AND RE-ESTABLISH IT CLOSE TO THE FORM AND THEN WE CAN LINK THIS PROTEIN WITH THE EFFECT OF THE BINDING DOMAIN AND SOME OTHER COMPONENTS TO SEPARATE THE MOLECULE. AND HE COULD OBSERVE THE CHANGE THE IMAGING. THIS IS WHERE WE ESTABLISH THE PLATFORM FOR OUR PROJECT SO WE TRY TO USE THIS TO DEVELOP THE NUCLEOTIDE SUGAR. MOST INTERESTING TO US IS IT'S SYNTHESIZED AS THE GLYCOSEIS PATHWAY AND NUCLEOTIDES ARE DIFFERENT TYPES OF MOLECULES TO FORM THIS METABOLITES WHICH CAN BE FURTHER USED BY ENZYMES TO FORM MODIFICATION OR MAYBE MODI MODIFY IN SOME STRUCTURES GET IT ON THE CELL SURFACE. SO WE'RE LOOKING AT A CULE WHICH CONNECT S -- AT A MOLECULE WITH A SIGNAL AWE STARTED WITH THE TRANSFERASE AND THIS LOOKS AT THE GLYCAN SYNTHESIS PATHWAY AND MODIFY THE LIPID DERIVED GLYCAN STRUCTURE. WE TOOK THE TRANSFERASE AND WE LOOKED AT THE FLUORESCENCE OF THE GDP AND TO ACHIEVE THAT WE FIRST EXAMINED TO THE STRUCTURE OR THE PROTEIN. UNFORTUNATELY FOR THIS PARTICULAR ENZYME HAS BEEN STUDIED. WE REPORTED A STRUCTURE AND COMPARED THE STRUCTURE AND AND WE STARTED WITH AN OPEN LOOP AND IT WAS CONVERTED TO THE STRUCTURE. WE CAN STARTED BY THE GFP IN THE LOOP AND WE WERE ABLE TO FIND A RESPONSE. AT ONE POINT WE TRIED TO INCLUDE THE SPECIFICITY. AND THIS IS FAR FROM THIS BUT THIS LOCATION HAS A LARGE CONFIRMATION CHANGE WHICH CAN BE USED TO DEVELOP THE SENSOR. AND THIS IS A FLORUORESCENT EXPRESSION OF THE PROTEIN AND PURIFIED PROTEIN AND OBSERVED AN INCREASE. AND THERE WAS ANOTHER PEAK AND A NANOMETER DECREASE. THIS IS COMMON FOR THE SENSOR. AND THIS WAS TESTED FOR NUCLEOTIDE SUGARS AND THIS WAS CLOSE TO EACH OTHER. IN THE LITERATURE THIS REACTION WORKS AS A NFL INHIBITOR TO SELF-REGULATE THE ENZYME AND IMPROVE THE SPECIFICITY. WE WERE ABLE TO IMPROVE THIS BUT UNABLE TO AWARD THE INTERFERENCE. AND WE COMBINED THIS. WE WERE ABLE TO HAVE THE GLCNAC AND THIS SHOWS THE FIRST EXPERIMENT IN TRYING TO MODULATE THE GLUCOSE ENZYMES. AFTER THE 2-D WE WERE ABLE TO DECREASE THE GLCNAC AND SINCE IT WAS THE FIRST EXPERIMENT WE WANTED TO VALIDATE THIS WITH AND LOOKED AT THE LITERATURE AND WE ESTABLISHED PEOPLE CAN USE SOME SORT OF ASSAY METHOD USING THE GLCNAC AND DETECT AND QUANTIFY THIS AS MASS SPECT. WE CAN LOOK AT THE 606 AND 608 AND CAN LOOK AT THE GLCNAC IN THE CELL LINE. WE THOUGHT IT WAS GOOD ENOUGH TO CONFIRM AND WE USED THIS MASS SPECT METHOD AND COULD OBSERVE THE DECREASE OF GLCNAC AND CAN LOOK AT WHAT WAS CONTRIBUTED IN STANDARD. [TECHNICAL DIFFICULTIES] AND OVER EXPRESSION CAN DECREASE GLCNAC CONCENTRATION AND OVER EXPRESSION SLIGHTLY DECREASED THE XPS BUT UTP IS CONFIRMED IN THE PATHWAY. WE CHOSE THE MIN 6 CELLS BECAUSE IT'S METABOLIC SENSITIVE AND CAN SENSE GLUCOSE AND RESPOND TO GLUCOSE CONCENTRATION. SO WE WANTED TO SEE IF THE CELLS WERE MORE SENSITIVE AND THIS IS QUITE SENSITIVE TO GLUCOSE. IS GLUCOSE AS EXPECTED WE OBSERVE ED THE INCREASE IN THE RATIO INDICATING THE CONCENTRATION INCREASE. AND THIS IS AS EXPECTED. WE THERE'S DIFFERENT CONCENTRATION OF GLUCOSE. THIS IS GLUCOSE AND HIGH GLUCOSE AND CELLS FROM THE SAME SOURCE IN THREE DIFFERENT AMOUNTS AND PERFORM IMAGING AND AS EXPECTED WITH NO GLUCOSE AND THE CONCENTRATION UDP DECREASE DRASTICALLY. BUT BETWEEN THE TWO WE OBSERVED A SMALL INCREASE BUT IT'S NOT STATISTICALLY SIGNIFICANT. AND THERE WAS A RECENT PUBLICATION AND USED MASS SPECT TO STUDY HEART TISSUE AND THE DATA INDICATES THE CELLS BUT THE GLCNAC CONCENTRATIONS WERE REGULATED. AND THERE'S MULTIPLE ENZYMES AND METABOLITES REGULATING THE PATHWAY AND YOU CAN INCREASE THE PATHWAY. THIS IS NOT TOO MUCH OF AN INCREASE AS WE WOULD EXPECT. >> CAN YOU WRAP UP IN ABOUT TWO MINUTES. >> WE USED AN ALTERNATE STRATEGY AND IT WORKS WITH CELLS. WE TRIED TO WRITE A PAPER AND THE THINK WE CAN FURTHER PERFORM THIS MEASUREMENT AND THE NUCLEUS HAS HIGH CONCENTRATIONS AND WE DON'T KNOW THE MECHANISM BUT WE HAVE TO CONFIRM. TO SUMMARIZE WE ENGINEERED A GENETIC SENSOR AND WE MONITORED THIS AND SUGGESTS METABOLITES THROUGH THE PATHWAYS ENTIRELY UNREGULATED AND HAS BEEN PUT TO THE ARCHIVE AND WE HAVE TWO STRATEGY AND TWO SENSORS FOR THE GLCNAC AND WE'RE EXPLORING THE SENSORS AND CORRELATE THE DATA WITH THE SENSORS AND HAVE OTHER CELL TYPES SUCH AS NEURONS AND BETA CELLS I WANT TO THANK THE COMMON FUND FOR THE SUPPORT AND WE SHARED DATA WITH RESEARCHERS AND COLLABORATED TO VALIDATE OUR SENSORS. I EXPECT PROBABLY YOU CAN DROP ME AN E-MAIL IF YOU HAVE ANY SUGGESTIONS AND THANK YOU FOR YOUR TIME. >> THANK YOU. IS THERE ANY BURNING QUESTION OUT THERE? IF NOT WE SHOULD MOVE ON TO OUR NEXT SPEAKER. THAT WOULD BE LAURA. WOULD YOU LIKE TO COME ON? >> I'LL START AND LAURA WILL TAKE OVER. >> GREAT, THANKS, BARBARA. >> NOT A PROBLEM. WE'LL GO FOR 15 MINUTES BETWEEN US. AND I WANTED TO DECLARE WE HAVE NO CONFLICTS OF INTEREST AND THE TEAM I'M REPRESENTING IS SHOWN ON THE SLIDE. MY GROUP PRESENT A FAR RICHER AND BROAD LIKE GLYCAN THERE'S OVER 700 SUGAR BUILDING BLOCKS AROUND YOU CAN'T IMAGINE HOW MANY COMMON FUNDS THAT WOULD KEEP BUSY AND QUAL FOR INTERACTIONS AMONGST DIFFERENT BACTERIA AND ALSO BETWEEN THE BACTERIA AND HOST THEY INFECT. IT'S CRITICAL FOR US TO HAVE TOOLS TO STUDY THE INTERACTION AND UNDERSTAND THE BACTERIA AND CARBOHYDRATES. I'LL PRESENT ON THE MICROBIAL GLYCAN ANALYSIS PROBES. WE TOOK OUR INSPIRATION FROM THE LECTIN FORM PART OF THE INNATE IMMUNE RESPONSE AS UNDERSTANDING EARLY INFECTIONS. THERE'S MANY SOL HUMAN LECTINS OFTEN CHARACTERIZED BY STRUCTURE AND MANY WITH UNCLEAR PARTNERSHIPS BUT ALSO MANY DISTRIBUTED IN CERTAIN LOCATIONS AND OUR CHOICES HAVE BEEN TO ACHE THE IMPORTANT HUMAN LECTINS AND DEPLOY THEM OVER FOR THE MICROAL ANALYSIS PROBE. -- MICROBIAL ANALYSIS PROBES. SO WHAT WE DEVELOPED WAS A WORK FLOW MANY HAVE SEEN BEFORE TO GROW FROM HAVING THE PROTEIN THAT IS THE HUMAN LECTIN TO TRY TO DEVELOP CONSTRUCTS THAT CAN BE EXPRESSED WELL IN BACTERIAL NON-MAMMALIAN SYSTEMS AND DEVELOP A PLATFORM FUNCTIONALIZING AND CHARACTERIZING THE M GAP TOOLS TO BE DEPLOYED FOR APPLICATION. ACROSS THE BOTTOM OF THE SLIDE I GIVE THE STATUS OF THE VARIOUS GAPS DEVELOPED AND WHICH ONES ARE COME TO STAGES WHERE THEY'RE DEPLOYED FOR APPLICATION. IF YOU SEE ANY FAVORITES, PLEASE LET US KNOW. ONE TROOP IS MITIGATION THAT WORKS WELL FOR PROTEINS THAT ARE OFTEN MONOMERIC AND BACTERIA BUT WHAT I SHOW ON THE SLIDE IS A LITTLE BIT ABOUT THE REACTION WHICH HAS MINIMAL DETERMINATES AND CONVENIENT AND BELOW I SHOW THE SORTS OF THINGS THAT COULD BE DEPLOYED AND WHAT TYPES OF EXPERIMENTS THAT COULD BE DEPLOYED WITH LABEL AND GAPS IN HAND THAT HAVE BIOTIN OR FLUORESCENT SLIDES. WHAT I WANT TO PRESENT SAY BRIEF STORY OF A CASE STUDY THAT CAME OUT OF THE PREVIOUS MEETING WHERE WE SPOKE WITH STEFAN RUHL AND IT'S EXCLUSIVE TO THE OLD CAVITY BEHIND THERE'S ABUNDANT FUNCTION BUT SOME BINDING HAS BEEN SHOWN TO DIVERSE ORGANISMS. IT'S ONE OF THE GAPS WE'RE ABLE TO MAKE IN GOOD AMOUNTS. HERE'S THE PREPARATION. WHAT I WANT TO TELL YOU IS WITH SOME M GAPS WE HAVE ROBUST PRODUCTION METHODS AND HAPPY TO SHARE THE RE-AGENTSES WITH PEOPLE AND YOU CAN SEE ON THE LEFT SIDE THE LABELLING OF THE M GAPS WITH THE PROCEDURE THAT WORKS WELL THROUGH A FLOW-BASED MEDIATED LIGASE. [AUDIO DIGITIZING] WE SAW CLEARLY FROM EACH SIDE OF THE SCREEN SOME CLEAR RESPONSES. IN THIS CASE WE USED SOME OF OUR OTHER MGAPS AS CONTROLS AND YOU SAW NO COMPARABLE RESULTS. WE WERE VERY EXCITED TO GET THESE MICROBES OUT AND ASSESS THEM. CHRISTINE ISABELLA IN LAURA'S GROUP HAS DONE FLOW ANALYSIS AND HAS BEEN ABLE TO SHOW CLEARLY THAT CG16B DOES BIND CERTAIN MICROBES AT DIFFERENT LEVELS. SOME VERY WELL, SOME MORE WEAKLY. THERE IS NO INFLUENCE FROM MULTIVALENT PRESENT AND THESE WERE BROUGHT BACK TO OUR LAB FOR CULTIVATION AND FOUND SEVERAL IDENTIFIED SPECIES IN THE STREPSTREP STREPTO COCCHUS SPECIES AND WE SAW THE LARGE BLOB UP FROM THE BASELINE AND IN NEGATIVE SAMPLES WAS ABSENT THAT STAIN. I WANTED TO SHOW YOU THE FRACTIONATION OF THE ORGANISM SHOWING THE DIFFERENT CONDITIONS AND HOW WE CAME TO ASSIGN THE TARGET. NOW WE HAVE GREAT EXPERIMENTAL PATH TO DO WHOLE GENOME SEQUENCE FOR EXACT CANDIDATES, CARRY ON INVESTIGATION OF THE INTERACTIONS WITHIN THE ORAL CAVITY AND WE WOULD LIKE TO DETERMINE THE MINIMAL GLYCAN EPITOPE AND CARRY OUT MICROSCOPY. OVER TO YOU, LAURA. >> THANK YOU, BARBARA. WHAT YOU JUST HEARD FROM BARBARA IS HOW POWERFUL THE LECTIN S ARE FOR LOOKING AT MICROBES IN MICROBIOME SETTINGS. AND WE HAVE SHOWN PREVIOUSLY WE CAN LABEL MICROBES IN THE GUT. IS THIS A MOUSE SMALL INTESTINE. THAT LED US TO THINK ABOUT USING THE LECTINS AND DEMONSTRATING THEIR NEW USE AS A WAY TO PROFILE MICROBIOMES WITHIN LARGER COMMUNITIES. YOU KNOW THE MICROBIOME IS EMERGING AS AN IMPORTANT COMPONENT AS REGULATION OF HEALTH AND HEALTH INDICATOR. THINK OF HOW OTHER PEOPLE WERE PROFILING MICROBIOMES. ONE WAY THEY TEND TO DO THIS IS USING AN IMMUNOGLOBULIN WHICH IS AN IMMUNOGLOBULIN FOUND IN YOUR GUT AND DEVELOPED A METHOD CALLED IGA SEQ TO GIVE A SENSE OF THE IMMUNE STATUS OF THE GUT. SO LECTINS AS BARBARA MENTIONED ARE IMPORTANT IN INNATE IMMUNITY AND UNLIKE HERE WHERE WE DON'T KNOW WHAT THE ANTIGEN IS, HERE WE KNOW WHAT THE GLYCANS THEY RECOGNIZE ARE AND THESE WOULD BE COMPLIMENTARY TOOLS. WE BEGIN TO DEPLOY THIS USING TWO LECTINS. AN A MANNOSE LECTIN IS KNOWN AND THE OTHER WE SUSPECT IS FOUND IN COMMENCEAL BINDING. YOU CAN SEE FROM THE GLYCAN ARRAY THEY HAVE DIFFERENT SPECIFICITY. I SHOW THAT AT THE LEVEL OFF THE MOLECULES THEY BIND AND DETECT IT AT THE ARRAY LEVEL. OUR STRATEGY IS TO USE THE FLUORESCENT LECTINS AND THOUGHT WE COULD LABEL SPECIFIC MICROBES AND PULL THEM DOWN BASED ON THEIR FLUORESCENT SIGNATURES AND ANALYZE THEM OR SORT THIS OUT AND ANALYZE THE NUCLEIC ACID SEQUENCE. FIRST, IF WE LOOK AT THE FLOW CYTOMETRY DATA AND TAKE A STOOL SAMPLE AND LOOK AT THE LECTIN VERSUS HUMAN LECTIN THAT WOULD BE HERE. YOU CAN SEE HUMAN INTELECTIN DON'T HAVE MUCH OVERBINDING SPECIFICITY. NOW WE AGAIN APPLIED THIS TO SEQUENCING AND I WANT TO POINT OUT WE'RE EXCITED ABOUT THIS APPLICATION BECAUSE IT'S THE FIRST STEP TOWARDS RELATING CELL SURFACE GLYCANS DIRECTLY TO SEQUENCE. YOU ALREADY HEARD ABOUT THE PATHWAYS STARTING WITH MICROBES AS A PLACE TO DO THAT. I'LL SHOW YOU A FEW EXAMPLES. THREES ALL HEALTHY INDIVIDUALS AND WE CAN SEE MANNOSE FINDING LECTIN AND THE SPECIES IT BINDS. WE CAN GET SPECIFIC INFORMATION WHICH IS VERY DIFFICULT TO GET USING MANY OF THE TOOLS OUT THERE AND SEE THE LECTIN ARE VERY DISTINCT. ONE THING THAT WE ALSO FOUND AND IT'S ILLUSTRATED IN THE SLIDE WE ALSO QUANTIFIED IS MANNOSE BINDING LECTIN IS NARROWER IN ITS SPECIFICITY FOR DIFFERENT MICROBES. ONE THING I WANT TO HIGHLIGHT AND GOES TO THE STUDY OF HUMAN SAMPLES WHEN WE LOOKED AT BINDING OF HUMAN INTELLECTIN TO ISOLETS MANY -- ISO LATES AND SEE THE SAME WITH MAN NOSE BINDING LECTIN AND THEY DON'T ALWAYS REPRESENT THE GLYCANS THERE IN THE HUMAN HOST AND HIGHLIGHTS THE IMPORTANCE OF THIS METHOD FOR RAPIDLY PROFILING. THE OTHER THING WE BEGAN TO LOOK AT A HEALTHY INDIVIDUAL VERSUS AN INDIVIDUAL THAT HAS A DIS DISBIOTDIS BUY OT BIOME. THE MICROBES ARE BETTER IN A HEALTHY THAN DISEASED PATIENT. WE CAN LOOK AT THE DETECTION USING PRINCIPLE COMPONENT ANALYSIS AND YOU CAN SEE THAT MANNOSE BINDING LECTIN WHICH IS A PATHOGEN SPECIFIC LECTIN IS NOT A VERY GOOD IT DOESN'T SHOW MUCH CHANGE BETWEEN HEALTHY AND DISEASE WHERE HUMAN INTELLECTIN SHOWS CHANGE BETWEEN HEALTHY STATES AND DISEASE STATES. THAT HIGHLIGHTS WHAT WE CAN DO AND THE TOOLS WE'RE DEVELOPING. I WANT TO POINT OUT WE PROVIDED THESE TO MANY INDIVIDUALS AND OUR FEATURE DIRECTION ARE TO EXPLORE LECTIN FUNCTION FURTHER IN THE ORAL CAVITY IN THE GUT AND USING THE TOOLS I DESCRIBED TO ASSESS LECTIN CELL TYPE SELECTIVITY SO OTHERS MAY DEPLOY THEM AND DISSEMINATION OF THESE FUNCTIONALIZED LECTINS. TO THAT END WE'RE DEVELOPING THIS COMBINED OF STRATEGY OF A PRODUCT DATA SHEET THANKS TO AMANDA DUGAN AND THE IDEA IS TO PROVIDE USERS WITH INFORMATION THEY WOULD NEED INCLUDING PROTOCOLS FOR USING THESE LECTINS UNDER DIFFERENT CONDITIONS. I WANT TO HIGHLIGHT THAT WE ARE SENDING THEM OUT TO LABS THAT ARE USING THEM TO ADDRESS NEW SORTS OF QUESTIONS. I WANT TO THANK THOSE FOR THE PROGRAM TO CATALYZE INTERACTIONS AND GIVE ME A CHANCE TO HANG OUT WITH BARBARA AND BARBARA'S LAB AND THANK THEM FOR THE SUPPORT OF THE PROJECT AND HAPPY TO ANSWER QUESTIONS. >> THANK YOU. DO WE HAVE QUESTIONS FOR EITHER OF THE TWO? >> I GUESS NOT. >> OKAY. WE'LL TAKE ADVANTAGE AND TRY TO CATCH UP. STEFAN, YOUR NEXT. -- YOU'RE NEXT. >> I WAS HAPPY TO HEAR IN THE TWO PRECEDING TALKS THE WORD ORAL CAVITY SEVERAL TIMES MENTIONED. I'LL CONTINUE BUT NOT WITH MAMMALIAN LECTINS BUT MICROBIAL LECTINS WE HARVEST AND HARNESS FROM THE ORAL MICROBIOME. WHY? IT'S ARGUABLY ONE OF THE MOST DIVERSE COMMUNITIES IN MAMMALIAN HOSTS AND HAS BEEN KNOWN FOR A LONG TIME GLYCAN MEDIATED INTERACTIONS PLAY A ROLE IN BACTERIAL COADHESION AND ADHESION TO HOST CELL SURFACES IN THE ORAL CAVITY. THIS HAS BEEN SHOWN IN EARLY ELECTRON MICROSCOPIC ASSAYS WHERE TWO STRAINS OF BACTERIA ARE PUT TOGETHER IN A VIAL AND SHAKEN AND IF YOU SEE THESE CO-AGGREGATES DEVELOP YOU NOW ONE BINDS TO THE OTHER AND THESE COULD BE DISSOLVE BY THE APPROPRIATE SHUL AR SHOWING A LECTIN CARBOHYDRATE INTERACTION TOOK PLACE. IT BECAME CLEAR ORAL BIOMES ARE RECOGNIZED ON THE SURFACE OF OTHER BACTERIA AND BACTERIA BINDING TO HOST CELL GLYCANS ON ORAL SURFACES ON THE TWO SURFACE OR EPITHELIAL SURFACES. MY LAB IS INTERESTED IN THESE EARLY PIONEERING, BINDING BACTERIA THAT ARE MOSTLY COMPOSED BACTERIA. THEY'RE NORMAL HABITAT IS THE ORAL CAVITY AND THEY GET INTO CONTACT WITH OTHER MEMBERS OF ORAL BIOFILMS AND THEIR REGULAR MILIEU IS IN SALIVA AND BIND TO SALIVARY GLOBULINS AND MANY ARE MEDIATED BY ADHESIONS ON THE STREPTO COCCI AND CARRY GLYCANS ON THE SURFACE OF THE GLYCOPROTEINS. THEY BIND TO STREPTOCOCCI AND PHAGEOCYTES AND IF THEY MAKE IT PAST THE BARRIER THEY CAN ENTER THE BLOODSTREAM AND CAN INTERACT WITH GLYCOPROTEINS ON BLOOD CELLS AND CAN CAUSE ENDO CARDITIS AND THIS BROUGHT US TOGETHER AND THE CREW IN PARTICULAR HAVE STUDIED ADHESIONS ON STREPTOCOCCI FOR A LONG TIME AND THEY'RE ELONGATED RICH REPEAT PROTEINS THAT EXTEND SEVERAL HUNDRED NANO METERS FROM THE SURFACE OF THE BACTERIA AND CARRY A NON-REPEAT BINDING REGION THAT IN THE CASE OF THESE BACTERIA THAT BIND TO SYLIC ACID COMES WITH A UNIQUE MOTIF. WHEN THE NON-REPEAT BINDING REGIONS ARE BEING RECOMBINANTLY EXPRESSED WE CAN TEST THEM AND WE CAN FIND DIFFERENT PROFILES BUT THEY HAVE UNDERLYING GLYCAN MOTIFS AND LINKAGES. AMONG THE LAB STRAINS WE DID NOT FIND ANY THAT BOUND TO THE PREVALENT FORM OF ACID. A STUDENT IN MY LAB WENT OUT WITH THE HYPOTHESIS SUCH STREPTOCOCCI WOULD PROBABLY NOT EXIST IN THE ORAL CAVITY OF HUMANS. AND BECAUSE OF A DEFECTIVE ENZYME THAT CANNOT CONCERT AND TURNS INTO A MINI ACID. THE HYPOTHESIS WAS WE'D FIND THESE IN THE IN THE GREAT APES HOMOLOGOUS TO US HOWEVER, AFTER TESTING STREPTOCOCCI SIGLECS ON THE DIFFERENCE BETWEEN HUMAN AND CHIMPANZEE THE FOUND THE MAJORITY OF SKRETREPTOCOCCI WAS FOUND IN HUMANS AND VERY LITTLE TO NUN IN THE GREAT APES AND MAY HAVE EVOLUTIONARY IMPLICATION WE CAN DISCUSS LATER BUT THE IDEA OCCURRED US THERE MAY BE MANY MORE OUT HERE AND WE JOINED THE TOOL SUB GROUP HERE AND NEW HERE IS A BIG JUMP FORWARD. AFTER THEY ISOLATE THE BACTERIAL STRAINS WE SELECTED A FEW BASED ON THE STRENGTHS OCCUR IRING AND THIS IS IN DIFFERENT BRANCHS AND HERE ON THE LEFT SIDE YOU SEE THE WHOLE REPEAT PROTEINS THAT MOSTLY CLUSTERS ACCORDING TO THEIR STRAIN. ON THE RIGHT-HAND SIDE YOU SEE A TREE OF THE SIGLEC DOMAINS. AND THIS CARRIES DIFFERENT CANONICAL PEPTIDE MOTIFS OF DIFFERENT AMINO ACID COMPOSITION AND ALL THE STREPTOCOCCI THAT BIND AND THIS IS SOMETHING TO BE REMEMBERED FOR LATER. AND WE SUBJECTED THEM TO GLYCAN ARRAY TESTING AND I'M SHOWING YOU TWO EXAMPLES. ONE EXAMPLE OF A TOOL THAT BINDS TO THE ACID. THIS BINDS TO A PARTICULAR LINKAGE AND SUB TERMAL GLYCAN. HERE WE HAVE A HANDFUL MORE ALREADY. AND IN COLLABORATION WITH VANDERBILT UNIVERSITY WE'RE GETTING EXPERIMENTAL 3-D STRUCTURES OF THE BINDING REGIONS AND WE HAVE TWO NEW ONES THIS YEAR FROM SY10 AND ANOTHER THEY'RE DE VOID OF BINDING AS YOU CAN SEE HERE THERE'S NO EXCHANGE MUTANTS WHERE THE ACID HAS BEEN EXCHANGED FOR AN LYSIN AND WE CAN USE THEM AS CONTROL TOOLS FOR APPLICATIONS LIKE FLOW CYTOMETRY THAT IS PURSUED BY A POST-DOC IN MY LAB AND HERE WE USED LEPTINS AND ALSO WE ARE IN THE PROCESS OF USING THE TOOL TO IMAGE BIO MILL MABIO -- BIO FILM AND FROM WHAT'S AVAILABLE IN THE DATABASE YOU SEE A FILO GENETIC TREE BASED ON WHOLE GENOME INFORMATION. SPLITTING UP IN THE DIFFERENCE SPECIES OF STREPTOCOCCI. THESE SPECIES HAVE REPEAT PROTEINS. HOWEVER, ONLY THE LONG STICKS IN RED ARE THOSE THAT CARRY A SIGLEC AND UNIQUE DOMAIN. IT'S INTERESTING TO SEE THEY ARE NOT DISTRIBUTED ACROSS AND ONLY APPEAR IN A CERTAIN GROUP OF STREPTOCOCCI AND THOSE HAPPEN TO BE IN THE ORAL CAVITY. MY HYPOTHESIS IS THIS IS BECAUSE THEY'RE EXPOSED TO SALIVA. IT'S AN ADVANTAGE TO POSSESS THE BIND AND THERE'S A LARGE NUMBER WE'RE NOT TAPPED INTO AND A POST-DOC TOOK THE SEQUENCES AND BUILT IT BASED ON THE SIGLEC AND UNIQUE DOMAIN SEQUENCE. WHEN HE DID THAT THE CLUSTERING OCCURS ACCORDING TO THE BINDING SPECIFICITY AND TO ASSOCIATION FOR CERTAIN SPECIES. WHAT BECOMES EVIDENT IS THE ACID BINDING STREPTOCOCCI BINDING STRUCTURE IN THIS EXAMPLE. I THINK THIS MAY BE OF EVOLUTIONARY SIGNIFICANCE AND SOMETHING TO BE FOLLOWED UP. AND WE KNOW SOME BIND TO ACID AND SIALIC ACID. HERE'S A GREAT POTENTIAL FOR A COMPANY TO DEVELOP THIS INTO A TOOL SET OF TOOLS THE MILESTONES WE'RE DOING CROSS VALIDATION OF ALL TOOLS WITH THE MODIFIED ACID ARRAY. WE HAVE THIS WONDERFUL COLLABORATION WITH BARBARA AND LAURA TESTING CG16B. WE'LL BE OPEN TO ANY COMPANY INTERESTED IN OUR BACTERIAL LECTINS. THANK YOU VERY MUCH. >> THANK YOU. DOES ANYBODY HAVE QUESTIONS FOR HIM? >> DO THEY ALL SHOW SPECIFICITY FOR THE ALPHA 3 LINKAGE OR WOULD YOU BE FORTUNATE ENOUGH TO FIND SOME THAT MIGHT HAVE SPECIFICITY FOR ALPHA OR DON'T YOU KNOW? >> I THINK WE KNOW. THEY ALL HAVE PREFERENCE FOR A LINKAGE BUT I THINK WE INTRODUCED THE BIAS IN OUR SEARCH PROCESS BECAUSE WE USED HUMAN AND CHIMPANZEE ON THE LINKAGE. SO I WONDER WHAT WOULD HAVE HAPPENED IF WE HAD TAKE E-REEGE ROW SIGHTS. >> I WOULD LOVE DO THIS I JUST NEED ANOTHER PERSON IN MY LAB TO DO IT AND MAY BE ALPHA 6 BINDERS WE'RE NOT AWARE OF. WE ONLY HAVE ALPHA 2-3 LINKED LECTINS. >> DOES ANYBODY HAVE A QUESTION? >> STEFAN, GREAT AS ALWAYS. WHEN YOU DID THE PHILO GENETIC ANALYSIS IF YOU SEE SOME OF THE ONES THAT BIND LIKE THE TYPES OF ACID IF THOSE INFECT OTHER MAMMALS? >> I DON'T WANT TO BE -- IT SEEMS LIKE THE BINDING STREPTOCOCCI CLUSTER AND ALL BELONG TO STREPTOCOCCUS AND THERE'S LITERATURE SHOWING ONE TYPE IS PREDOMINANTLY PREVALENT IN HUMANS. IT MAY BE A STREPTO COCCUS THAT EVOLVED ALONG WITH THE DRAMATIC CHANGE IN DIET WHICH GOES WITH CONSUMPTION OF STARCH AND MEAT. WE'RE BASICALLY THE MEAT EATING PRIMATE AS EVERYBODY KNOWS WHICH WAS FACILITATED WITH THE INVENTION OF FIRE. SOME SAY THIS MAY HAVE HAPPENED 2 MILLION YEARS AGO. WE'RE NOT SURE. BUT IF IT'S THE MEAT EATING WE COME BACK TO THE HYPOTHESIS THERE MAY BE EXPRESSION OF THE ACID IN THE HUMAN CAVITY BUT PROBABLY NOT ON HOST TISSUE BECAUSE WE TESTED THAT. MAYBE ON THE SURFACE OF OTHER BACTERIA. THERE ARE SAVE EN -- SCAVENGERS THAT MAY EXPRESS THE ACID AND WOULD BE THE COADHESION PARTNERS FOR THE ACID BINDING BACTERIA. THAT'S SOMETHING MY LAB FOLLOWS RIGHT NOW. >> IT WOULD EXPAND THE VOCABULARY OF LEPTINS. >> THANK YOU. WE'LL HAVE IT TO MOVE ON. THE NEXT PRESENTATION IS BY ONE OF OUR SUPPLEMENT AWARDEES, DR. CHIEKO MINO. >> THANK YOU FOR THE INVITATION. I'M COMING FROM ONE OF THESE KNOWN BIOLOGY LAB AND WOULD LIKE TO GIVE A BRIEF INTRODUCTION OF THE RESEARCH AND WHAT WE'RE DOING IN THE REALM. SO THE FOCUS OF OUR RESEARCH IS ON THE HUMAN DISEASE CALLED THE VASCULAR ENDOTHELIAL DYSFUNCTION AND INCLUDES DISEASES SUCH AS ATEAR YAL SCLER OWES -- ARTERIAL SCLEROSIS AND OTHER DISORDERS TO STUDY THE HUMAN DISEASE WE TURN TO MOUSE DISEASE MODELS. WE GIVE THEM AND GIVE THEM A DIET TO DEVELOP TYPE 2 DIABETES. WHAT WE'RE WORKING ON A MOUSE MODEL OF THE METABOLOMIC SYNDROME TO GIVE THEM HIGH CHOLESTEROL AND HIGH CARB DIET TO GIVE TYPE 2 DIABETES. AS THEY GET FAT THE HIGH CARBOHYDRATE DIET FOR A LONG TIME THEY START TO LOSE THIS. TWO WEEKS ON HIGH FAT DIET AND TWO WEEKS AND EIGHT WEEKS AS THEY PROGRESS THEY TEND TO LOSE I IGG SIALATION AND WE FOUND THAT SIALTION HAPPENS AND FOUND THE ADDITIONAL MOUSE MODEL AND IT PLAYS A CAUSAL ROLE TO A RECEPTOR TO THE SURFACE OF ENDOTHELIAL CELLS RESULTING IN A HUGE DYSFUNCTION OF ENDOTHELIAL CELLS CAUSING TYPE 2 DIABETES AND LIVER RESISTANCE IN THE END. WE LOOKED AT WHY HIGH FAT DIET CAUSES THE HYPOSIALTION. WE'RE FOCUSSING ON THE LIVER. DEF WE FOCUSSED ON TWO ENZYMES. A AND SEE WHAT HAPPENS TO THE HIPE HYPOSIALYLATION. SO WHEN THEY'RE FAT WE GET IGG AND WHEN THEY LOSE THE WEIGHT AND WHAT HAPPENS TO THE HYPO HYPOSIALATION AND DOING THE TRANSCRIPT OME ANALYSIS AND THEY'RE ISOLATE FROM THE HUMAN BEINGS WHICH ARE AND COMPARING THE TRANSCRIPTOMES OR GENES REGULATING THAT IN THE PATIENTS OR ALTERED BASED ON OBESITY. YOU KNOW RHEUMATOID ARTHRITIS IS AN INFLAMMATORY DISEASE THAT IS DESTRUCTIVE TO YOUR JOINTS AND THE WAY WE'RE OPEN TO THESE DISEASES FIRST THE PATIENTS HAVE AN INCREASED RISK FOR INCREASED CARDIOVASCULAR EVENTS. HEART ATTACKS, STROKES AND IT'S NOT KNOWN WHY RA PATIENT HAVE INCREASED RISK FOR CARDIOVASCULAR DISEASES. THAT PIQUED OUR INTEREST. AT THE SAME TIME WE ALSO KNOW FOR MORE THAN THREE DECADES IN RA PATIENTS THE SERUM IGG TENDS TO HAVE INCREASED CONCENTRATION AND IT'S NOT KNOWN HOW IT'S HAPPENING IN THE RA PATIENTS AND WE USE THE MOUSE LINE THAT DEVELOPED RHEUMATOID ARTHRITIS AND WE SEE THE INJECTED MISSING YOU SEE THIS IS A NORMAL JOINT BUT WITHIN A WEEK THE MICE RECEIVING THE RA SERUM DEVELOPED SEVERE SWELLING. USING THE MOUSE MODEL AND WE GIVE THEM AND SEE IF WE CAN LIMIT THE SITUATION OF CARDIOVASCULAR DISEASE. YOU'RE LOOKING AT AFTER 12 AND 8 WEEKS ON THE DIET THE MOUSE INJECTED WITH RA SERUM HAVE SEVERE ATHEROSCLEROSIS. YOU SEE WITH THE RED STAINING AND LIPID DEPOSITION AND SEE THAT MIMICS THE ATHEROSCLEROSIS IN HUMANS AND LOOKED AT THE SAME MOUSE MODELS. YOU CAN SEE BY LOOKING AT THE ARTERIES YOU SEE THE MICE WHO INJECT WHO GOT RECEIVED THE RA SERUM WITH A LOT OF THROMBOSIS GOING ON. ALSO TIME TO OCCLUSION BECAUSE THE THROMBI IS SO LARGE IT'S A SHORT TIME AND WE MIMIC THE SITUATION IN THE RA MODELS THEY INCREASED THROMBOSIS AND SCLEROSIS AND USING THE MODEL YOU CAN SEE THERE ARE SIX ANIMALS IN EACH GROUP AND YOU CAN SEE THE IMMUNOGLOBULIN MIMICS THE HUMAN SITUATION. OUR GOAL IS TO USE THE MOUSE MODEL AND ASK A QUESTION WHETHER THE IGG HYPOSIALATETION IS CAUSED IN THE MOUSE AND WHY THEY HAVE LOW HYPOSIALYATION AND WE HAVE A LARGE COHORT AND CAN LOOK AT THE IGG PUT TO DO THAT WE NEED TO GO TO THE GLYCANS AND THEN COMES THE NIH COMMON FUND AND WE RECEIVED A SUPPLEMENT TO LOOK AT THE ASSESSMENT OF GLYCANS DEVELOPED BY THE GROUP AND SHE'LL PRESENT LATER THE METHOD AND WE CAN ASSESS THE TARGET PROTEIN IN OUR CASE IGG WITH THE SIALIC ACID OR HOW MUCH OF THE IGG IS CONTAINING THESE GLYCAN GLYCANS SO WE APPLIED THE METHOD AND IT'S HUMAN I GGs. WE EXPECT A LOT OF VARIATION BUT DIRECTIONALITY IS ALL CORRECT AND WE LOOKED AT THE ALPHA 26 IS LOWER AND THE GALACT OSE EXPOSURE AND A LEVEL OF GLUTAMATE IN HERE. WE'RE EXPECTING TO RUN THIS OF THIS EASY AND USING MOUSE MODELS AS WELL AS HUMAN PATIENTS AND I HOPE WE CAN GET THE INTERESTING RESULTS NEXT YEAR. WE HAVE TO HAVE OUR LOCAL MENTOR AND THE FUNDING AND THE METHOD. THANK YOU. >> THANK YOU VERY MUCH. IS THERE A QUICK QUESTION FOR HER? IF NOT WE'LL MOVE ON TO THE NEXT TALK WHICH WILL BE CATHERINE GRIMES. GO AHEAD. YOU'RE ALL SET TO GO. >> I MISS SEEING EVERYONE IN PERSON BUT I'M EXCITED TO TELL YOU WHAT MY GROUP IS DOING WORKING ON THE BUILDING OF BACTERIA CELL WALL BUILDING BLOCKS AND MY LAB IS INTERESTED IN THE GLYCAN AS THEY SLED FRAGMENT WHICH IS ARE IMMUNE MODULATORS. WE'RE INTERESTED IN STUDYING THIS PART OF THE BACTERIAL GLYCAN AND WE WANT A WAY TO ACCOUNT FOR ALL OF THESE AND THERE'S BUILDING BLOCKS SHOWN IN BLUE AND GREEN AND THERE'S DIFFERE STREPTOCOCCUS ORIUS AND OTHERS WHO LINK THEIR GLYCAN AND MODIFY THEM AS WELL AS. S AND IT WILL CHANGE THE AMINO ACIDS. SO MY GROUP AND I WANTED TO UNDERSTAND DO THESE DIFFERENT VAGMENTS HAVE DIFFERENT RESPONSES AND GIVE A QUICK SAMPLING TEST. WE TOOK THE FRAGMENT MANY PEOPLE USE COMMERCIALLY AVAILABLE AND SYNTHESIZED THE FRAGMENTS FOUND IN YOUR YOGURT AND DO THEY ACTIVATE DIFFERENT IMMUNE SIGNALLING PATHWAYS IN DIFFERENT RESPONSES INSTEAD OF LOOKING AT ONE QUICK RESPONSE WE TEAMED UP WITH LOOKED AT DIFFERENT RESPONSES FROM THE THREE SEEMINGLY SIMILAR FRAGMENTS. AND WE WONDERED WHAT ARE THE RELATIVELY RELEVANT FRAGMENTS AND THIS IS WHERE OUR WORK COMES IN AND INTERESTED IN THE PEPTIDOGLYCAN AND WANT TO CONTAIN CONVENIENT CHEMICAL BIOLOGY HANDLES AND WE WONDERED HOW DO WE ENGINEER THE SYSTEM. AND THEY START WITH SEEMING SUGARS AND IT GETS POLYMERIZED AND WE THOUGHT USING THIS FIRST COMMITTED STEP WE COULD BUILD THE SUGARS. WE TOOK A PAGE FROM KINASE IN THE TRANSFERASE AND CAN BUILD THIS IN SITU AND THEN WE CAN EVEN SEE THEM INSIDE OF MACROPHAGE CELLS WITH HIGH RESOLUTION AND WONDERED IF WE CAN EXPAND THIS TO OTHER FRAGMENTS AND OTHER TYPES OF BACTERIA. AND WE WOULDN'T HAVE TO RELY ON THE GENETIC METHOD TO INSTALL THESE IN THE GENOMES OF CERTAIN BACTERIA. WE WERE HELPED TO DRIVE THE MICROBIOLOGY ASPECT OF THE PROJECT. AND MY LAST UPDATE AND YOU CAN MAKE THESE ON A SCALE WHICH IS NICE AND CAN USE THESE TO LABEL THE BACILLUS AND WE INCLUDED THE LABELLING APPROACH AND WE'RE DEVELOP THESE THAT ARE SMALLER THAN THE FIRST GIN REACTION TETRAZINES. AND WE CAN BUILD THEM OUT AND WE'RE COLLABORATING AND DETERMINING IF WE CAN UNDERSTAND WHERE THESE ENZYMES INTERACT WITH THE LIPID TO SUBSTRATES. WE WOULD HAVE THIS AND CAN BEGIN TO UNDERSTAND WHERE THEY BIND. WE DID THIS IN AN E. COLI SYSTEM AND LOOKED AT OTHER BACTERIA WE COULD LABEL AND I'M NOT A MICRO BIOLOGIST BY TRAINING AND WE'VE ALSO FARMED THIS OUT TO OTHER LABS FROM THE COMMON FUND AND WE'VE BEEN ABLE TO DELIVER THE PROBE TO OTHER LABS ACROSS THE WORLD AND YOU CAN SEE THE STUDY OF H.PYLORI AND WE ARE LOOKING AT HOW IT'S BUILT IN A MODEL AND THEN WE'VE ALSO LABELLED MICROBACTERIA AND WE'RE EXCITED WE CAN START TO LABEL BACTERIA AND NOW WHAT WE WANT TO DO IS GAIN INFORMATION OF WHAT THE FRAGMENTS RELEASED FROM THE MATERIALS. WE'RE USING THESE ISO TAGS THAT WERE DEVELOPED AS PART OF THE COMMON FUND AND WE'LL TREAT WHAT WAS LABELLED WITH MACROPHAGES ON THE LIFE SPAN AND WHICH WE WILL ENRICH AND HAVE THE ISO TAG WE CAN START TO DIG DOWN TO GATHER WHAT FRAGMENT ARE RELEASED. WE CAN SEE DIFFERENT FRAGMENTS BEING RELEASED FROM E. COLI AND LOOKING TO DIFFERENT FRAGMENTS. AND WE WONDER HOW CAN WE PERHAPS DO THIS IN A MORE EFFICIENT WAY. THE BASIC STRATEGY OF HOW WE LABEL OUR BACTERIA IS WE'RE GOING INSERT WITH OUR PROBE AND IF IT NEEDS THE BIND WE'LL GET IT THERE AS WELL AND THEN USE CHEMISTRY AND CAN USE THIS APPLICATION AND POTENTIALLY IDENTIFY THE CELL WALL FRAGMENTS POTENTIALLY POST INCUBATION WITH THE MACROPHAGE. WE WONDERED ABOUT THE FIRST PLACE TO OPTIMIZE WAS THE SYNTHESIS OF THIS PROBE. WHEN THEY FIRST STARTED THIS THEY USED A TRANSFER REACTION TO INSTALL THIS ON THE MOLECULE AND THEN PROTECTED AND INSTALLED HER ACID AND HYDROGENATED TO YIELD THE AMINO DERIVATIVE TO THEN DECORATE WITH THE APPROACH. WE LIKE DOING THIS HOWEVER, ON SCALE IN ACADEMIA LAB WE WERE NERVOUS TO GO MUCH ABOVE 5 GRAMS AND THEY ESTABLISHED A WAY TO DO THIS ON A LARGE SCALE NOW ON OU GROUP TO CREATE THE DERIVATIVES TO THEN DECORATE. WE LIKE WE DON'T HAVE TO DO THE TRANSFER REACTION SO WE WONDERED POTENTIALLY WE CAN GET OUR PROBES INTO BACTERIA A LITTLE BIT BETTER I THINK GLYCOBIOLOGISTS KNOW THEY CAN ACETYL THESE. AND WHAT THEY FOUND IS THAT E. COLI DON'T LIKE THIS AND THEY CAN'T TAKE IT OFF AND DON'T APPEAR TO HAVE THE CORRECT ACETYLASES TO REMOVE THE ACETATES BUT HAVE AN ASTERASE AND CAN LABEL IT. WE USED A PROBE THAT APPEARS TO BE MORE EFFICIENT WHEN WE CAP WITH THE METHYLESTER AND CAN REMOVE THOSE SPECIFIC SUGARS. WE'RE EXCITED WE LEARNED A LOT ABOUT THE SUGARS AND WE CAN DO THIS ON SCALE AND CAN DO LOTS OF DIFFERENT THINGS AND GROUPS AND EXPANDED THIS ACROSS THE VARIETY OF DIFFERENT TYPES OF ORGANISMS THAT SHOWED THIS AS AN EFFICIENT WAY TO LABEL THE GLYCAN. WE'RE PACKAGING OUR METHODOLOGY TO LABS ACROSS THE WORLD WE SHARED THIS WITH MANY DIFFERENT LABS ACROSS THE WORLD AND WE'VE ALSO BEEN LUCKY TO PUBLISH WITH OUR OWN AND OTHER COLLABORATORS. WE PUBLISHED A VARIETY OF METHODS ON PAPERS BOTH ON CENTRAL BIOLOGY AND SHOWING TO MAKE THE SUBSTRATES. WE'VE ALSO PUBLISHED ONE IN HOW TO GROW IN DIFFERENT MICRO ENVIRONMENTS WITH MACROPHAGES AND POTENTIALLY THE RIGHT SURFACES. IN ADDITION WE'VE BEEN BUSY ADVERTISING OUR METHODOLOGY AND NATASHA AND I HAVE BEEN ORGANIZING WORKSHOPS AND EXCITED THAT IT SEEMS AS WE MAY HAVE FOUND A PARTNER TO POTENTIALLY DISTRIBUTE THIS FOR US WHICH IS IMPORTANT BECAUSE WE WON'T HAVE TO KEEP SENDING IT OUT. FINALLY WE'D HAVE TO ACKNOWLEDGE THE GREAT STUDENTS WHO DID ALL THE WORK AND IN ADDITION, JENNING TAYLOR AND DOING PILOT STUDIES AND SHOWING WE CAN USE A VARIETY OF SETTINGS. THANK YOU VERY MUCH FOR YOUR TIME AND ATTENTION AND I CAN'T WAIT TO GET TOGETHER NEXT YEAR IN BETHESDA AND WILL TAKE QUESTIONS IF THERE'S TIME. >> THANK YOU, CATHERINE. WE HAVE A COUPLE MINUTES. >> WHAT'S IT SAY ABOUT THE TRANSFERASES AND HOW IT'S UTILIZED? >> IT'S A PRETTY ESSENTIAL SYSTEM. I THINK WE ALL KNOW WHERE BACTERIA LIEKDZ TO LIVE. -- LIKES TO LIVE. >> ANYBODY ELSE? IF NOT YEAR PRETTY WELL ON -- IF NOT WE'RE PRETTY WELL ON SCHEDULE. THERE WAS A GLITCH WITH DR. STOWELL AND WE'LL NOW HAVE OUR NEXT PRESENTATION FROM DR. CHRISTY PETERSEN. ARE YOU READY AT THIS POINT TO GIVE YOUR PRESENTATION? >> SORRY TO PUT YOU ON THE SPOT BUT IF YOU'RE READY TO GO? >> SURE. ACTUALLY DR. GRIMES DID A GREAT JOB OF INTRODUCE WHAT WE'LL BE TALKING ABOUT. THAT IS SYNTHESIZING FRAGMENTS OF BORRELIA AND WE HAVE SHOWN YOU CAN USE E. COLI TO EXPRESS A BORRELIA PROTEIN THAT'S BEEN USED AS A HUMAN VACCINE AGAINST FIGHT AGAINST BORRELIA PULLED OFF THE MARKET FOR A VARIETY OF REASONS. PEOPLE DON'T SEEM TO MIND GIVING VACCINE TO MICE ESPECIALLY WHITE FOOTED MICE AND IF THEY EAT THIS BAIT THEY THEN DEVELOP ANTIBODIES WHICH THEN IS TAKEN UP BY THE TICK VECTOR THE BLACK LEGGED TICK THE VECTOR FOR LYME DISEASE AND IN THAT PROCESS THEY ARE ABLE TO ELIMINATE THE BORRELIA FROM THE TICK AND MAKES IT SO YOU STILL HAVE TICKS BUT YOU NO LONGER HAVE TICKS WITH BORRELIA IN THEM. WHAT THEY FOUND OVER MULTIPLE YEARS BECAUSE I KNOW THIS ISN'T NEWS TO ANYBODY WHO WANTS TO HEAR BUT TICKS CAN LIVE SEVERAL YEARS TO GO THROUGH THEIR FULL LIFE CYCLE. IN ORDER TO SEE THE EFFECT AF THIS VACCINE AND THE ECOLOGY OF LYME DISEASE WE HAD TO LOOK ACROSS FIVE YEARS AND IN THE CONTROL PLOTS THE AMOUNT OF BORRELIA WENT OVER THAT PERIOD OF TIME THEN DROPPED IN THE FIELD WITH TARGETED VACCINE. THIS SHOWS THAT WHEN MICE ARE GIVEN THE VACCINE THEY CAN GET RID OF THE BORRELIA IN THE TICKS BUT HASN'T SHOWN THE NEXT STEPS. I HAVE SHOWN THE NEXT SECTION AND WHAT WE ARE DOING IS WE ARE USING HUNTING DOGS WHO HAVE KENNELS AND USE THAT TO SHOW THIS WILL NOT ONLY GET THE BORRELIA OUT OF THE TICK BUT THEN PREVENT THESE DOGS AND PROXY FOR PEOPLE TO GET INFECTED WITH LYME DISEASE. AS A PART OF THAT, WE NEED MORE SENSITIVE AND OUR GROUP AND THERE'S BEEN VARIOUS GLYCO ARRAYS. WE'LL BE ABLE TO DO THE STANDARDS ALL THE WAY THROUGH THE EPIDEMIOLOGY AND FIELD ASSESSMENT OF THE DIAGNOSTICS. THE PEPTIDOGLYCAN IS WHAT ALLOWS THE VECTOR TO LIVE AND IF YOU LOOK CLOSELY AT THE STRUCTURE AS IT WAS SAID YOU'VE GOT THIS LONG STRAND THEN HAVE YOU THE BREADS AND THAT'S WHERE THE DIFFERENCE BETWEEN DIFFERENT SPECIES COMES IN. WE SPECIFICALLY WANT TO LOOK AT THIS WEIRD ONE THAT WAS MENTION ED IN THE LAST TALK. WHAT WAS SHOWN PREVIOUSLY IS OVER GENERATIONS OF BACTERIA YOU CAN SEE PEPTIDOGLYCAN ARE BEING SHED AND FIND THEM IN CULTURE AND SHOWN HERE BY INCORPORATING RADIO ACTIVITY THROUGH THE CARBON OR THE HYDROGEN IN THESE L ORNATHINE AND AS THE GOING OUT IN THE CULTURE AND TO SHOW THE OPPOSITE OF THAT AND THIS WAS AGAIN THE PATHWAY WAS SHOWN NICELY BY CATHERINE LIKE I ASKED HER TO SHOW ALL THIS STUFF. USING A BIO ASSAY OF THE DIFFERENT RECEPTORS ABLE TO SENSE THE NEURAL PEPTIDES OVER TIME AND CULTURE THE RECEPTORS ARE GOING TO BIND TO THOSE DIFFERENT PEPTIDOGLYCANS AND LEAD TO THE ACTIVATION WITHIN THE CELL AND YOU WILL LOSE THE ACTIVITY. IN SYNOVIAL FLUID OF MICE WHO HAVE LYME ARTHRITIS YOU WITH IS THE GLYCANS CONCENTRATING IF YOU GIVE THEM THE BORRELIA PEPTIDOGLYCAN ONE DAY AFTER INJECTION YOU SEE THE JUMP IN THE ARTHRITIC SCORE AND IN THEIR PREVALENCE ACROSS THE GROUP. IT'S NOT JUST A ONE-MOUSE THING BUT ALL OF THEM AND THEY ALL SEEM TO GET IT FROM PEPTIDOGLYCAN. AND THEY CAN BE DETECTED BEING SLOUGHED OFF THE BACTERIA OF THE BORRELIA AND THAT IS WORK WE STARTED WITH AN UNDERGRAD AND THIS IS MATERIALS WE'LL BE ABLE TO USE IN IMMUNOLOGICAL STUDIES AS WE FOUND SPECIFICALLY NATURAL KILLER T CELLS ARE VERY IMPORTANT. THE IMMUNE RESPONSE TO LYME DISEASE AND THEY HAPPEN TO BE THE EXACT IMMUNE CELLS THAT ARE RECOGNIZING THE STRUCTURES. WE HAVE SPECIFIC TARGETS TO PUT ON FOR DETECTION OF MONOCLONAL ANTIBODIES OR ANTIBODIES AS A WHOLE OUT OF PERHAPS JOINT FLUID. THIS IS THE PEPTIDOGLYCAN AND EACH NUMBER THEN REFERS TO THE DIFFERENT CHEMICAL SPECIES SHOWN HERE. IT WAS OUR GOAL TO PICK THIS IN THE BOX AND SEVERAL OTHERS AND USE THEM AS WHAT WE'LL HAVE FOR THOSE DIFFERENT MOLECULES TO DETECT AS STANDARDS BUT TO DETECT AND THEY'RE CREATING AMINO ACID BUILDING BLOCKS SPECIFIC FOR BORRELIA AND THEN THEN WE'LL HAVE MY POST-DOC GO NEXT WEEK AND START WITH THE BASICS OF SYNTHESIS AND IDENTIFICATION OF THESE AND THEN WORKING TO USE THEM IN TARGETS TO MAKE SURE WE CAN DETECT THEM IN CULTURE AND LASTLY OUR GOAL IS TO USE IT IN THIS ACTUAL TRIAL WE'RE DOING IN THE FIELD TO FIND TO BE ABLE TO BOAR EEL INFECTION EARLIER. AND THESE ARE OUR DOGS WHO HAVE HIGH TITERS FOR THE PEOPLE AT RISK OF THESE DISEASE. >> THANK YOU VERY MUCH. ANYBODY HAVE QUESTIONS FOR DR. PETSEN? PETERSEN? >> IF NOT WE'LL MOVE ON TO THE NEXT PRESENTATION WHICH WILL BE BY LORI YANG. >> HI, I'M LORI, FOUNDER AND CEO WITH LECTENZ BIO AND I'LL SUMMARIZE WHAT WE'VE ACCOMPLISHED AND PRESENT ADDITIONAL DATA. A DISCLOSURE WE ARE A FOR PROFIT COMPANY. WE ARE AN ACADEMIC SPIN OFF AT EGA AND PART OF AN ACADEMIC LAB. WE ARE HEADQUARTERED IN ATHENS, GEORGIA AND HAVE A SATELLITE FACILITY IN SAN DIEGO WHERE I AM BASED AND LIKE ANYONE ELSE THE PANDEMIC IS DISRUPTIVE IN DIFFERENT WAYS AND WE'RE TRYING TO KEEP GOING WHILE KEEPING SAFE AND THINGS ARE PICKING BACK UP. WHAT IS GLYCOSAN? THANK YOU FOR INTRODUCE THE ASSAY AND INTRODUCING FLOW CYTOMETRY AND GO INTO DETAIL ABOUT BOTH. IT'S ESSENTIALLY A MIX AND RUN ASSAY. BREADS ARE MULTI-PLEX MICROSPHERES WITH INTENSITY. EACH IS CONJUGATED TO A DIFFERENT GLYCAN SPECIFIC RE-AGENTS AND HAVE A GREEN FLUORESCENT GRIK FLORFLOO FLUORESCENT PROTEIN AND THEY FLOW IN A SINGLE FILE. EACH HAS A RATE OF HUNDREDS PER SECOND. LET ME DESCRIBE WHAT HAPPENED IN THE FLOW CELL TO GET THE DATA FOR THE FINAL GLYCOPROTEINS. FIRST LIGHT SCATTERED IS MEASURED IN RELATION TO THE CELL AND EACH DOT REPRESENTS AN EVENT. SO A TIGHT POPULATION BECAUSE THEY'RE VERY UNIFORM. AND THIS IS DRAWN AROUND SO ONLY SINGLE BREADS ARE ANALYZED AND NOT DEBRIS AND THE BEADS ARE MEASUREDED IN THE HISTOGRAM AND HERE THEY ARE SHOWN AS FIVE DISTINCT PEAKS. EACH PEAK HAS A MARKER SO THE GREEN FLUORESCENT CAN BE SEPARATELY MEASURED. WE SEE HIGH LEVELS OF GREEN FLUORESCENCE. IN OTHER WORDS, A HIGH AMOUNT OF GLYCOPROTEIN BECAUSE THIS HAS ONLY [INDISCERNIBLE]. WE CAN COMBINE THE TWO FLUORESCENT PARAMETERS INTO ONE LIGHT SCATTER. EACH DOT REPRESENTS A BEAD AND THEY'RE HIGH ON THE AXIS. THIS IS COMPARED TO THE OTHERS BECAUSE THEY ONLY HAVE GLYCOPROTEINS. AND HERE'S THE FLUORESCENCE OF EACH GLYCAN DETECTING BEAD. THEY CAN BE TAKEN OFF THE FLOW CYTOMETRY OUR CAN ANALYZE IT WITH THE SOFTWARE AND WE HAVE PROTOCOLS FOR THIS AND NOTE THIS DATA IS NOT ABSOLUTELY QUANTITATIVE. DIFFERENT GLYCAN BINDING PROTEINS HAVE DIFFERENT AFFINITY FOR THE RESPECTIVE GLYCAN. IT STARTS DETECTING CHANGES. GLYCOPROTEIN MADE OUT OF MORE THAN ONE GLYCOSYLATION AND WE TREATED FLUORESCENT LABEL WITH ENZYMES TO OBSERVE AND IT GOES UP BECAUSE IT WAS EXPOSED. SIMILARLY THIS WENT UP BECAUSE IT'S CONSISTENTLY WE SEE THE CHANGE. WE CROSS VALIDATED OUR ASSAYS ON OTHER CYTOMETRY AND WE HAVE A SIGH -- CYTOMETER THAT MEASURES THIS AND THE ANALYSIS AND IT ALSO SHOWED ENGINEERING OF PROTEINS THAT IS MONITORING THE ACTION OF GLYCOSYLATES. AND THIS WAS LABELLED WITH THE ALPHA 2 OR 3 STYLE TRANSFER. UNTREATED IT SHOWS HIGH LEVELS. AND TREATMENT OF THE T CELL TRANSFERS RESULTS IN AN INCREASE BECAUSE OF THE ACID BEING ADDED. AND THIS RESULTS IN AN INCREASE AND AS A REMINDER THIS IS NOT ABSOLUTELY QUANTITATIVE. AND THE RESPECTED ACID CAN DETECT LEAKAGE SPECIFIC ADDITION. SO HERE'S ANOTHER EXAMPLE. THIS TIME WE'RE MONITORING THE MOLECULES AS SEEN IN THE ENZYME AND WHEN TREATED WITH ACTIVE THE ORANGE BAR THERE IS AN INCREASE IN THE SIGNAL. NOT ABSOLUTELY QUANTITATIVE. IN SUMMARY THERE'S GLYCAN ANALYSIS THAT IS NOT A SUBSTITUTE FOR BUT COMPLEMENTS FULL CHARACTERIZATION AND IS MULTI PLEX. YOU CAN RUN THEM AT ONCE INTO MULTIPLE FEATURES AT THE SAME TIME AND MIX AND MATCH AND MAY COS TOMORROWIZE THEM IF THERE'S A PARTICULAR GLYCAN BINDING RE CENTER YOU'D LIKE. THERE'S NO NEED FOR RELEASE OF THE GLYCANS AND GLYCOPROTEIN PRIOR TO THE ANALYSIS SEPARATION OR EXTENSIVE WASHES AND EASY TO USE. DOES NOT REQUIRE A HIGHLY TRAINED PERSONNEL ONLY A BASIC BENCH TOP FLOW CYTOMETER OR OFTEN ACCESSIBLE. AND IT CAN BE MONITORED AFTER A SHORT BINDING TIME. WE PARTICIPATED IN THE LARGEST BIO INDUSTRY CONVECTION AND PARTICIPATED IN THE BIOLOGY DISCUSSION AND THE WHITE PAPER WAS SUBMITTED FROM LAST MAY. AND THANK YOU FOR THE PARTICIPANTS FOR THEIR WRITING CONTRIBUTIONS. HOPEFULLY THIS REACHES A WIDER AUDIENCE AS WELL. AND ALSO AS YOU HEARD NIH GRANTIS HAVE RECEIVED ADMINISTERED DUADMINISTER DOCUMENTS AND ONE WAS TO DR. MINEO AND DR. MOHAMED AND THEY'RE GENERATING DATA AND WILL BE ABLE TO DEVOTE MORE TIME TO THE PROJECT THIS SUMMER AND HERE'S A MIXTURE OF THE FIVE AND THE CRUISE CONTROL PROTEINS AND THIS IS AVAILABLE AND HOPEFULLY THINGS CONTINUE TO PICK UP. LASTLY, I'D LIKE TO THANK EVERYONE ON THE LECTENZ BIO TEAM AND THOSE KEY TO THE COMMERCIALIZATION AND THANKS OUR FUNDING SOURCES, COMMON FUND AND COMPANION FBIRR TO DEVELOP THE APPLICATION AND WOULD LIKE TO THANK MY CUSTOMERS, COLLABORATORS AND FOR ALL CREDIT CHECK OUT OUR WEBSITE AND I'D BE HAPPY TO ANSWER ANY QUESTIONS NOW IF THERE'S TIME. >> ANYONE HAVE QUESTIONS ABOUT HER SIMPLE PLATFORM HERE? >> LOOKS LIKE WE'RE RIGHT ON TIME. THE NEXT PRESENTATION WILL BE BY ANOTHER AWARDEE, DR. ECKMAN. >> THANK YOU FOR THE OPPORTUNITY TO TALK TO YOU. I'M THE ULTIMATE NON INSIDER. IT'S INTERESTING FOR ME TO SEE HOW FAR THE GLYCOSCIENCE FIELD HAS GONE. THE WORK I'M TALKING ABOUT IS A TIGHT COLLABORATION WITH A GROUP AT UCSD ABOUT THE ROLE OF SIALIC ACID IN THE GUT. IT'S LINKAGE TO THE CARBOHYDRATE CHANGE WE TOUCHED ON AND SHOW UP ON LIPIDS AND PROTEIN. IS THE REASON WE GOT INTERESTED IN FATTY ACIDS PARTICULARLY THE INTESTINE IS BECAUSE THEY'RE COMMON IN THE INTESTINE. THERE WAS EVIDENCE SOME OF THE IS ACETYLATIONS ARE INVOLVED AND THE INGREDIENTS ALONG THE GUT AXIS CHANGE IN INFLAMMATORY BOWEL DISEASE THAT CAN OCCUR. WE KNOW THERE'S A NUMBER OF ENZYMES INVOLVED BUT ONE EXISTS IN MICE AS AN ESTERASE AND MAY MAY HAVE A ROLE IN IBD, INFLAMMATORY BOWEL DISEASE AND KNOW GUT BACTERIA HAVE SIMILAR ENZYMES SO THEY CAN NIBBLE AWAY AT THE SIALIC ACID OF THE HOST. THE GENES ARE NOT FULLY UNDERSTOOD. WHAT SYNTHETIC GENES ARE NOT ON ENTIRELY KNOWN AND WE NEED TO DETERMINE THE FUNCTIONS OF THE ACETYLATEDD SIALIC ACID. NOW I THINK THE NEXT STEP WAS TO HAVE PROBES THAT CAN BE USED IN VARIOUS SYMPTOMS. THOSE ARE THE PROBES VALIDATED USING THE PROBES. IT SHOULD REFLECT THE SPECIFICITY AND THEN OTHER DETECTIONS. THIS ALLOWED US TO USE THE GLYCOSCIENCE TOOLS AS A NON-GLYCOSCIENCE EXPERT AND STUDY THEM. AND IT'S VERY MUCH FOCUSSED ON GUT INFLAMMATION AND HOST MICROBIAL INTERACTIONS IN THE GUT WITH THE TOOLS IN THE LAB. ONE QUESTION WE STARTED ASKING THEM ABOUT THE DEVELOPMENT EXPRESSION AND THE POTENTIAL OF THE MICROBIOTA WITH THE RESEARCH CENTER HAS GERM-FREE MICE TO DETERMINE WHAT THE LEVELS OF THE SIALIC ACIDS WERE IF WE DISSECT THIS MORE CAREFULLY IN THE MODIFIED FORM THERE'S NO SIGNIFICANT DIFFERENCE AND THEY ALL LOOK SIMILAR. WE DON'T THINK THE MICROBIOTA IS REQUIRED FOR THE PRODUCTION OR EXPRESSION OF THE PARTICULAR MODIFICATIONS AND HERE THE CONVENTIONAL AND GERM-FREE MICE BEFORE THEY'RE WEANED HAVE HIGHER LEVELS AND IT'S VERY SIMILAR TO THE CONVENTIONAL MICE. WE ALL THINK AGE PLAYS A ROLE IN THIS SYSTEM. THERE MUST BE A FUNCTION OF SOME SORT WE'RE TRYING TO DISCOVER. SO WE THEN LOOKED AT NORMAL EXPRESSION IN THE ADULT MOUSE FOR THE ASSIMILATED FORM USING THE PROBE. THIS IS THE 7-9 ACETYLATED FORM USING THE PROBE. AND YOU CAN SEE HERE THIS IS AT THE BASE OF THE STRUCTURE AND SO ON THE BOTTOM IS ROUGHLY THE BOTTOM THERE AND THE SAME SO AT THE BASE OF WHERE MOST THE STEM CELLS ARE THIS IS WHERE IT'S EXPRESSED. AND THIS IS NOT AT THE BASE SO THIS IS A DISTINCT EXPRESSION IN DIFFERENT REGIONS OF THE EPITHELIUM AND WE ALSO SEE SOME OF THE MODIFIED SIALIC ACIDS COME OUT AS MUTANTS. THIS IS SECRETED OUT AND LIES ON THE COLONIC SURFACE AND THEN WE WERE INTERESTED IN THE QUESTION WHAT CAN BE MODIFIED IN MANY FORMS IN ORDER TO GET TO A FUNCTIONAL ISSUE. SO THERE'S STUDIES GOING BACK SOME YEARS NOW THAT THIS ENZYME INITIALLY FOUND FROM THE HUMAN GENOME MINING EFFORT FINDING ENZYMES WITH TRANSFERASE ACTIVITY AND WHETHER IT'S IMPORTANT. ANOTHER GROUP GENERATE THE MICE AND SHOWED IN RED BLOOD CELLS THE NINE OSCILLATION DISAPPEARS IN THE KNOCKOUT. THE MICE HAVE ESSENTIAL L ESSENTIALLY AND HERE WE USE THE PROBE AND THE KNOCKOUTS DON'T SHOW THE STAINING. THIS IS COMPLETELY DEPENDENT ON THIS AND IF YOU FEEL THIS IS DEPENDING ON THE CASD1 AND WE CONFIRMED THIS THEN WE FIRST LOOKED AT A MODEL OF COLITIS BECAUSE AS OFTEN IN GUT FUNCTION AND IN OTHER SYSTEM TOO IF NOTHING HAPPENS YOU HAVE TO CHALLENGE THE SYSTEM TO SEE IF IT'S MORE LIKELY. THE WAY WE STRESS IT HERE IS THAT WE USE A CHEMICAL THAT'S GIVEN ORALLY IN THE DRINKING WATER AND THAT INDUCES ULCERATIONS AND IN INDUCES COLITIS AND CAN THEN EXPRESSION THE FORMS OF SIALIC ACID. YOU CAN SEE IN THE WILD TYPE AND KNOCKOUTS THIS IS THE TOTAL AND THIS GOES DOWN A LITTLE BIT BUT THERE'S THE KNOCKOUTS WHICH HARDLY HAVE ANYTHING AND THE WILD TYPE MICE DO AND IF YOU COMPARE THEM TO THE DSS THE LEVELS GO DOWN BY TWOFOLD OR SO. AND THIS AGAIN WAS CONFIRMED BY AND USED A NUMBER OF OTHER COLITIS MODELS NOT CHEMICALLY INDUCED AND FOUND THE LEVELS GO DOWN. AND HERE YOU SEE THE STAINING AND IN THE WE SEE SOME HERE BUT NOT IN THE CONTROL MICE AND THE CAS -- THE CASD1 DON'T HAVE STAINING AND HERE WE USED THE PROBE USED IN THE ACID. TELLING US THE CHANGE IS SPECIFIC TO THE 9 FORM. THE PHENOTYPE OF THE MICE WAS INTERESTING. HERE'S THE CLINICAL PHENOTYPE AND THE BODY WEIGHT MEASUREMENTS AND THE DSS WILD TYPE MICE IN RED AND THE KNOCKOUT MICE USED CANTLY MORE SO THERE'S AN OVERALL CLINICAL MEASURE AND IF WE LOOK AT THE HISTOLOGY IT CAUS CAUSES INFLAMMATION HERE AND THERE BUT THERE'S MORE EPITHELIAL LOSS AND IF WE MEASURE THEM AND ARE MORE SENSITIVE TO THE COLITIS. THAT'S WHAT WE FOUND WHEN IT WAS STRONGLY EXPRESSED AND THE DISTRIBUTION AND GERM-FREE MICE HAVE NORMAL LEVELS. AND THE COLITIS REDUCES IT OOJD THE CASD IS RESPONSIBLE AND INTESTINE IN GENERAL AS IT IS IN OTHER ACETYLATION AND THE CASD KNOCKOUT HAVE NO TEENT BUT SENSITIZES MICE TO INDUCED COLITIS. WE CONCLUDE THE ACETYLATION OF SIALIC ACID IS PROTECTIVE AND THERE'S NEW OPPORTUNITIES. WE DIDN'T THINK ABOUT IT WHEN WE FIRST STARTED TALKING ABOUT THIS AND IT'S MORE THAN JUST THE PROBE US NEED SOMEBODY TO TALK YOU THROUGH THE PROBLEMS SAYING TO BE AWARE OF THIS AND THAT AND IT'S A WONDERFUL COLLABORATION AND THEN THANK YOU TO THE NIH FOR THE ORIGINAL GRANT THAT MADE THE PROBE POSSIBLE AS WELL AS THE SUPPLEMENT THAT KICKED OFF THIS WHICH MAY HAVE NOT EXISTED OTHERWISE. THANK YOU. >> THANK YOU, DR. ECKMANN. YOU HAD A PRODUCTIVE STINT. >> HE LITERALLY HAS HIS OFFICE BELOW MY OFFICE. >> DOES ANYBODY HAVE QUESTIONS FOR DR. ECKMANN? >> I HAVE A QUESTION. >> SO THERE'S A LOT OF DATA ON CHANGES IN THE MICROBIOME TO COLITIS. I FEEL LIKE YOU CAN GO IN AND MINE THAT DATA AND LOOK FOR SIALIC ACID BINDING LECTINS ESPECIALLY IN THE MICROBES UP REGULATED. HAVE YOU DONE THAT? I THINK THAT MAY BE A COOL WAY TO LINK WHAT YOU FOUND TO SPECIFIC CHANGES IN THE MICROBIOME. >> WE HAVE NOT DONE THAT BUT WE'RE THINKING ABOUT MICROBES NOT SURPRISINGLY. ONE OF THE THINGS WE'RE DOING NOW WE'RE JUST STARTING ON IS TO LOOK AT THE MICROBIOMICS OF THE CHANGE IN THE CASD KNOCKOUT WITH AND WITHOUT INFLAMMATION. YOU'RE SAYING THE OPPOSITE. AND WE KNOW THERE'S A WAR OUT THERE FOR SIALIC ACID. BOTH THE MICROBES AND ALL ARE FIGHTING FOR IT. MY MAIN ISSUE SAND I NEED THE GUIDANCE OF THE EXPERTS, HOW DO YOU IDENTIFY THE MICROBIAL GENES. >> THERE ARE SOME SIALICASS AND SEE IF THEY'RE TAKING ADVANTAGE OF THE CHANGE IN THE ACCESSIBILITY OF THE NON-MODIFIED ACID. >> WE NEED METAGENOMICS. >> YES. >> I THINK IT MATE BE OUT THERE FOR THE CSS MODELS. >> LIKE THAT. I'LL KEEP THAT IN MIND. THANKS. >> ANY OTHER QUESTIONS. ? -- IF NOT. I'LL TAKE THE OPPORTUNITY TO SAY DR. DESAI IS STUCK OUT OF TOWN. HE WASN'T ABLE TO MAKE IT BACK. WE HAVE A VIDEO BUT WE CAN'T PLAY THE VIDEO. TECHNICALLY SOMETHING'S WRONG WITH THAT AND WE HAVE MORE TIME. GIVEN THAT WE'RE SUPPOSED TO HAVE A BREAK NOW. LET'S COME BACK AT 4:00 AND WE'LL RESUME WITH DR. SCHNAAR. >> WE'RE INTO 4:00. DO YOU WANT TO GET START? >> AS YOU WISH. >> FIRST, I WANT TO THANK ALL THE SPEAKERS FOR A GREAT DIVERSITY OF TECHNOLOGY. I ALSO WANT TO THANK THOSE WHO USED THE WORD GLYCOLIPID AND THANK YOU FOR THE USE OF OTHER TERMS. DON'T GET ME WRONG, I LIKE PROTEOMICS AND IN SOME TISSUES GLYCOLIPIDS DOMINATE. THEY'RE THE MAJOR GLYCAN CONSTITUENTS IN THE VERTEBRATE BRABE. THEY DRIVE PHYSIOLOGY AND PATHOLOGY WHICH IS WE'RE PURSUING THE TOOLS WE'RE PURSUE PURSUING. TO THE MOTIVATION FOR GANGLIO GANGLIOSIDES EXTEND OUT. THE IMPACT STRUCTURE REGULATE CELL DIVISION AND DIFFERENTIATION AND SUPPORT CELL INTERACTION. THEY'RE IMPLICATED IN CANCER PROGRESSION AND INTELLECTUAL DISABILITY AND WOUND HEALING AND PROTEINOPATHIES AND NEURODEGENERATIVE DISEASES. THEY HAVE GANGLIOSIDES IN DIFFERENT CONTEXTS. TOOL CREATION AND APPLICATION DONE BY A TEAM AND THEY'VE BEEN DEDICATED TO TOOLS AND SUCCESSFUL IN DOING SO THE TOOL KIT GANGLIOSIDES WERE CHOSEN AND MAYBE ABOUT 100 OR MORE GANGLIOSIDES HAVE BEEN TRACED AND IN THE EXAMPLE I'M SHOWING ARE GLYCANS AND SEROMIDE AND HAS SACCHARIDE WITH ONE OR MOHR SIALIC ACIDS DISTRIBUTED. AND GM3 REGULATES CELL DIVISION AND GM1 MODULATES NERVE GROWTH. THERE'S A THERAPEUTIC OR THE CANCER AND I'M PRESENTING THIS ON AS AN EXAMPLE OF GM1 SHOWN HERE. THEY HAVE CREATED THERE'S THREE TAGGED PROBES THE LACTOSE CAN ALSO BE DERIVEITIZED AND THIS CAN MAINTAIN A MINIMALLY DISRUPTIVE DUAL ACTION PROBE. SO I'M GOING TO WALK THROUGH THE CHEMISTRY. THESE HAVE INDIVIDUAL STEPS TO GENERATE THE MOLECULES. GM 1 CAN BE OXIDIZED AT THE SIALIC ACID TO TRIGGER A UNIQUE ALDEHYDE WITH AGENTS TO BE USED TO WITH A LINKAGE AND WE CAN TAKE THIS AND SEE ONE SPECIES AND THROUGH A MIXED MODE WITH A 43% WE HAVE TAG A. IT'S BEEN CHARACTERIZED BY MASS SPECT. THIS GOES FROM NATURAL GANGLIOSIDES AND BY PROTON. TAG B IS A TWO-STEP REACTION. ONE FROM A UNIVERSITY WAS KIND ENOUGH TO PROVIDE US WITH THE PLASMID FOR AN ENZYME THAT DOES A SINGLE STEP REMOVAL OF THE FATTY ACID. LEAVING THE TAGGING HERE YOU CAN EVEN SEE THE LENGTHS OF THE TAG B. THE REASON AND AGAIN THE STRUCTURES APPEAR WITH MATRIX AND BOTH PROTON AND CARBON. WE NEED TO KNOW IF THE GLYCOSE NEEDS TO BE INTACT AND BIND TO DIFFERENT SITES. WE HAVE SOME MODIFICATIONS FOR TOOL KIT EXPANSION WE HAVE GENERATED GANGLIOSIDES FROM NATURAL SOURCES AND SYNTHETICALLY. WE HAVE DIFFERENT VERSIONS AND HAVE DIFFERENT ONES THROUGH LARGE SCALE EXTRACTION AND WE HAVE NICE PURIFIED GANGLIOSIDES IN DIFFERENT QUANTITIES FOR MAKING THE TOOL KIT. WE HAVE A NUMBER WE'RE PURSUING. WE CAN PURCHASE THIS AND BY COLLABORATION WITH THE PURIFICATION OF GLYCOLIPIDS WILL HAVE THESE GD3. GM 1 CAN BE CONVERTED TO T12. OR BY TST1 SOLUBLE TRANSFERS PROVIDED AS A PLASMID FROM THE UNIVERSITY OF ALBERTA. LIKEWISE GT1B CAN BE CONVERTED AND THEN RECONVERTED BACK TO G21B FOR MODIFICATION. OR GD2 BY THE SAME. SO OUR ENTIRELY KIT IS NOW IN SITE FOR GENERATING MULTIPLE TAG FORMS OF EIGHT GANGLIOSIDES DRIVING MUCH OF THE GANGLIOSIDE FUNCTION. IN PAST WORK AT MY SITE WE HAD CELLS AND ARE QUICKLY TAKEN UP IN THE CELLS AND DEGRADED IN THE LICE -- LYSOSOME AND THEY ADDED THIS MIXTURE AND WE HAD A DEMONSTRATION PROJECT TO HUMAN EPIDERMOID CARCINOMA AND YOU CAN SEE IT'S GOT VERY NICE SURFACE DISTRIBUTION OF THE COBBLESTONE LIKE CELLS. THAT STEP IS NOW COMPLETE IN TERMS OF VALIDATING AND TO WRAP UP THE DEMONSTRATION PROJECTS I'M DOING CROSS VALIDATION AND WE'RE CHARGING THE CELLS BECAUSE THEY HAVE EPIDERMAL GROWTH FACTOR MODULATED BY THE CELLS AND THE NEURONAL CELLS WHICH HAVE TRACK A KNOWN TO BE MODULATED BY GANGLIOSIDES AND MOVING ON TO PRIMARY NEURONS TO TAG NEW ASPECTS. WE'LL ADDRESS THE PROTEINS AND SOLUBLIZE THE PROTEINS AND USE MASS SPECTOMETRY TO IDENTIFY INTERACTING PROTEINS AND THE BINDING SPECIFICITY. THAT'S WHERE WE ARE NOW AND HAVE THE KIT AVAILABLE AVAILABLE SHORTLY. I'LL THANK THE LABORATORY AND DAVE MIERS AND THE ASSISTANCE OF THE CONVERSIONS AND THE GROUP HERE AND WITH THAT HAPPY TO TAKE ANY QUESTIONS. >> THANK YOU, RON. I HAVE A QUESTION OFF THE BAT. I ASSUME YOU DON'T WANT TO SPEND THE REST OF YOUR LIFE MAKING THE TOOL KIT TO SUPPLY REST OF THE WORLD. DO YOU HAVE SOMEBODY THAT MAY WANT TO COMMERCIALIZE THIS FOR YOU? >> WE HAVEN'T LINED THAT UP YET. IT'S A RESEARCH TOOL AND WE'RE TRYING TO RAISE PROTOCOL TO CONVERT THIS TO SOMETHING WE HOPE TO BE COMMERCIALIZED. >> DOES SOMEONE ELSE HAVE A QUESTION? I LIKE HOW YOU'RE ADDING THE FUNCTIONALIZATION ON THE GANGLIOSIDE. DO YOU THINK IT WILL BE APPLIED TO THE LIPIDS? >> ABSOLUTELY. WE GO THROUGH SOME SOLUBLITY ISSUES AND LOSE SOME IS SIALIC ACID. IT MAY REQUIRE SOME TWEAKS TO SOLUBLIZE. I DON'T THINK THERE'LL BE ANY PROBLEMS WITH MOVING TO THE LIPIDS ESPECIALLY WITH THE ABILITY TO REMOVE FATTY ACID. WE'LL REPLACE IT. >> THANK YOU. >> HOW DO YOU GO ABOUT VALIDATING THE LIPIDS ARE BEHAVING JUST LIKE -- >> WE CAN'T VALIDATE THAT. WE CAN VALIDATE THE DISTRIBUTION AND THEY'RE ACCESSIBLE TO OTHER PROBES ON THE CELL SURFACE. GANGLIOSIDES SHOULD BE SIMILAR. NOW, ONE WAY WE CAN PERHAPS DO THIS IS IF MULTIPLE PROBES WORKED ON THE SAME TARGETS. THE REASON I SAY THAT IS BECAUSE ONCE THEY'RE IN THE MEMBRANE THEY DIFFUSE RAPIDLY. IF THEY HAVE THE NATURAL SEROMIDE I EXPECT THEM TO ACT NATURAL. IT'S A GREAT QUESTION. >> LOOK AT THE CONFIRMATION OF THE GROUP AND SEE IF THEY CAN VALIDATE WHETHER THEY'RE EQUIVALENT TO WILD TYPE. >> THAT'S A GREAT IDEA. ALREADY WE'VE SEEN INTERESTING TWEAKS. FOR EXAMPLE, WE GENERATED MONOCLONAL ANTIBODIES TO THE GANGLIOSIDES. AND SOME OF OUR ANTIBODIES WILL NOT RECOGNIZE THE SUGAR IF THE INTERNAL SIALIC ACID IS CARRYING THE TAG. AND WE'RE MAKING MULTIPLE FORMS. >> GREAT SET OF QUESTIONS. HANNES BUELOW IS NEXT. >> REMEMBER, WE WON'T BE HOSTING DR. DESAI SO YOU'LL BE AFTER HAN HANNES. >> I'LL MOVE ON TO THE TITLE OF THE GRANT THAT FUNDS US AND THE PRESENTATION. I WANT TO PREAMBLE MY PRESENTATION WITH THE FACT I'M HONORED TO BE PART OF THIS GROUP FROM WHICH I GAIN MUCH MORE THAN I CAN CONTRIBUTE AND THIS IS A CONSORTIUM OF FOUR LABS OR PEOPLE YOU SEE HERE AND I'M ONLY THE MESSENGER. SO HEPARAN ARE ALWAYS ATTACHED TO CELLS AND WHAT'S PARTICULAR ABOUT THESE HEPARAN SULFATES IS THEY CAN BE MODIFIED IN VARIOUS POSITIONS, FIVE TO BE EXACT. THEY ARE NOT ALWAYS MODIFIED AND THEY'RE HIGHLY SPECIFIC ENZYMES TRANSFERASES AND SO FORTH THAT INTRODUCE THE MODIFICATIONS DURING BIO SYNTHESIS. THESE HEPARAN SULFATE PRESENT ON ALL CELLS AND REALLY IMPORTANT. THEY'RE INVOLVED IN CELL SIGNALLING PATHWAYS AND MANY OTHERS. THEY'RE CHARACTERIZED BY AN EXTRAORDINARY MOLECULAR DIVERSITY DUE TO THE POSSIBILITY OF DIFFERENTIAL MODIFICATIONS AND HAVE CELLULAR SPECIFICITY. I'LL SHOW YOU IN A FEW MINUTES ACCOUPLE EXAMPLES TO SHOW YOU WHAT I MEAN BY THAT. THE ISSUE WITH THEM IS THEY'RE INCREDIBLY DIVERSE IN STRUCTURE EVEN THOSE FROM HOMOGENOUS TISSUES AND THE BIOCHEMICAL ANALYSIS REMAINS CHALLENGING AND ONLY POSSIBLE BY SPECIALIZED LABS WHICH WE DO NOT BELONG. IT REQUIRES ADVANCE MASS SPECT AND SO FORTH AND WE HEARD ABOUT THAT AND NOT AVAILABLE TO THE GLYCAN BIOLOGIST WHO WANTS TO STUDY SUCH MOLECULES. DESPITE THIS THE RESOLUTION REMAINS LIMITED BECAUSE OF THE HETEROGENEITY FROM NATURAL SOURCES. THE NETHERLANDS LOOKED AT THE ANTIBODIES AGAINST THE SULFATES AND ISOLATED 36 DIFFERENT ONES IS THAT RECOGNIZE DIFFERENT HEPARAN SULFATE AND THEY DO NOT RECOGNIZE AN INDIVIDUAL MODIFICATION A 6-0 OR 3-0 SULFATE BUT RECOGNIZE AN EPITOPE. THE SECOND IMPORTANT THING TO APPRECIATE IS THEY RECOGNIZE MODIFICATION COMBINATIONS AND WE KNOW IN SOME CASES THAT THEY REQUIRE 3-0 OR 6-0 SULFATION BUT THE REMAINDER REMAINS ELUSIVE. AS I MENTIONED THE CELLULAR SPECIFICITY IS QUITE ASTOUNDING AND ALREADY RECOGNIZED IN THE 1998 PAPER AND STAINED RAT KIDNEY WITH THREE DIFFERENT ANTIBODIES AND YOU CAN IMMEDIATELY APPRECIATE THE ANTIBODIES STAINED THE TISSUE DIFFERENTLY. THIS ONE STAINS THE GLUMERROUS AND THE SAME IS TRUE IN WORMS WHICH IS MY PRIMARY RESEARCH AND IN THE C ELEGANS YOU CAN SEE WHAT IS A CELLULAR SPECIFICITY OF THE ANTIBODIES. AND LED TO THE IDEA OF WHETHER YOU CAN HARNESS THESE AND CREATE A BAR CODE FOR A CELL AND WOULD CREATE A BAR CODE TO IDENTIFY A CELL. AND THIS HAS FRAGMENTED ANTIBODIES TO DEFINED CELLS OF CHOICE AND MAYBE USE THEM TO ISOLATE SUCH CELLS AND THEN CAN WE DEFINE THE EPITOPES RECOGNIZED BY THE FRAGMENTS AND SO IN THIS WAY AT LEAST GARNER SOME STRUCTURAL INSIGHT WITH REGARD TO THE HEPARAN SULFATE ON OUR CELLS OF INTEREST. THAT'S THE PREMISE FOR THE GRAND FOR THE UL 1 WITH THREE SPECIFIC AIMS OR STILL HAS TO DEVELOP A FLUORESCENT SPACE HIGH THROUGHPUT PLATFORM TO DETERMINE THE BINDING PROTEEFS RECOGNIZED BY DIFFERENT ANTIBODIES AND DEFINE THE CELL POPULATIONS IN THE CELL LINEAGES. AND I'M SHOWING YOU THIS TABLE NOT TO READ ET REALLY BUT SEE WE'RE MAKING PROGRESS AND HAVE SOME SPECIFIC ANTIBODIES AND HAVE PROTEIN PREPS AND NOW HAVE THE MAJORITY OF TAGGED VERSIONS AND STARTING TO FILL UP THE COLUMN FOR THE FC CONSTRUCTS. HAVING THE TOOLS IN HAND WE DECIDED TO TEST THE HYPOTHESIS AND WHETHER WE CAN USE THE APPROACH AND USE IT FIRST ON FOUR DIFFERENT LUKEMIC CELL LINE AND A LEUKEMIA CELL LINE AND MYELOID CELL LINE IN THE MOUSE AND WHAT YOU CAN SEE ERYTHROLEUKEMIA AND YOU CAN SEE ALL THE CELL LINES IN FACT SHOW PATTERNS THEY MAY BE SIMILAR AND SO WE WITH AIM ONE TO FILL IN THE TABLE, IF YOU WILL, AND THE SECOND AIM IS TO DETERMINE THE BINDING MOTIFS THAT ARE RECOGNIZED BY DIFFERENT HEPARAN SULFATE SPECIFIC ANTIBODIES. AND USING IT IN COLLABORATION AND DEVELOP SOFTWARE TOOLS AND YOU HAVE THE PROGENITOR HERE AND THE ERYTHROCYTE PROGENITOR AND THEN A MACROPHAGE PROGENITOR AND ANOTHER STEM CELL THAT GIVES RISE TO THIS BRANCH. AND USE MARKERS TO PURIFY THE THREE CELL POPULATIONS. THEN TESTED WHICH OF THE ANTIBODIES FOUND WHERE. AND WHAT BECAME APPARENT WAS THAT IT WAS WITH THE ANTIBODIES WE USED IT WAS OF PARTICULAR THE MEGA CARRIER POPULATION SITE SHOWED STAINING. THIS IS REFLECTED IN THE HEAT MAP HERE WHERE YOU SEE THE DIFFERENT POPULATIONS AND PROGENITOR BRANCHES AND MOST OF THE STAINING BUT NOT ALL WAS PRESENT IN THE MEGA CARRIER SITE ERYTHROCYTE PROGENITORS AND THE EXPERIMENT WE DID WAS FIRST ISOLATED THE MEPs AND THEN DIVEDED THEM INTO HS3A8 AND THE HIGH BINDING POPULATION AND THEN THE LOW BINDING POPULATION. THEN SUBJECTED BOTH TO RNA SEQUENCING TO SEE HOW THE POPULATIONS ARE DIFFERENT. WHAT BECAME REALLY APPARENT ALMOST IMMEDIATELY IS THAT THE HIGH BINDING CELLS OBVIOUSLY THEY CLUSTER DIFFERENTIALLY BUT THE HIGH BINDING CELLS EXPRESSED MARKED GENES OF ERYTHROCYTES WHERE THE LOW BINDING GENES EXPRESSED GENES THAT LED TO CARRIER CHANGES YOU SEE ON THE RIGHT SIDE. WE THEN USED A HETEROGENEOUS POPULATION OF HEMATOPOIETIC CELLS AND THEN RNA CELL SEQUENCING AND YOU NOW SEE THE FAMILIAR PLOTS THAT IDENTIFY THE CELL POPULATION ARE PRESENT IN SUCH A HETEROGENEOUS POOL BUT YOU SEE THE ARM THAT LEADS TO THE MEP BRANCH IS CLEARLY MISSING IN THOSE CELLS LOW-BINDING COMPARED TO THOSE THAT ARE HIGH-BINDING. THEN WE WENT A STEP FURTHER AND LOOKED AT THE EXPRESSION OF THE SET OF HEPARAN SULFATE RELATED GENES INCLUDING SOME OF THE MODIFICATION ENZYMES. AND WHAT YOU CAN SEE IN THIS POPULATION OF CELLS HERE THESE ARE THE MEGA CARRIER SITES AND ERYTHROCYTE PROGREN -- PROGENITOR WAS HIGHLY EXPRESSED. THE TWO ENZYMES WERE PARTICULARLY HIGHLY EXPRESSED. WE KNOW OF SOME OF THE CHARACTERISTICS OF THE BINDING SITE AND I DON'T WANT TO SPEND TOO MUCH TIME EXPLAINING EXACTLY WHAT THIS POSITION WEIGHT MATRIX SIGNIFIES BUT SUFFICE TO SAY THE S REFLECTS AN N SULFATE AND THE 6 REFLECTS THE SULFATE AND THIS IS RICH IN ENSULFATE AND SULFATE AND THIS TELLS US OUR ANTIBODIES THAT ISOLATE THOSE CELLS ARE A WAY OF LOOKING AT THE TRANSCRIPT TRANSCRIPTOME IN A NON-DESTRUCTIVE WAY AND IT'S AN APPROACH TO ISOLATE CELLS FROM SOMETIMES HIGHLY HETEROGENEOUS POOLS. TO SUMMARIZE WE CAN USE THE PANEL OF FRAGMENT ANTIBODIES TO GLYCOTYPE CELLS AND NO REASONS TO THINK YOU COULDN'T APPLY IT. THERE'S EXPERIMENTS AND ONE OF THE ADVANTAGES AND I THINK THAT'S IN THE VEIN OF THE WHOLE PURPOSE OF THIS GROUP IS YOU CAN USE THESE IF YOU WANT TO AND BE AGNOSTIC TO THE GLYCAN STRUCTURE AND USE THEM AS MARKERS IF YOU WANT TO. YOU CAN ALSO USE THEM TO GET INSIGHT IN THE STRUCTURAL CHARACTERISTICS OF THE SURFACE OF THE CELLS YOU'RE INTERESTED IN AND WITH THE IDENTIFICATION OF THE EPITOPES AND MORE ADDITIONAL RE-AGENTS WE'LL GET FURTHER AND MORE INSIGHT AND ALLOW A HIGH RESOLUTION PURIFICATION OF SPECIFIC CELLS OR CELL TYPES. IN COMPARISON TO RNA SEQ. I HAVE TO ACKNOWLEDGE MY COLLABORATORS WHO ARE DOING THE COM COMPUTATIONAL WORK AND OTHERS AND HAPPY TO TAKE ANY QUESTIONS. >> THANK YOU. ANY QUESTIONS? >> HAVE YOU TRIED USING THESE AT ALL IN IMAGING? >> YOU MEAN THE IMMUNOHISTOCHEMISTRY? >> YEAH. >> WHAT I'VE DONE OR WHAT WE'VE DONE IS USED THEM IN WORMS. WE FUSED THEM AND SECRETED THEM IN THE BODY CAVITY AND CREATED LIVE ANIMALS THAT ANTIBODY STAIN THEMSELVES. THAT'S WHAT THE IMAGE WAS FROM WHERE YOU HAD A SINGLE NEURO IDE STAIN. >> JUST WONDERING IF YOU'VE DONE TISSUES. >> I HAVEN'T BUT PEOPLE HAVE. THE LITERATURE IS FULL OF PEOPLE USING THEM ON TISSUES. >> YEAH, JUST WONDERED ABOUT YOURS. >> YOU MEAN ON THE HEMATOPOIETIC CELL IMAGE? >> YEAH. I'D LOVE TO TALK TO YOU OFF-LINE. I'M JUST ASKING BECAUSE NOT ALL RE-AGENTS WORK IN ALL CONTEXTS AS YOU KNOW. THAT'S WHY I AM ASKING. >> YEAH. >> I HAVE A QUICK ONE. ARE THEY INHIBITING? >> THAT'S SOMETHING I CAN'T ANSWER AND SOME ARE. WE SHOWED EXPRESSING SOME ANTIBODIES IN WORMS HAS AFFECTS ON THE DEVELOPMENT OF SYNAPSES IN THAT CASE. SOME MAY VERY WELL BE INACTIVATING BUT WE DON'T KNOW THAT SYSTEMATICALLY LIKE WE DO NOT REALLY KNOW WHAT THE EPITOPES ARE FOR THE MOST PART. S >> THANK YOU, VERY MUCH, HANNES. WE'LL MOVE ON TO THE NEXT TALK, JONATHAN AMSTER. >> ALL RIGHT. HOPEFULLY YOU CAN SEE MY SLIDES. YES? >> YEP. >> VERY GOOD. I'LL KEEP TALKING ABOUT THE GLYCANS. OUR PROJECT HAS BEEN THE LAST FEW YEAR TO DEVELOP A PLATFORM FOR AUTOMATING THE GLY AMINO GLYCANS AND GLYCOSE AMINO GLYCANS. WE HAVE NO CONFLICTS OF INTEREST TO DISCLOSE. FOCUS HAS BEEN ON GLYCOSAMINOGLYCANS AND WE'RE INTERESTED IN THEM. FOR MANY REASONS ONE IS THEIR ORDNANCE TO DISEASE AND THEIR STRUCTURAL FEATURES PRESENT CHALLENGES AND OPPORTUNITIES AND THEIR LINEAR POLYMERS OPPOSED TO THE GLYCANS WE'VE BEEN HEARING ABOUT TODAY. THAT MAKES THEM EASIER TO ANALYZE BY MASS SPECTOMETRY AND THE MODIFICATION OCCUR IN THEM THEY'RE PRODUCED BY ENZYMATIC PROCESSES THAT DON'T GO TO COMPLETION. THE ONLY WAY ONE CAN FIND THESE THINGS IS TO MEASURE THEM. I CAN'T USE INFORMATICS AND WE'VE BEEN WORKING ON THE STRUCTURES ABOUT A DOZEN YEARS NOW. SOME METHODS WE HAVE ARE SPECIALIZED BUT THOUGHT IT WOULD BE NICE TO BUILD A PLATFORM THAT BIOLOGISTS COULD USE TO ANSWER IMPORTANT PROBLEMS. SO WE BROKE THIS DOWN TO FOUR GENERAL CATEGORIES OF EFFORT. ONE IS SO HAVE METHODS THAT ARE COMPATIBLE WITH MASS SPECTO METRY AND THERE'S MODIFICATIONS THAT LEAD TO TARGET PROTEINS. ANOTHER IS TO HAVE ON LINE SEPARATION METHODS WE USE CAPILLARY ELECTROPARESIS FOR THIS PURPOSE TO SEPARATE WHATEVER PICTURES AND INTRODUCE THEM IN THE MASS SPECTROMETRY AND TRIED OPTIMIZE IT FOR GLYCOSAMINOGLYCANS. WHAT WE GET IS COMPLICATED AND INFORMATIONALLY RICH. IT WOULD BENEFIT DISSEMINATION OF THESE METHODS TO PEOPLE WHO ARE NOT MASS SPECTRO METERS. THESE ARE THE FOUR PARTS OF THE PROJECT WE'VE WORKED ON WITH A GOAL OF CREATING THE TECHNOLOGY THAT CAN THEN BE USED BY OTHERS LIKE GLYCOGUYOLOGIST -- GLYCOBIOLOGISTS AND I'LL QUICKLY GO THROUGH THE ACCOMPLISHMENTS WITHOUT DIVING INTO THE DETAILS THAT ONLY MASS SPECT PEOPLE WOULD CARE ABOUT. IN TERMS OF ISOLATING SEQUENCES WHAT WE WANT IS A PROCEDURE WITH OLIGOMER THAT HAVE BEEN RELEASED IN A LARGER CHAIN 50 TO 200 SUGARS IN LENGTH BUT THE REACH THAT BINDS TO THE PROTEIN COULD BE 4 OR 5 OR 6 SUGARS OR AS MANY AS 10 OR 20. WE POLYMERIZED THE PIECES AND WANT A METHOD THAT FINDS THE PARTS THAT BIND SPECIFICALLY TO WHATEVER TARGET PROTEIN WE'RE INTERESTED IN STUDYING. SO THE CHALLENGES ARE THE SAMPLE HAS TO BE PRESENT IN AN AMOUNT AND CONCENTRATION SUITABLE FOR THE MASS SPECT AND SEPARATION THAT WILL FOLLOW IN SOLVING COMPOSITION HAS TO MATCH THE REQUIREMENT. THINGS YOU MIGHT THINK OF DOING THAT WORK WELL FOR OTHER TYPES OF COMPOUNDS FOR INSTANCE USING MAGNETIC FEED TECHNOLOGY DOESN'T GIVE YOU ENOUGH. THERE WAS A FILTER DEVELOPED TO PRODUCE ENOUGH SAMPLE FOR MASS SPECT STUDIES. THAT WAS PUBLISHED JUST LAST YEAR. WITH RESPECT TO SEPARATION WE'VE BEEN USING GAGS FOR SEPARATION. THEY'RE HIGHLY IONIC AND NOT WELL SUITED FOR DOING THINGS LIKE REVERSE PHASE MAMMOTOGRAPHY AND THERE'S BEEN THINGS THAT EASILY IONIZE AND AND HAVE TO MODIFY THE SURFACE AND SO WE PUBLISHED ON THIS AND THAT IS WORKING QUITE WELL. WHERE WE PUT MOST OF OUR ENERGY THOUGH IS AFTER ONE HAS ISOLATED L LIGOSACCHARIDES OF INTEREST AND WE GET MOLECULAR WEIGHTS BECAUSE THE IONIZATION IS STABLE AND YOU CAN DETERMINE THE COMPOSITION AND DEGREE OF POLYMERIZATION AND ASSCETYLS ARE PRESENT BUT DON'T HAVE THE SEQUENCE INFORMATION AND WE HAVE TO FRAGMENT THE IONS AND PUT ENERGY AND BREAK GLYCOSITTIC BONDS AND GIVES INFORMATION ABOUT THE STRUCTURE OF GAGS BUT WE DON'T WANT TO DO THOUGH IS HAVE A LOSS OF THE MODIFICATION. YET, THAT'S THE GROUP MOST PRESENT AND THE BASIC IDEA IS THERE'S COMPONENTS ONE CAN BUY COMMERCIALLY SO IT CAN BE DUPLICATED AND IT WILL BE AN INSTRUMENT AND ORBIT MASS SPECK AND COMMERCIAL INTERPHASE AND THESE THREE PARTS GO TOGETHER. AND IN THE INTERFACE THE BASIC IDEA IS WE HAVE A SEPARATION CAPILLARY THAT GOES THROUGH THE SPRAY EMITTER FOR DOING ELECTROSPRAY. AS COMPOUNDS COME OUT OF THE SEPARATION CAPILLARY THEY ENTER THE CONICAL END OF THE SPRAY TIP AND THERE'S A VERY SMALL MIXING VOLUME AND SO THE PARAMETERS WE HAVE CONTROL OF THAT AFFECT THE SEPARATION ARE THE COATING ON THE SEPARATION CAPILLARY AND THE EXPIRATION OF THE ELECTROLYTE AS WELL AS THE SAMPLE SOLUTION AND A SHEATH LIQUID THAT PASSES AROUND THE OUTSIDE OF THE SEPARATION CAPILLARY. THESE DETAILS HAVEN'T BEEN PUBLISHED BUT WE HAVE GOTTEN GOOD CONTROL OF THESE AND SO AN EXAMPLE OF A SEPARATION THAT WE WERE ABLE TO DO TWO OR THREE YEARS AGO IS THE SEPARATION OF ENOXAPARIN. YOU CAN SEE THERE'S NOT TOTAL BASELINE SEPARATION OF ALL THE COMPONENTS BUT WE WERE ABLE TO CHARACTERIZE THE COMPOSITION FROM THE MOLECULAR WEIGHT OF THESE AND THERE WERE TRIMERS AND UP TO 20 PRESENT. WE IMPROVED OUR ABILITY TO DO SEPARATION. THIS IS THE STATE OF THE ART FOR SEPARATION. THIS IS AN EXAMPLE OF HEPARAN SULFATES FROM SERUM. IT'S A PROJECT WITH COLLABORATORS WITH THE UNIVERSITY IN THE NETHERLANDS STUDYING HEPARIN BIOMARKERS OF KIDNEY DISEASE. THESE ARE SULFATES THAT HAVE BEEN EXTRACTED AND PUT ON THE CZE. SO THE NEXT STEP IS ION ACT A -- ACTIVATION AND 10 YEARS AGO WE HAD A CHOICE IN WHICH YOU BARRED YOUR SPECIES WITH ELECTRONS AND ACTIVATE THEM AND THEY FALL APART IN WAYS TO GIVE YOU GOOD INFORMATION FOR CHARACTERIZING THEIR STRUCTURES BUT THIS ONLY WORKS SOMETIMES AND HAD TO USE ELECTROMETHOD TRANSFORMATION AND THIS SHOWS VARIOUS COMPONENTS WITHIN ENOXAPARIN AND CAN GET THE MOST ABUNDANT COMPONENTS HERE AND THE NOT SO GOOD NEWS IS WE ONLY GET GLYCOSIDD IDI IDI IDIC CLEAVAG E AND THIS COULD BE A SUCCESS. NS WE KNOW BUT 6S OR 3S IS A QUESTION. AND THE ELECTROPARESIS LEADS TO THESE HEAVILY SULFATED MOLECULES. IT'S BEEN KNOWN FOR A LONG TIME IF YOU DON'T HAVE THE PROTONS YOU GET SUL VATE COMPOSITION. THIS HAS BEEN AN ISSUE IN THE PLATFORM. WE CONTINUED LOOKING AT DIFFERENT METHODS OF VAKT VASION AND LOOKED AT VIRTUALLY ALL THE ONES WE SEE ON THE LIST EXCEPT FOR THE BLUE ONES WHICH ONLY WORK HERE AND WE WENT TO THE LAB AT THE UNIVERSITY OF TEXAS TO DO PRELIMINARY EXPERIMENTS ON A TRAP EQUIPPED WITH A UV LASER. SO THIS IS THEPHERESIS THIS THIS IS A SACCHARIDE WITH EIGHT SULFATE GROUPS. WE'VE ONLY REMOVED FOUR PROTONS AND TWO CARBOXYL GROUPS AND IN THE PAST IF WE WERE TO DO TRANSFER ASSOCIATION THIS WOULD HAVE GIVEN US A SERIES OF DEPOSITION. -- DECOMPOSITION BUT NOW GETTING THE AMOUNT OF FRAGMENTATION TO TELL US WHERE THE SULFATES ARE WITHIN THE LIGAND. AND THIS IS A METHOD OF CHOICE AND PURSUING AN APPLICATION ON THIS. TO WRAP UP OUTREACH AND DISSEMINATION WE PUBLISHED QUITE A BIT ON SETTING THIS GRANT ON GAGS AND THESE HAVE GENERATED INTERESTING COLLABORATIONS WITH PEOPLE AROUND THE WORLD AND IN THE U.S. ON VARIOUS DISEASE RELATED AND INTERESTING BIOLOGICAL QUESTIONS THAT NEED THIS SORT OF TECHNOLOGY. AND WANT TO THANK THOSE WHO HELPED US OUT AND VERY THANKFUL FOR THE FUNDING WE'VE GOTTEN THROUGH THE COMMON FUND THAT BEGAN AS AN R21 AND CONTINUED. THANK YOU I'LL BE HAPPY TO TAKE QUESTION. >> I NOTICE IT SEEMS LIKE YOU FINALLY DEDUCED THE STRUCTURE OF THE WEIRD EPITOPE THOMAS CLAUSSEN HAD. >> I CAN'T SAY WE DID IT OURSELVES. THERE WAS A LOT OF DIFFERENT ASPECTS OF THE ASPECT. THEY DID SOME WORK. >> VERY INTERESTING EPITOPE. ANY OTHER QUESTIONS? >> YES. I'M WORKING ON SULFATES AND WHAT KEEPS THEM IN FACT IN THEIR POSITIONS IN TERMS OF DISSEMINATION. WE REACH OUT TO YOU? ALWAYS HAPPY TO FORM COLLABORATIONS FOR INTERESTING PROJECTS. AS I WAS SAYING THE METHOD THAT SEEMS TO DO THE BEST JOB RIGHT NOW IS ULTRA VIOLET ASSOCIATION AND IS A COMMERCIALLY AVAILABLE OPTION. >> THEY'RE PROBABLY NOT TOO EXCITED ABOUT HAVING TO LEARN HOW TO DO THIS TYPE OF PHORESIS. >> IF THERE'S NO OTHER QUESTIONS WE'LL MOVE TO OUR LAST TALK BY XU WANG. >> I'M CONTINUE WITH THE GLYCOSAMINOGLYCANS. TO DEVELOP A TECHNIQUE TO ANALYZE THE STRUCTURE OF GLYCANS. I SHOULD TELL YOU I'M NOT AN EXPERT IN SINGLE MOLECULE TECHNIQUES. I'M A BIO CHEMIST AND INTERESTED IN GLYCAN PROTEINS. I HAVE TO GIVE MY COLLABORATORS FOR DOING MOST THE WORK ON THE PROJECT. AND THEY'RE VERY IMPORTANT BIOLOGICALLY AND WE'RE INTERESTED IN THE STRUCTURE ACTIVITY RELATIONSHIPS. IT'S NOT EASY TO STUDY GAGS BECAUSE IT'S NOT EASY TO FIGURE OUT WHERE THEIR STRUCTURE IS. I LIST SOME OF THE CHALLENGES FOR DOING THAT. AND A SINGLE MOLECULE TECHNIQUE IS MOST SUITABLE FOR DOING THIS. AND WANTED TO LOOK AT A SINGLE MOLECULE TECHNIQUE THAT CAN BE USED TO SEQUENCE LINEAR BIO POLYMER. IT'S A SIMPLE CONCEPT. HAVE YOU THIS AND DRIVE THE ELECTRODES ON TOP. MAYBE ON BOTTOM BUT THE ELECTRODES ARE INSULATED BY A THIN LAYER. WHEN THEY ANALYZE IT INTERACTS WITH THE MOLECULES ON THE ELECTRODES. THIS CAN BRIDGE THE GAP AND HAVE YOU ONE ELECTRODE TO THE OTHER. THE IDEA IS THEY'LL INTERACT WITH THE MOLECULES DIFFERENTLY AND THE RESULT IS DIFFERENT PATTERN OF SPIKES. USING THE DIFFERENT PATTERNS OF SPIKES THESE ARE ELECTRONIC NOISE AND NOT HUMANLY INTERPRETABLE. IF YOU PUT THIS INTO A MACHINE LEARNING ALGORITHM USUALLY YOU CAN FIGURE OUT WHICH SIGNAL COMES FROM WHICH COMPOUND. AND HAVE FABRICATED THE DEVICE THAT CAN DO THE SEQUENCING. THE THING WE FIGURED OUT IS GLYCOSAMINOGLYCAN GOES THROUGH THIS EASILY AND WE CAN MONITOR AND EVERY TIME THE POLYMER GOES THROUGH WE CAN SEE THE CURRENT GOES DOWN. WE CAN SHOW IT GOES THROUGH EASILY AND THIS IS A SACCHARIDE AND ON AVERAGE WE SPENT LESS THAN 21 SECONDS ON THE PORE AND IT TURNS OUT TO BE A PROBLEM BECAUSE IT'S IN CONTACT WITH THE ELECTRODE TO COLLECT ENOUGH INFORMATION TO IDENTIFY THEM. IN THE SECOND PHASE OF THE PROJECT WE HAD TO OVERCOME HOW DO WE SLOW DOWN THE TRANS LOCATION OF THE POLYMERS. THEY'RE IN CONTACT WITH THE ELECTRODES FOR A CONSISTENT AMOUNT OF TIME. AND TO SOLVE THE PROBLEM MY COLLABORATE COLLABORATOR CAME UP WITH THE IDEA AND ESSENTIALLY WHAT WE DO IS WE ATTACH WHAT WE WANT TO ANALYZE TO THE PRIMER. AND THEN ON THE OTHER SIDE WE IMMOBILIZE AND GIVE THE PRIMER AND THE TEMPLATE TO THIS POLYMERASE. AS IT IT GROWS LONGER AND LONG IT'S GRADUALLY LOWERED AND BECAUSE IT'S TETHERED IT CANNOT GO THROUGH ANY FASTER THAN THE ENZYME. AND WE KNOW THE KINETICS OF THE ENZYME. THE SPEED SHOULD BE SUFFICIENTLY SLOW TO ALLOW THE CONTACT BETWEEN THE ELECTRODES AND THE SUGAR INJECTION. THAT'S THE IDEA FOR CONTROLLING IT. FOR THE NEXT FEW YEARS THE LAST FEW YEARS WE BASICALLY WORKED ON IMPLEMENTING THOSE AND TESTING THEM AND THIS IS HOW WE TESTED. WE HAVE IT HERE AND ATTACH THE ENZYME AND IMMOBILIZED IT ON ONE SIDE AND THEN WE GIVE IT A TEMPLATE AND THEN WE MONITORED THE CURRENT THROUGH THE NANOPORE AND INITIATE THE REACTION BY INJECTING TO THE SITE OF THE NANOPORE WHERE THE ENZYME IS AND WE SEE SOMETHING CHANGING APPROXIMATELY 10 MINUTES AFTER AND WE SEE THE CURRENT GOES DOWN INDICATING THERE'S SOMETHING IN THE PORE AND IT'S USUALLY SUSTAINED. THE LAST 10 SECONDS BEFORE IT RECOVERS AND IT REPRESENTS TRANS LOCATION OF THE SINGLE STRAND. HOW CAN WE CONFORM THAT? THERE'S A COUPLE THINGS WE DID TO CONFORMITY. -- CONFORM IT. WE THEN SEE THE TRANS LOCATION WE SAW IS OBVIOUSLY MUCH MORE THAN THAT. WE CLEARLY IT'S NOT UNCONTROLLED TRANS LOCATION. EVERY TIME WE SAW THE TRANS LOCATION SIGNAL WE SUBJECTED IT TO THAT SAMPLE AND SHOWED EVERY TIME WE SAW THE TRANS LOCATION SIGNAL WE ALSO SEE PRESENCE AND AIF WE DON'T SEE THAT WE DON'T SEE OUR PRODUCT. THAT SHOWS WE HAVE TRANS LOCATION AND NOT JUST [INDISCERNIBLE]. EVERY TIME WE SEE THE DECREASE WE SEE HOW WIDE IT IS IT'S CONSISTENT WITH TRANS LOCATION OF THE SINGLE STRAND MADE BY THE POLYMERASE. THOSE THREE TESTS BASICALLY TELL US YES THE CONTROLLED TRANS LOCATION APPARATUS DOES WORK. THE OTHER CHALLENGE TURNS OUT TO BE TOUGHER AND THE DIFFICULTY IS THE ELECTRODES NEED TO BE REALLY CLOSE. THE INSULATION LAYER HAS TO BE LESS THAN 2 NANO METERS. AND WE WERE ABLE TO PRODUCE A TRIP WITH THE SIGNALS AND THEY HAVE A LOT OF DISTINGUISHING CHARACTERISTICS THAT ALLOWS US TO TELL. BUT UNFORTUNATELY WE NEVER COULD PRODUCE THIS DEVICE WITH HIGH YIELD. THE GLYCAN WILL NEVER BECOME A REALITY. AND ONE THING IN THE ANALYSIS OF THE DIFFERENT MOLECULES IS TURNS OUT PROTEINS CAN PRODUCE ELECTRICITY. A SINGLE PROTEIN ACROSS ELECTRODE ELECTRODES WHAT IS INTERESTING IS THE CONNECTIVITY OF THE PROTEIN DEPENDING ON THE CONFIRMATION AND SO HE'S TRYING TO USE THIS DISCOVERY TO COME UP WITH A VALIDLY SINGLE MOLECULE DNA SEQUENCING DEVICE BY ATTACHING TO DO ELECTRODES AND THIS TYPE OF DEVICE TURNS OUT TO BE EASY TO MANUFACTURE BECAUSE THE PROTEINS DON'T NEED TO BE CLOSER OR CLOSE TO EACH OTHER. WE'RE USING A PROTEIN SENSOR TO BRIDGE ELECTRODES AND USING THE PROTEIN TO SENSE WHATEVER GLYCAN IS AVAILABLE. WE'RE GOING DEVELOP THE IDEA FOR THE GLYCAN AS WELL AND WE'RE TRYING DIFFERENT ENZYMES KNOWN TO PROCESS THE GLYCAN AND BY NOW WE'RE TRYING HEPARANASE TOOL AND USING THE TECHNIQUES WE DEVELOPED AND CREATED THIS HEPARANASE TOOL AND PUT IT ACROSS THE ELECTRODES TO LOOK UNDER THE MICROSCOPE. YOU DON'T SEE ANY WHEN YOU PASS ELECTRICITY THROUGH THE ENZYME AND WHAT YOU ADD IS THE SACCHARIDE AND YOU SEE AN EXTRA [INDISCERNIBLE] POP OUT IN THE DISTRIBUTION AT A HIGHER CONNECTIVITY. WHAT THIS MEANS AND I'M SHOWING IT HERE IS THAT WHEN HAVE YOU THE ENZYME IT STARTS TO HAVE THIS HIGHER SPECK. AND TRYING TO FIGURE OUT IF THERE'S A PATTERN YOU SEE IN THE CHEMICAL STRUCTURES. WE DON'T HAVE TO NARROW THE IMPACT. HERE'S PUBLICATIONS AND WE'RE WORKING TO TEST THE WORK AND WE HAVE SOME DOCTORS AND WE KNOW THE STRUCTURE OF THE CS CHANNEL SO WE CAN USE IT AS WELL. SOME OF THE WORK IN THE POSTERS AND SOME TECHNOLOGY WE USED TO FROM THE PROJECT AND DISCOVERED SOMETHING INTERESTING THE SIGNALS WE SEE CAN BE USED AND HERE'S POST-DOCS WE HAD WORK ON THE PROJECT AND AS WELL AS THE LOCATIONS AND FOR GIVING US SOME STANDARDS FOR TESTING THE EXPERIMENTS AND OBVIOUSLY WE WANT TO THANK THE COMMON FUND FOR FUNDING SUCH A RISKY PROJECT AND WE WOULDN'T BE ABLE TO [INDISCERNIBLE] WITHOUT THEIR SUPPORT. ALL RIGHT. THAT'S IT. >> THANK YOU, XU. ANYBODY HAVE QUESTIONS FOR HIM? IF NOT I WANT TO THANK YOU ALL FOR HANGING WITH US THROUGH THIS LONG AFTERNOON HERE. WE CERTAINLY HAVE SEEN A LOT OF GREAT SCIENCE. APPRECIATE THAT YOU STUCK THROUGH IT. I HOPE YOU ENJOYED IT ALL AS MUCH AS I DID. ARE YOU STILL HERE? >> I'M ALWAYS HERE. OKAY WE'RE GOING TO START AGAIN TOMORROW MORNING AT 8:00 SO IF YOU CAN SIGN ON AND TEST YOUR SYSTEMS IF YOU HAVEN'T ALREADY. AND TEST BEFORE YOU HAVE TO PRESENT AND TOMORROW'S GOING TO BE THE GLYCAN DEMOS ONE WITHIN THIS ZOOM ROOM AND THREE IN THEIR OWN ROOMS. GOT COPIES OF THE AGENDA AND THERE'S COPIES UP ON TO THE WEBSITE YOU CAN PLAN WHICH TALKS AND YOU CAN EITHER STAY IN THIS ROOM OR CLICK OVER TO ONE OF THE ZOOM ROOMS TO TAKE PART IN THOSE. AND EXPECT TO FINISH THE TALKS AROUND 3:00 TOMORROW.